AU753099B2 - Extended cDNAs for secreted proteins - Google Patents

Description

WO 99/25825 PCT/IB98/01862 1 EXTENDED cDNAs FOR SECRETED PROTEINS Background of the Invention The estimated 50.000-100,000 genes scattered along the human chromosomes offer tremendous promise for the understanding, diagnosis, and treatment of human diseases. In addition, probes capable of specifically hybridizing to loci distributed throughout the human genome find applications in the construction of high resolution chromosome maps and in the identification of individuals.

In the past, the characterization of even a single human gene was a painstaking process, requiring years of effort. Recent developments in the areas of cloning vectors, DNA sequencing, and computer technology have merged to greatly accelerate the rate at which human genes can be isolated, sequenced, mapped, and characterized. Cloning vectors such as yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) are able to accept DNA inserts ranging from 300 to 1000 kilobases (kb) or 100-400 kb in length respectively, thereby facilitating the manipulation and ordering of DNA sequences distributed over great distances on the human chromosomes. Automated DNA sequencing machines permit the rapid sequencing of human genes. Bioinformatics software enables the comparison of nucleic acid and protein sequences, thereby assisting in the characterization of human gene products.

Currently, two different approaches are being pursued for identifying and characterizing the genes distributed along the human genome. In one approach, large fragments of genomic DNA are isolated, cloned, and sequenced. Potential open reading frames in these genomic sequences are identified using bioinformatics software. However, this approach entails sequencing large stretches of human DNA which do not encode proteins in order to find the protein encoding sequences scattered throughout the genome. In addition to requiring extensive sequencing, the bio-informatics software may mischaracterize the genomic sequences obtained. Thus, the software may produce false positives in which non-coding DNA is mischaracterizcd as coding DNA or false negatives in which coding DNA is mislabeled as non-coding

DNA.

An alternative approach takes a more direct route to identifying and characterizing human genes. In this approach, complementary DNAs (cDNAs) are synthesized from isolated messenger RNAs (mRNAs) which encode human proteins. Using this approach, sequencing is only performed on DNA which is derived from protein coding portions of the genome. Often, only short stretches of the cDNAs are sequenced to obtain sequences called expressed sequence tags (ESTs). The ESTs may then be used to isolate or purify extended cDNAs which include sequences adjacent to the EST sequences. The extended cDNAs may contain all of the sequence of the EST which was used to obtain them or only a portion of the sequence of the EST which was used to obtain them. In addition, the extended cDNAs may contain the full coding sequence of the gene from which the EST was derived or, alternatively, the extended cDNAs may include portions of the coding sequence of the gene from which the EST was derived. It will be appreciated that there may be several extended cDNAs which include the EST sequence as a result of alternate splicing or the activity of alternative promoters.

WO 99/25825 PCT/IB98/01862 2 In the past, the short EST sequences which could be used to isolate or purify extended cDNAs were often obtained from oligo-dT primed cDNA libraries. Accordingly, they mainly corresponded to the 3' untranslated region of the mRNA. In part. the prevalence of EST sequences derived from the 3' end of the mRNA is a result of the fact that typical techniques for obtaining cDNAs. are not well suited for isolating cDNA sequences derived from the 5' ends of mRNAs. (Adams et al.. Nature 377:174. 1996. Hillier et al., CGnome Res. 6:807-828. 1996).

In addition, in those reported instances where longer cDNA sequences have been obtained, the reported sequences typically correspond to coding sequences and do not include the full 5' untranslated region of the mRNA from which the cDNA is derived. Such incomplete sequences may not include the first exon of the mRNA. particularly in situations where the first exon is short. Furthermore, they may not include some exons, often short ones, which are located upstream of splicing sites. Thus, there is a need to obtain sequences derived from the 5' ends of mRNAs which can be used to obtain extended cDNAs which may include the 5' sequences contained in the 5' ESTs.

While many sequences derived from human chromosomes have practical applications, approaches based on the identification and characterization of those chromosomal sequences which encode a protein product are particularly relevant to diagnostic and therapeutic uses. Of the 50,000-100,000 protein coding genes, those genes encoding proteins which are secreted from the cell in which they are synthesized, as well as the secreted proteins themselves, are particularly valuable as potential therapeutic agents. Such proteins are often involved in cell to cell communication and may be responsible for producing a clinically relevant response in their target cells.

In fact, several secretory proteins, including tissue plasminogen activator, G-CSF, GM-CSF, crythropoietin, human growth hormone, insulin, interferon-a. interferon-3, interferon-y, and interleukin-2, are currently in clinical use. These proteins are used to treat a wide range of conditions, including acute myocardial infarction, acute ischemic stroke, anemia, diabetes, growth hormone deficiency, hepatitis, kidney carcinoma, chemotherapy induced neutropenia and multiple sclerosis. For these reasons, extended cDNAs encoding secreted proteins or portions thereof represent a particularly valuable source of therapeutic agents. Thus, there is a need for the identification and characterization of secreted proteins and the nucleic acids encoding them.

In addition to being therapeutically useful themselves, secretory proteins include short peptides, called signal peptides, at their amino termini which direct their secretion. These signal peptides are encoded by the signal sequences located at the 5' ends of the coding sequences of genes encoding secreted proteins.

Because these signal peptides will direct the extracellular secretion of any protein to which they are operably linked, the signal sequences may be exploited to direct the efficient secretion of any protein by operably linking the signal sequences to a gene encoding the protein for which secretion is desired. This may prove beneficial in gene therapy strategies in which it is desired to deliver a particular gene product to cells other than the cell in which it is produced. Signal sequences encoding signal peptides also find WO 99/25825 PCT/IB98/01862 3 application in simplifying protein purification techniques. In such applications, the extracellular secretion of the desired protein greatly facilitates purification by reducing the number of undesired proteins from which the desired protein must be selected. Thus. there exists a need to identify and characterize the portions of the genes for secretory proteins which encode signal peptides.

Public information on the number of hunan genes for which the promoters and upstream regulatory regions have been identified and characterized is quite limited. In part, this may be due to the difficulty of isolating such regulatory sequences. Upstream regulatory sequences such as transcription factor binding sites are typically too short to be utilized as probes for isolating promoters from human genomic libraries.

Recently, some approaches have been developed to isolate human promoters. One of them consists of making a CpG island library (Cross, S.H. et al.. Purilication of CpG Islands using a Methylated DNA Binding Column. Nature Genetics 6: 236-244 (1994)). The second consists of isolating human genomic DNA sequences containing Spel binding sites by the use of Spel binding protein. (Mortlock et al., Genome Res. 6:327-335, 1996). Both of these approaches have their limits due to a lack of specificity or of comprehensiveness.

5' ESTs and extended cDNAs obtainable therefrom may bejused to efficiently identify and isolate upstream regulatory regions which control the location, developmental stage, rate, and quantity of protein synthesis, as well as the stability of the mRNA. Theil et al., BioFactors 4:87-93 (1993). Once identified and characterized, these regulatory regions may be utilized in gene therapy or protein purification schemes to obtain the desired amount and locations of protein synthesis or to inhibit, reduce, or prevent the synthesis of undesirable gene products.

In addition, ESTs containing the 5' ends of secretory protein genes or extended cDNAs which include sequences adjacent to the sequences of the ESTs may include sequences useful as probes for chromosome mapping and the identification of individuals. Thus, there is a need to identify and characterize the sequences upstream of the 5' coding sequences of genes encoding secretory proteins.

Summnry of the Invention The present invention relates to purified, isolated, or recombinant extended cDNAs which encode secreted proteins or fragments thereof. Preferably, the purified, isolated or recombinant cDNAs contain the entire open reading frame of their corresponding mRNAs, including a start codon and a stop codon. For example, the extended cDNAs may include nucleic acids encoding the signal peptide as well as the mature protein. Alternatively, the extended cDNAs may contain a fragment of the open reading frame. In some embodiments, the fragment may encode only the sequence of the mature protein. Alternatively, the fragment may encode only a portion of the mature protein. A further aspect of the present invention is a nucleic acid which encodes the signal peptide of a secreted protein.

The present extended cDNAs were obtained using ESTs which include sequences derived from the authentic 5' ends of their corresponding mRNAs. As used herein the terms "EST" or EST" refer to the WO 99/25825 PCT/IB98/01862 4 short cDNAs which were used to obtain the extended cDNAs of the present invention. As used herein, the term "extended cDNA" refers to the cDNAs which include sequences adjacent to the 5' EST used to obtain them. The extended cDNAs may contain all or a portion of the sequence of the EST which was used to obtain them. The term "corresponding mRNA" refers to the mRNA which was the template for the cDNA synthesis which produced the 5' EST. As used herein. le teerm "purified" does not require absolute purity; rather, it is intended as a relative definition. Individual extended cDNA clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total huumn DNA. The extended eDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection. Thus, creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 10-l106 fold purification of the native message. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.

As used herein, the term "isolated" requires that the material be removed from its original environment the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.

As used herein, the term "recombinant" means that the extended cDNA is adjacent to "backbone" nucleic acid to which it is not adjacent in its natural environment. Additionally, to be "enriched" the extended cDNAs will represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid backbone molecules. Backbone molecules according to the present invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. Preferably, the enriched extended cDNAs represent 15% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. More preferably, the enriched extended cDNAs represent 50% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. In a highly preferred embodiment, the enriched extended cDNAs represent 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. "Stringent", "moderate," and "low" hybridization conditions are as defined in Example 29.

Unless otherwise indicated, a "complementary" sequence is fully complementary. Thus, extended cDNAs encoding secreted polypeptides or fragments thereof which are present in cDNA libraries in which one or more extended cDNAs encoding secreted polypeptides or fragments thereof make up 5% or more of the number of nucleic acid inserts in the backbone molecules are "enriched recombinant extended cDNAs" WO 99/25825 PCT/IB98/01862 as derined herein. Likewise, extended cDNAs encoding secreted polypeptides or fragments thereof which are in a population of plasmids in which one or more extended cDNAs of the present invention have been inserted such that they represent 5% or more of the number of inserts in the plasmid backbone are enriched recombinant extended cDNAs" as defined herein. However. extended cDNAs encoding secreted polypeptides or fragments thereof which are in cDNA libraries in which the extended cDNAs encoding secreted polypeptides or fragments thereof constitute less than 5% of the number of nucleic acid inserts in the population of backbone molecules, such as libraries in which backbone molecules having a cDNA insert encoding a secreted polypeptide are extremely rare. are not "enriched recombinant extended cDNAs." In particular, the present invention relates to extended cDNAs which were derived from genes encoding secreted proteins. As used herein, a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal peptides in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g.

soluble proteins), or partially receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.

Extended cDNAs encoding secreted proteins may include nucleic acid sequences, called signal sequences, which encode signal peptides which direct the extracellular secretion of the proteins encoded by the extended cDNAs. Generally, the signal peptides are located at the amino termini of secreted proteins.

Secreted proteins are translated by ribosomes associated with the "rough" endoplasmic reticulum.

Generally, secreted proteins are co-translationally transferred to the membrane of the endoplasmic reticulum. Association of the ribosome with the endoplasmic reticulum during translation of secreted proteins is mediated by the signal peptide. The signal peptide is typically cleaved following its cotranslational entry into the endoplasmic reticulum. After delivery to the endoplasmic reticulum, secreted proteins may proceed through the Golgi apparatus. In the Golgi apparatus, the proteins may undergo posttranslational modification before entering secretory vesicles which transport them across the cell membrane.

The extended cDNAs of the present invention have several important applications. For example, they may be used to express the entire secreted protein which they encode. Alternatively, they may be used to express portions of the secreted protein. The portions may comprise the signal peptides encoded by the extended cDNAs or the mature proteins encoded by the extended cDNAs the proteins generated when the signal peptide is cleaved off). The portions may also comprise polypeptides having at least consecutive amino acids encoded by the extended cDNAs. Alternatively, the portions may comprise at least consecutive amino acids encoded by the extended cDNAs. In some embodiments, the portions may comprise at least 25 consecutive amino acids encoded by the extended cDNAs. In other embodiments, the portions may comprise at least 40 amino acids encoded by the extended cDNAs.

Antibodies which specifically recognize the entire secreted proteins encoded by the extended cDNAs or fragments thereof having at least 10 consecutive amino acids, at least 15 consecutive amino WO 99/25825 PCT/IB98/01862 6 acids. at least 25 consecutive amino acids, or at least 40 consecutive amino acids may also be obtained as described below. Antibodies which specifically recognize the mature protein generated when the signal peptide is cleaved may also be obtained as described below. Similarly, antibodies which specifically recognize the signal pcptides encoded by the extended cDNAs may also be obtained.

In some embodiments, the extended cDNAs include the signal sequence. In other embodiments, the extended cDNAs may include the full coding sequence for the nature protein the protein generated when the signal polypeptide is cleaved off). In addition, the extended cDNAs may include regulatory regions upstream of the translation start site or downstream of the stop codon which control the amount.

location, or developmental stage of gene expression. As discussed above, secreted proteins are therapeutically important. Thus, the proteins expressed fromt the cDNAs may be useful in treating or controlling a variety of human conditions. The extended cDNAs may also be used to obtain the corresponding genomic DNA. The term "corresponding genomic DNA" refers to the genomic DNA which encodes mRNA which includes the sequence of one of the strands of the extended cDNA in which thymidine residues in the sequence of the extended cDNA are replaced by uracil residues in the mRNA.

The extended cDNAs or genomic DNAs obtained therefrom may be used in forensic procedures to identify individuals or in diagnostic procedures to identify individuals having genetic diseases resulting from abnormal expression of the genes corresponding to the extended cDNAs. In addition, the present invention is useful for constructing a high resolution map of the human chromosomes.

The present invention also relates to secretion vectors capable of directing the secretion of a protein of interest. Such vectors may be used in gene therapy strategies in which it is desired to produce a gene product in one cell which is to be delivered to another location in the body. Secretion vectors may also facilitate the purification of desired proteins.

The present invention also relates to expression vectors capable of-directing the expression of an inserted gene in a desired spatial or temporal manner or at a desired level. Such vectors may include sequences upstream of the extended cDNAs such as promoters or upstream regulatory sequences.

In addition, the present invention may also be used for gene therapy to control or treat genetic diseases. Signal peptides may also be fused to heterologous proteins to direct their extracellular secretion.

One embodiment of the present invention is a purified or isolated nucleic acid comprising the sequence of one of SEQ ID NOs: 134-180 or a sequence complementary thereto. In one aspect of this embodiment, the nucleic acid is recombinant.

Another embodiment of the present invention is a purified or isolated nucleic acid comprising at least 10 consecutive bases of the sequence of one of SEQ ID NOs: 134-180 or one of the sequences complementary thereto. In one aspect of this embodiment, the nucleic acid comprises at least 15, 25, 30, 75, or 100 consecutive bases of one of the sequences of SEQ ID NOs: 134-180 or one of the sequences complementary thereto. The nucleic acid may be a recombinant nucleic acid.

Another embodiment of the present invention is a purified or isolated nucleic acid of at least WO 99/25825 PCT/IB98/01862 7 bases capable of hybridizing under stringent conditions to the sequence of one of SEQ ID NOs: 134-180 or a sequence complementary to one of the sequences of SEQ ID NOs: 134-180. In one aspect of this embodiment, the nucleic acid is recombinant.

Another embodiment of the present invention is a purified or isolated nucleic acid comprising the full coding sequences of one of SEQ ID NOs: 134-180. wherein the full coding sequence optionally comprises the sequence encoding signal peptide as well as the sequence encoding mature protein. In one aspect of this embodiment, the nucleic acid is recombinant.

A further embodiment of the present invention is a purified or isolated nucleic acid comprising the nucleotides of one of SEQ ID NOs: 134-180 which encode a mature protein. In one aspect of this embodiment, the nucleic acid is recombinant.

Yet another embodiment of the present invention is a purified or isolated nucleic acid comprising the nucleotides of one of SEQ ID NOs: 134-180 which encode the signal peptide. hi one aspect of this embodiment, the nucleic acid is recombinant.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of one of the sequences of SEQ ID NOs: 181-227.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a mature protein included in one of the sequences of SEQ ID NOs: 181- 227.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a signal peptide included in one of the sequences of SEQ ID NOs: 181- 227.

Yet another embodiment of the present invention is a purified or isolated protein comprising the sequence of one of SEQ ID NOs: 181-227.

Another embodiment of the present invention is a purified or isolated polypeptide comprising at least 10 consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227. In one aspect of this embodiment, the purified or isolated polypeptide comprises at least 15, 20, 25. 35, 50, 75, 100, 150 or 200 consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227. In still another aspect, the purified or isolated polypeptide comprises at least 25 consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227.

Another embodiment of the present invention is an isolated or purified polypeptide comprising a signal peptide of one of the polypeptides of SEQ ID NOs: 181-227.

Yet another embodiment of the present invention is an isolated or purified polypeptide comprising a mature protein of one of the polypeptides of SEQ ID NOs: 181-227.

A further embodiment of the present invention is a method of making a protein comprising one of the sequences of SEQ ID NO: 181-227, comprising the steps of obtaining a cDNA comprising one of the sequences of sequence of SEQ ID NO: 134-180, inserting the cDNA in an expression vector such that the WO 99/25825 PCT/IB98/01862 8 cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the protein encoded by said cDNA. In one aspect of this embodiment, the method further comprises the step of isolating the protein.

Another embodiment of the present invention is a protein obtainable by the method described in the preceding paragraph.

Another embodiment of the present invention is a method of making a protein comprising the amino acid sequence of the mature protein contained in one of the sequences of SEQ ID NO: 181-227, comprising the steps of obtaining a cDNA comprising one of the nucleotides sequce eqence of sequence oSEQ ID NO: 134- 180 which encode for the mature protein, inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the mature protein encoded by the cDNA. In one aspect of this embodiment, the method further comprises the step of isolating the protein.

Another embodiment of the present invention is a mature protein obtainable by the method described in the preceding paragraph.

Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the sequence of one of SEQ ID NOs: 134-180 or a sequence complementary thereto described herein.

Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the full coding sequences of one of SEQ ID NOs: 134-180, wherein the full coding sequence comprises the sequence encoding signal peptide and the sequence encoding mature protein described herein.

Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the nuclcotides of one of SEQ ID NOs: 134-180 which encode a mature protein which are described herein.

Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the nucleotides of one of SEQ ID NOs: 134-180 which encode the signal peptide which are described herein.

Another embodiment of the present invention is a purified or isolated antibody capable of specifically binding to a protein having the sequence of one of SEQ ID NOs: 181-227. In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 10 consecutive amino acids of the sequence of one of SEQ ID NOs: 181-227.

Another embodiment of the present invention is an array of cDNAs or fragments thereof of at least nucleotides in length which includes at least one of the sequences of SEQ ID NOs: 134-180, or one of the sequences complementary to the sequences of SEQ ID NOs: 134-180, or a fragment thereof of at least 15 consecutive nucleotides. In one aspect of this embodiment, the array includes at least two of the sequences of SEQ ID NOs: 134-180, the sequences complementary to the sequences of SEQ ID NOs: 134- 180, or fragments thereof of at least 15 consecutive nucleotides. In another aspect of this embodiment, the array includes at least five of the sequences of SEQ ID NOs: 134-180, the sequences complementary to the sequences of SEQ ID NOs: 134-180, or fragments thereof of at least 15 consecutive nucleotides.

A further embodiment of the invention encompasses purified polynucleotides comprising an insert from a clone deposited in ATCC accession No. 98619 or a fragment thereof comprising a contiguous span of at least 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200 nucleotides of said insert. An additional embodiment of the invention encompasses purified polypeptides which comprise, consist of, or consist essentially of an amino acid sequence encoded by the insert from a clone deposited in ATCC accession No. 98619, as well as polypeptides which comprise a fragment of said amino acid sequence consisting of a signal peptide, a mature protein, or a contiguous span of at least 5, 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200 amino acids encoded by said insert.

An additional embodiment of the invention encompasses purified polypeptides which comprise a contiguous span of at least 5, 8, 10, 12, 15, 25, 40, 60, 100, or 200 amino acids of SEQ ID NOs: 185, 186, 191, 192, 200, 20 201, 213, 214, 215, or 227, wherein said contiguous span comprises at least one of the amino acid positions which was not shown to be identical to a public sequence in any of Figures 9 to 16. Also encompassed by the invention are purified polynucleotides encoding said polypeptides.

In another embodiment, the present invention provides a purified or isolated polypeptide comprising a sequence of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

In another embodiment, the present invention provides a purified or isolated polypeptide comprising a mature protein of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

In another embodiment, the present invention provides a purified or isolated polypeptide comprising a signal peptide of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

In another embodiment, the present invention provides a purified or isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) at least 58 consecutive amino acids of SEQ ID NO:186; 9A b) at least 33 consecutive amino acids of SEQ ID NO:197; c) at least 64 consecutive amino acids of SEQ ID NO:201; d) at least 62 consecutive amino acids of SEQ ID NO:214; e) at least 23 consecutive amino acids of SEQ ID NO:223; f) at least 10 consecutive amino acids of SEQ ID NO:227; and g) any fragment comprising at least 10 amino acids of SEQ ID NO: 186, 197, 201, 214, 223, or 227, wherein the polypeptide has at least one biological activity of a polypeptide of the invention.

In another embodiment, the present invention provides a purified or isolated polypeptide comprising a sequence which is at least 95% identical to any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227, wherein the polypeptide has at least one biological activity of a polypeptide of the invention.

Preferably, the biological activity is cell adhesion.

In another embodiment, the present invention provides a purified or isolated nucleic acid sequence encoding a polypeptide of the invention, or a sequence complementary thereto.

Preferably, the nucleic acid comprises a sequence selected from SEQ ID NOs: 139, 154, 150, 167, 176 or 180.

20 In another embodiment, the present invention provides a method of making a polypeptide comprising one of the sequences of SEQ ID NO: 186, 197, 201, 214, 223 or 227, the method comprising the steps of: obtaining a nucleic acid comprising one of the sequences of SEQ ID NO: 139, 154, 150, 167, 176 or 180; inserting said nucleic acid in an expression vector such that said nucleic acid is operably linked to a promoter; and introducing said expression vector into a host cell whereby said host cell produces the polypeptide encoded by said nucleic acid.

Preferably, the method further comprises the step of isolating said polypeptide.

In another embodiment, the present invention provides a polypeptide obtainable by a method of the invention.

In another embodiment, the present invention provides a host cell containing a recombinant nucleic acid according to the invention.

9B In another embodiment, the present invention provides a purified or isolated antibody capable of specifically binding to a polypeptide having a sequence of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

In another embodiment, the present invention provides an array of polynucleotides comprising at least one polynucleotide selected from the group consisting of: a) a polynucleotide sequence of SEQ ID NOs: 139, 150, 154, 167, 176 or 180; b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: 186, 197, 201, 214, 223 or 227, wherein the polypeptide fragment has at least one biological activity of the polypeptide provided as SEQ ID NO: 186, 197, 201, 214, 223 or 227; c) a polynucleotide encoding a signal sequence of SEQ ID NO: 186, 197, 201, 214, 223 or 227, d) a polynucleotide encoding a polypeptide which is at least identical to any of one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227, wherein the polypeptide has at least one biological activity of the polypeptide provided as SEQ ID NO: 186, 197, 201, 214, 223 or 227; and e) a polynucleotide sequence complementary to any one of a) to d).

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Brief Description of the Drawings Figure 1 is a summary of a procedure for obtaining cDNAs which have been selected to include the 5' ends of the mRNAs from which they are derived.

Figure 2 is an analysis of the 43 amino terminal amino acids of all human SwissProt proteins to determine the frequency of false positives and false negatives using the techniques for signal peptide identification described herein.

Figure 3 shows the distribution of von Heijne scores of 5' ESTs in each of the categories described herein and the probability that these 5' ESTs encode a signal peptide.

Figure 4 shows the distribution of 5' ESTs in each category and the number of 5' ESTs in each category having a given minimum von Heijne's score.

Figure 5 shows the tissues from which the mRNAs corresponding to the 5' ESTs in each of the categories described herein were obtained.

Figure 6 is a map of pED6dpc2. PED6dpc2 is derived from pED6dpcl by insertion of a new polylinker to facilitate cDNA cloning. SST cDNAs are cloned between EcoRI and Notl. PED vectors are described in Kaufman et al.

(1991), NAR 19:4485-4490.

Figure 7 provides a schematic description of the promoters isolated and the way they are assembled with the corresponding 5' tags.

Figure 8 describes the transcription factor binding sites present in each of these promoters.

Figure 9 depicts an amino acid alignment between SEQ ID NO: 214 and 0 murine SH3BGRL (AF042081). Identities are shown by and conservative substitutions by Cell attachment motif (RGD) is in bold type and the proline rich region is underlined.

WO 99/25825 PCT/IB98/01862 Figure 10 depicts a multiple amino acid alignment between SEQ ID NOs: 185 and 215, and murine MEK binding partner (AF082526). Positions conserved in all three proteins are indicated by Figure 11 depicts an amino acid alignment between SEQ ID NO: 186 and murine claudin-2 (AF072128). Identities are shown by and conservative substitutions by Figure 12 depicts an amino acid alignment between SEQ [I NO: 213 and GMF-y (AB001993). In the alignment present the translation starts at position 2 of SEQ ID NO: 166. The actual start methionine of SEQ ID NO: 213 appears to be at position 13. Identities are shown by and conservative substitutions by Figure 13 depicts an amino acid alignment between SEQ ID NO: 191 and Derwent Protein Sequence Database Accession NO: W36955. Identities are shown by and conservative substitutions by Figure 14 depicts an amino acid alignment between SEQ ID NO: 200 and human Ring zinc finger protein (AF037204). Amino acids defining an EGF-like domain are highlighted. The region defining an almost perfect Ring Finger domain is boxed. Identities are shown by and conservative substitutions by Figure 15 depicts an amino acid alignment between SEQ ID NO: 192 and Y15286. Identities are shown by and conservative substitutions by Figure 16 depicts a multiple amino acid alignment between SEQ ID NOs: 201 and 227, and human stomatin (x85116). Positions conserved in all three proteins are indicated by The amino acid sequences in SEQ ID NOs: 201 and 227 differ in their N-terminal sequences: segment 1-76 (SEQ ID NO: 201) and segment 1-26 (SEQ ID NO: 227). The remainder of these 2 proteins are 99.5% identical. The band 7 protein family signature is boxed. The microbody C-terminal targeting signal appears in bold type.

Detailed Description of the Preferred Embodiment 1. Obtaining 5' ESTs The present extended cDNAs were obtained using 5' ESTs which were isolated as described below.

A. Chemical Methods for Obtaining mRNAs having Intact 5' Ends In order to obtain the 5' ESTs used to obtain the extended cDNAs of the present invention, mRNAs having intact 5' ends must be obtained. Currently, there are two approaches for obtaining such mRNAs.

One of these approaches is a chemical modification method involving derivatization of the 5' ends of the mRNAs and selection of the derivatized mRNAs. The 5' ends of eukaryotic mRNAs possess a structure referred to as a "cap" which comprises a guanosine methylated at the 7 position. The cap is joined to the first transcribed base of the mRNA by a 5'-triphosphate bond. In some instances, the 5' guanosine is methylated in both the 2 and 7 positions. Rarely, the 5' guanosine is trimethylated at the 2, 7 and 7 positions. In the chemical method for obtaining mRNAs having intact 5' ends, the 5' cap is specifically derivatized and coupled to a reactive group on an immobilizing substrate. This specific derivatization is WO 99/25825 PCT/IB98/01862 11 based on the fact that only the ribose linked to the methylated guanosine at the 5' end of the mRNA and the ribose linked to the base at the 3' terminus of the mRNA. possess 3'-cis diols. Optionally, where the 3' terminal ribose has a 3'-cis diol, the 3'-cis diol at the 3' end may be chemically modified, substituted, converted, or eliminated, leaving only the ribose linked to the methylated guanosine at the 5' end of the mRNA with a 3'-cis diol. A variety of techniques are available for climinating the 3'-cis diol on the 3' terminal ribose. For example, controlled alkaline hydrolysis may be used to generate mRNA fragments in which the 3' terminal ribose is a 3'-phosphate. 2'-phosphate or 3')-cyclophosphate. Thereafter, the fragment which includes the original 3' ribose may be eliminated from the mixture through chromatography on an oligo-dT column. Alternatively, a base which lacks the 3'-cis diol may be added to the 3' end of the mRNA using an RNA ligase such as T4 RNA ligase. Example I below describes a method for ligation of pCp to the 3' end of messenger RNA.

EXAMPLE 1 Lication of the Nucleoside Diphosphate pCp to the 3' End of Messenger RNA.

1 pg of RNA was incubated in a final reaction medium of 10 Al in the presence of 5 U of T4 phage RNA ligase in the buffer provided by the manufacturer (Gibco BRL), 40 U of the RNase inhibitor RNasin (Promega) and, 2 pil of "pCp (Amersham #PB 10208). The incubation was performed at 37°C for 2 hours or overnight at 7-80C.

Following modification or elimination of the 3'-cis diol at the 3' ribose, the 3'-cis diol present at the 5' end of the mRNA may be oxidized using reagents such as NaBl-., NaBI-hCN. or sodium periodate, thereby converting the 3'-cis diol to a dialdehyde. Example 2 describes the oxidation of the 3'-cis diol at the 5' end of the mRNA with sodium periodate.

EXAMPLE 2 Oxidation of 2' 3'-cis diol at the 5' End of the mRNA 0.1 OD unit of either a capped oligoribonucleotide of 47 nuclcotides (including the cap) or an uncapped oligoribonucleotide of 46 nucleotides were treated as follows. The oligoribonucleotides were produced by in vitro transcription using the transcription kit "AmpliScribe T7" (Epicentre Technologies).

As indicated below, the DNA template for the RNA transcript contained a single cytosine. To synthesize the uncapped RNA, all four NTPs were included in the in vitro transcription reaction. To obtain the capped RNA, GTP was replaced by an analogue of the cap, m7G(5')ppp(5')G. This compound, recognized by polymerase, was incorporated into the 5' end of the nascent transcript during the step of initiation of transcription but was not capable of incorporation during the extension step. Consequently, the resulting RNA contained a cap at its 5' end. The sequences of the oligoribonucleotides produced by the in vitro transcription reaction were: +Cap: WO 99/25825 PCT/IB98/01862 12 5'm7GpppGCAUCCUACUCCCAUCCAAUUCCACCCUAACUCCUCCCAUCUCCAC-3' (SEQ ID NO: 1) -Cap: 5'-pppGCAUCCUACUCCCAUCCAAUUCCACCCUAACUCCUCCCAUCUCCAC-3' (SEQ ID NO:2) The oligoribonucleoides were dissolved in 9 ;tl of acetate buffer (0.1 M sodium acetate. pH- 5.2) and 3 tpl of freshly prepared 0. I M sodium periodate solution. The mixture was incubated for I hour in thle dark at 4CC or room temperature. Thereafter, the reaction was stopped by adding 4 ~l of 10% ethylene glycol. The product was ethanol precipitated, resuspended in 10il or more of water or appropriate buffer and dialyzed against water, The resulting aldehyde groups may then be coupled to molecules having a reactive amine group.

such as hydrazine, carbazide, thiocarbazide or semicarbazide groups, in order to facilitate enrichment of the ends of the mRNAs. Molecules having reactive amine groups which are suitable for use in selecting mRNAs having intact 5' ends include avidin, proteins, antibodies, vitamins, ligands capable of specifically binding to receptor molecules, or oligonucleotides. Example 3 below describes the coupling of the resulting dialdehyde to biotin.

EXAMPLE 3 Coupling of the Dialdehyde with Biotin The oxidation product obtained in Example 2 was dissolved in 50 pil of sodium acetate at a pH of between 5 and 5.2 and 50 ptl of freshly prepared 0.02 M solution of biotin hydrazide in a methoxyethanol/water mixture of formula:

H

NH

2 _NH -(CH n- -4 I I In the compound used in these experiments, n=5, and the solid black dots represent oxygen.

However, it will be appreciated that other commercially available hydrazidcs may also be used, such as molecules of the formula above in which n varies from 0 to The mixture was then incubated for 2 hours at 370C. Following the incubation, the mixture was precipitated with ethanol and dialyzed against distilled water.

Example 4 demonstrates the specificity of the biotinylation reaction.

WO 99/25825 PCT/IB98/01862 13 EXAMPLE 4 Specificity of Biotinylation The specificity of the biotinylation for capped mRNAs was evaluated by gel electrophoresis of tile following samples: Sample I. The 46 nucleotide uncapped in vitro transcript prepared as in Example 2 and labeled with 3 "pCp as described in Example I.

Sample 2. The 46 nucleotide uncapped in vitro transcript prepared as in Example 2, labeled with "pCp as described in Example 1. treated with the oxidation reaction of Example 2. and subjected to the biotinylation conditions of Example 3.

Sample 3. The 47 nucleotide capped in vitro transcript prepared as in Example 2 and labeled with UpCp as described in Example 1.

Sample 4. The 47 nucleotide capped in vitro transcript prepared as in Example 2, labeled with 2 pCp as described in Example 1, treated with the oxidation reaction of Example 2, and subjected to the biotinylation conditions of Example 3.

Samples I and 2 had identical migration rates, demonstrating that the uncapped RNAs were not oxidized and biotinylated. Sample 3 migrated more slowly than Samples 1 and 2, while Sample 4 exhibited the slowest migration. The difference in migration of the RNAs in Samples 3 and 4 demonstrates that the capped RNAs were specifically biotinylated.

In some cases, mRNAs having intact 5' ends may be enriched by binding the molecule containing a reactive amine group to a suitable solid phase substrate such as the inside of the vessel containing the mRNAs, magnetic beads, chromatography matrices, or nylon or nitrocellulose membranes. For example, where the molecule having a reactive amine group is biotin, the solid phase substrate may be coupled to avidin or streptavidin. Alternatively, where the molecule having the reactive amine group is an antibody or receptor ligand, the solid phase substrate may be coupled to the cognate antigen or receptor. Finally, where the molecule having a reactive amine group comprises an oligonucleotide, the solid phase substrate may comprise a complementary oligonucleotide.

The mRNAs having intact 5' ends may be released from the solid phase following the enrichment procedure. For example, where the dialdehyde is coupled to biotin hydrazide and the solid phase comprises streptavidin, the mRNAs may be released from the solid phase by simply heating to 95 degrees Celsius in 2% SDS. In some methods, the molecule having a reactive amine group may also be cleaved from the mRNAs having intact 5' ends following enrichment. Example 5 describes the capture of biotinylated mRNAs with streptavidin coated beads and the release of the biotinylated mRNAs from the beads following enrichment.

WO 99/25825 PCT/IB98/01862 14 EXAMPLE Capture and Release of Biotinylated mRNAs Using Strepatividin Coated Beads The strcptavidin-coated magnetic beads were prepared according to the manufacturer's instructions (CPG Inc.. USA). The biotinylated mRNAs were added to a hybridization buffer (1.5 M NaCI, pH 5 6).

After incubating for 30 minutes, the unbound and nonbiotinylated material was removed. The beads were washed several times in water with 1% SDS. The beads obtained were incubated for 15 minutes at 950C in water containing 2% SDS.

Example 6 demonstrates the efficiency with which biotinylated mRNAs were recovered from the streptavidin coated beads.

EXAMPLE 6 Efficiency of Recovery of Biotinylated mRNAs The efficiency of the recovery procedure was evaluated as follows. RNAs were labeled with "pCp.

oxidized, biotinylated and bound to streptavidin coated beads as described above. Subsequently, the bound RNAs were incubated for 5, 15 or 30 minutes at 95 0 C in the presence of 2% SDS.

The products of the reaction were analyzed by electrophoresis on 12% polyacrylamide gels under denaturing conditions (7 M urea). The gels were subjected to autoradiography. During this manipulation, the hydrazone bonds were not reduced.

Increasing amounts of nucleic acids were recovered as incubation times in 2% SDS increased, demonstrating that biotinylated mRNAs were efficiently recovered.

In an alternative method for obtaining mRNAs having intact 5' ends, an oligonucleotide which has been derivatized to contain a reactive amine group is specifically coupled to mRNAs having an intact cap.

Preferably, the 3' end of the mRNA is blocked prior to the step in which the aldehyde groups are joined to the derivatized oligonucleotide, as described above, so as to prevent the derivatized oligonucleotide from being joined to the 3' end of the mRNA. For example, pCp may be attached to the 3' end of the mRNA using T4 RNA ligase. However, as discussed above, blocking the 3' end of the mRNA is an optional step.

Derivatized oligonucleotides may be prepared as described below in Example 7.

EXAMPLE 7 Derivatization of the Oliconucleotide An oligonucleotide phosphorylated at its 3' end was converted to a 3' hydrazide in 3' by treatment with an aqueous solution of hydrazine or of dihydrazide of the formula H2N(RI)NH2 at about I to 3 M, and at pH 4.5, in the presence of a carbodiimide type agent soluble in water such as 1-ethyl-3-(3dimethylaminoopryl)carbodiimide at a final concentration of 0.3 M at a temperature of 80C overnight.

The derivatized oligonucleotide was then separated from the other agents and products using a standard technique for isolating oligonucleotides.

WO 99/25825 PCT/IB98/01862 As discussed above, the nRNAs to be enriched may be treated to eliminate the 3' OH groups which may be present thereon. This may be accomplished by enzymatic ligation of sequences lacking a 3' OH.

such as pCp. as described above in Example 1. Alternatively, the 3' OH groups may be eliminated by alkaline hydrolysis as described in Example 8 below.

EXAMPLE 8 Alkaline Hydrolysis of mRNA The mRNAs may be treated with alkaline hydrolysis as follows. In a total volume of 10Otl of 0. IN sodium hydroxide, 1.5pg mRNA is incubated for 40 to 60 minutes at 40C. The solution is neutralized with acetic acid and precipitated with ethanol.

Following the optional elimination of the 3' OH groups, the diol groups at the 5' ends of the mRNAs are oxidized as described below in Example 9.

EXAMPLE 9 Oxidation of Diols Up to 1 OD unit of RNA was dissolved in 9 ul of buffer (0.1 M sodium acetate, pH 6-7 or water) and 3 ptl of freshly prepared 0.1 M sodium periodate solution. The reaction was incubated for I h in the dark at 4CC or room temperature. Following the incubation, the reaction was stopped by adding 4 up of cthylene glycol. Thereafter the mixture was incubated at room temperature for 15 minutes. After ethanol precipitation, the product was resuspended in 10l or more of water or appropriate buffer and dialyzed against water.

Following oxidation of the diol groups at the 5' ends of the mRNAs, the derivatized oligonucleotide was joined to the resulting aldehydes as described in Example EXAMPLE Reaction of Aldehydes with Derivatized Oligonucleotides The oxidized mRNA was dissolved in an acidic medium such as 50 ul of sodium acetate pH 4-6. il of a solution of the derivatized oligonucleotide was added such that an mRNA:derivatized oligonucleotide ratio of 1:20 was obtained and mixture was reduced with a borohydride. The mixture was allowed to incubate for 2 h at 37°C or overnight (14 h) at 10 0 C. The mixture was ethanol precipitated, resuspended in 10pl or more of water or appropriate buffer and dialyzed against distilled water. If desired, the resulting product may be analyzed using acrylamide gel electrophoresis, HPLC analysis, or other conventional techniques.

Following the attachment of the derivatized oligonucleotide to the mRNAs, a reverse transcription reaction may be performed as described in Example 11 below.

WO 99/25825 PCT/IB98/01862 16 EXAMPLE 11 Reverse Transcription of mRNAs An oligodeoxyribonucleotide was derivatized as follows. 3 OD units of an oligodcoxyribonucleotide of sequence ATCAAGAATrCGCACGAGACCATTA (SEQ ID NO:3) having and 3'-P ends were dissolved in 70 tl of a 1.5 M hydroxybenzotriazole solution. pH 5.3, prepared in dimethylformamide/water (75:25) containing 2 ltg of 1-elthyl.3-(3-dinmethlinyl opropy)carbodiimide. The mixture was incubated for 2 h 30 min at 220C. The mixture was then precipitated twice in LiCIO.acetone.

The pellet was resuspended in 2(X) pl of 0.25 M hydrazine and incubated at 80C from 3 to 14 h. Following the hydrazine reaction, the mixture was precipitated twice in LiCIOJacetone.

The messenger RNAs to be reverse transcribed were extracted from blocks of placenta having sides of 2 cm which had been stored at -800C. The mRNA was extracted using conventional acidic phenol techniques. Oligo-dT chromatography was used to purify the mRNAs. The integrity of the mRNAs was checked by Northern-blotting.

The diol groups on 7 pg of the placental mRNAs were oxidized as described above in Example 9.

The derivatized oligonucleotide was joined to the mRNAs as described in Example 10 above except that the precipitation step was replaced by an exclusion chromatography step to remove derivatized oligodeoxyribonucleotides which were not joined to mRNAs. Exclusion chromatography was performed as follows: ml of AcA34 (BioSepra#230151) gel were equilibrated in 50 ml of a solution of 10 mM Tris pH 8.0, 300 mM NaCI, 1 mM EDTA, and 0.05% SDS. The mixture was allowed to sediment. The supernatant was eliminated and the gel was resuspended in 50 ml of buffer. This procedure was repeated 2 or 3 times.

A glass bead (diameter 3 mm) was introduced into a 2 ml disposable pipette (length 25 cm). The pipette was filled with the gel suspension until the height of the gel stabilized at 1 cm from the top of the pipette. The column was then equilibrated with 20 ml of equilibration buffer (10 mM Tris HCI pH 7.4, mM NaCI).

.tl of the mRNA which had been reacted with the derivatized oligonucleotide were mixed in 39 pl of 10 mM urea and 2 tl of blue-glycerol buffer, which had been prepared by dissolving 5 mg of bromophenol blue in 60% glycerol and passing the mixture through a filter with a filter of diameter.

0.45 .m.

The column was loaded. As soon as the sample had penetrated, equilibration buffer was added. 100 ptl fractions were collected. Derivatized oligonucleotide which had not been attached to mRNA appeared in fraction 16 and later fractions. Fractions 3 to 15 were combined and precipitated with ethanol.

The mRNAs which had been reacted with the derivatized oligonucleotide were spotted on a nylon membrane and hybridized to a radioactive probe using conventional techniques. The radioactive probe used in these hybridizations was an oligodeoxyribonucleotide of sequence TAATGGTCTCGTGCGAATTCTTGAT (SEQ ID NO:4) which was anticomplementary to the derivatized WO 99/25825 PCT/IB98/01862 17 oligonucleotide and was labeled at its 5' end with P. 1/10th of the mRNAs which had been reacted with the derivatized oligonucleotide was spotted in two spots on the membrane and the membrane was visualized by autoradiography after hybridization of thile probe. A signal was observed. indicating that the derivatized oligonucleotide had been joined to thile mRNA.

The remaining 9/10 of the mRNAs which had been reacted with the derivatized oligonucleotide was rcverse transcribed as follows. A reverse transcription reaction was carried out with reverse transcriptase following thile Ilmainufacturer's instructions. To prime the reaction. 50 pmol of nonamers with random sequence were used.

A portion or the resulting cDNA was spotted on a positively charged nylon membrane using conventional methods. Thile cDNAs were spotted onil thile membrane after the cDNA:RNA heeroduplexes had been subjected to an alkaline hydrolysis in order to eliminate thile RNAs. An oligonucleotide having a sequence identical to that of the derivatized oligonucleotide was labeled at its 5' end with "P and hybridized to the cDNA blots using conventional techniques. Single-stranded cDNAs resulting from the reverse transcription reaction were spotted on the membrane. As controls, the blot contained I pmol, 100 fmol, fmol 10 fmol and I fmol respectively of a control oligodeoxyribonu.cleotide of sequence identical to that of thile derivatized oligonucleotide. The signal observed in thile spots containing the cDNA indicated that approximately 15 fmol of the derivatized oligonucleotide had been reverse transcribed.

These results demonstrate that the reverse transcription can be performed through the cap and, in particular, that reverse transcriptase crosses the bond of the cap of cukaryotic messenger RNAs.

The single stranded cDNAs obtained after thile above first strand synthesis were used as template for PCR reactions. Two types of reactions were carried out. First, specific amplification of the mRNAs for the alpha globin, dehydrogenase, pp1 5 and elongation factor E4 were carried out using the following pairs of oligodeoxyribonucleotide primers.

alpha-globin GLO-S: CCG ACA AGA CCA ACG TCA AGG CCG C (SEQ ID GLO-As: TCA CCA GCA GGC AGT GGC TTA GGA G 3' (SEQ ID NO:6) dchydrogenase 3 DH-S: AGT GAT TCC TGC TAC TTT GGA TGG C (SEQ ID NO:7) 3 DH-As: GCT TGG TCT TGT TCT GGA GTT TAG A (SEQ ID NO:8) pp 1 PP 15-S: TCC AGA ATG GGA GAC AAG CCA ATT T (SEQ ID NO:9) PP 15-As: AGG GAG GAG GAA ACA GCG TGA GTC C (SEQ ID NO: Elongation factor E4 EFAI-S: ATG GGA AAG GAA AAG ACT CAT ATC A (SEQ ID NO: 11) EFIA-As: AGC AGC AAC AAT CAG GAC AGC ACA G (SEQ ID NO:12) Non-specific amplifications were also carried out with the antisense (_As) WO 99/25825 PCT/IB98/01862 18 Soligodeoxyribonucleotides of the pairs described above and a primer chosen from the sequence of the derivatized oligodeoxyribonucleotide (ATCAAGAATTCGCACGAGACCATTA) (SEQ ID NO: 13).

A 1.5% agarose gel containing the following samples corresponding to the PCR products of reverse transcription was stained with ethidium bromide. (1/20th of the products of reverse transcription were used for each PCR reaction).

Sample 1: The products of a PCR reaction using the globin primers of SEQ ID NOs 5 and 6 in the presence of cDNA.

Sample 2: The products of a PCR reaction using the globin primers of SEQ ID NOs 5 and 6 in the absence of added cDNA.

Sample 3: The products of a PCR reaction using the dehydrogenase primers of SEQ ID NOs 7 and 8 in the presence of cDNA.

Sample 4: The products of a PCR reaction using the dehydrogenase primers of SEQ ID NOs 7 and S in the absence of added cDNA.

Sample 5: The products of a PCR reaction using the ppl5 primers of SEQ ID NOs 9 and 10 in the presence of cDNA.

Sample 6: The products of a PCR reaction using the ppl5 primers of SEQ ID NOs 9 and 10 in the absence of added cDNA.

Sample 7: The products of a PCR reaction using the EIE4 primers of SEQ ID NOs 11 and 12 in the presence of added cDNA.

Sample S: The products of a PCR reaction using the EIE4 primers of SEQ ID NOs 11 and 12 in the absence of added cDNA.

In Samples 1. 3, 5 and 7, a band of the size expected for the PCR product was observed, indicating the presence of the corresponding sequence in the cDNA population.

PCR reactions were also carried out with the antisense oligonucleotides of the globin and dehydrogenase primers (SEQ ID NOs 6 and 8) and an oligonucleotide whose sequence corresponds to that of the derivatized oligonucleotide. The presence of PCR products of the expected size in the samples corresponding to samples 1 and 3 above indicated that the derivatized oligonucleotide had been incorporated.

The above examples summarize the chemical procedure for enriching mRNAs for those having intact 5' ends. Further detail regarding the chemical approaches for obtaining mRNAs having intact 5' ends are disclosed in International Application No. W096/34981, published November 7, 1996.

Strategies based on the above chemical modifications to the 5' cap structure may be utilized to generate cDNAs which have been selected to include the 5' ends of the mRNAs from which they are derived. In one version of such procedures, the 5' ends of the mRNAs are modified as described above.

Thereafter, a reverse transcription reaction is conducted to extend a primer complementary to the mRNA to the 5' end of the mRNA. Single stranded RNAs are eliminated to obtain a population of cDNA/mRNA WO 99/25825 PCT/IB98/01862 19 heteroduplexes in which the mRNA includes an intact 5' end. The resulting heteroduplexes may be captured on a solid phase coated wiani olibei'i- ~able.f inering with the molecule, used .tolderivatize the 5' end of the mRNA. Thereafter, the straiidsofth lihetero 7 e stranded first cDNA strands which include the 5' end of the mnRNA. Seco rid^ d.c)N y C 'I then proceed using conventional techniques. For example, the procedures disclosed in WO 96/349 1 or in Carninci, P. et al. High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper. Genomics 37:327-336 (1996). may be employed to select cDNAs which include the sequence derived from the 5' end ol the coding sequence of the mRNA.

Following ligation of the oligonucleotide tag to the 5' cap of the mRNA, a reverse transcription reaction is conducted to extend a primer complementary to the mRNA to the 5' end of the mRNA.

Following elimination of the RNA component of the resulting heteroduplex using standard techniques, second strand cDNA synthesis is conducted with a primer complementary to the oligonucleotide tag.

Figure 1 summarizes the above procedures for obtaining cDNAs which have been selected to include the 5' ends of the mRNAs from which they are derived.

11. Enzvnmtic Methods for Obtaining mnRNAs hving Intact 5' Ends Other techniques for selecting cDNAs extending to the 5' end of the mRNA from which they are derived are fully enzymatic. Some versions of these techniques are disclosed in Dumas Milne-Edwards J.B.

(Doctoral Thesis of Paris VI University, Le clonage des ADNc complets: difficultes et perspectives nouvelles. Apports pour l'etude de la regulation de I'expression de la tryptophane hydroxylase de rat, Dec. 1993), EPO 625572 and Kato et al. Construction of a Human Full-Length cDNA Bank. Gene 150:243- 250(1994).

Briefly, in such approaches, isolated mRNA is treated with alkaline phosphatase to remove the phosphate groups present on the 5' ends of uncapped incomplete mRNAs. Following this procedure, the cap present on full length mRNAs is enzymatically removed with a decapping enzyme such as T4 polynucleotide kinase or tobacco acid pyrophosphatase. An oligonucleotide, which may be either a DNA oligonucleotide or a DNA-RNA hybrid oligonucleotide having RNA at its 3' end, is then ligated to the phosphate present at the 5' end of the decapped mRNA using T4 RNA ligase. The oligonucleotide may include a restriction site to facilitate cloning of the cDNAs following their synthesis. Example 12 below describes one enzymatic method based on the doctoral thesis of Dumas.

EXAMPLE 12 Enzymatic Approach for Obtaining 5' ESTs Twenty micrograms of PolyA+ RNA were dephosphorylated using Calf Intestinal Phosphatase (Biolabs). After a phenol chloroform extraction, the cap structure of mRNA was hydrolyzed using the Tobacco Acid Pyrophosphatase (purified as described by Shinshi et al., Biochemistry 15: 2185-2190, 1976) and a hemi 5'DNA/RNA-3' oligonucleotide having an unphosphorylated 5' end, a stretch of adenosine WO 99/25825 PCT/IB98/01862 ribophosphate at the 3' end, and an EcoRI site near the 5' end was ligated to the 5'P ends of mRNA using the T4 RNA ligase (Biolabs). Oligonucleotides suitable for use in this procedure are preferably 30-50 bases in length. Oligonucleotides having an unphosphorylated 5' end may be synthesized by adding a fluorochrome at the 5' end. The inclusion of a stretch of adenosine ribophosphates at tie 3' end of the oligonucleotide increases ligation efficiency. It will be appreciated that the oligonucleotide may contain cloning sites other than EcoRI.

Following ligation of the oligonucleotide to the phosphate present at the 5' end of the decapped mRNA, first and second strand cDNA synthesis may be carried out using conventional methods or those specified in EPO 625,572 and Kato et al. Construction of a Human Full-Length cDNA Bank. Gene 150:243- 250 (1994), and Dumas Milne-Edwards, supra. The resulting cDNA may then be ligated into vectors such as those disclosed in Kato et al. Construction of a Human Full-Length cDNA Bank. Gene 150:243-250 (1994) or other nucleic acid vectors known to those skilled in the art using techniques such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press (1989).

II. Characterization of 5' ESTs The above chemical and enzymatic approaches for enriching mRNAs having intact 5' ends were employed to obtain 5' ESTs. First, mRNAs were prepared as described in Example 13 below.

EXAMPLE 13 Preparation of mRNA Total human RNAs or PolyA+ RNAs derived from 29 different tissues were respectively purchased from LABIMO and CLONTECH and used to generate 44 cDNA libraries as described below. The purchased RNA had been isolated from cells or tissues using acid guanidium thiocyanatc-phenol-chloroform extraction (Chomczyniski, P and Sacchi, Analytical Biochemistry 162:156-159, 1987). PolyA+ RNA was isolated from total RNA (LABIMO) by two passes of oligodT chromatography, as described by Aviv and Leder (Aviv, H. and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972) in order to eliminate ribosomal RNA.

The quality and the integrity of the poly A+ were checked. Northern blots hybridized with a globin probe were used to confirm that the mRNAs were not degraded. Contamination of the PolyA+ mRNAs by ribosomal sequences was checked using RNAs blots and a probe derived from the sequence of the 28S RNA. Preparations of mRNAs with less than 5% of ribosomal RNAs were used in library construction. To avoid constructing libraries with RNAs contaminated by exogenous sequences (prokaryotic or fungal), the presence of bacterial 16S ribosomal sequences or of two highly expressed mRNAs was examined using

PCR.

Following preparation of the mRNAs, the above described chemical and/or the enzymatic procedures for enriching mRNAs having intact 5' ends discussed above were employed to obtain 5' ESTs WO 99/25825 PCT/IB98/01862 21 from various tissues. In both approaches an oligonucleotide tag was attached to the cap at the 5' ends of the mRNAs. The oligonucleotide tag had an EcoRI site therein to facilitate later cloning procedures.

Following attachment of the oligonucleotide tag to the mRNA by either the chemical or enzymatic methods, the integrity of the mRNA was examined by performing a Northern blot with 20 0-500ng of mRNA using a probe complementary to the oligonucleotide tag.

EXAMPLE, 14 cl)NA Synthesis U sine mRNA T'emnlates l aving Intact 5' Ends For the mRNAs joined to oligonucleotide tags using both the chemical and enzymatic methods, first strand cDNA synthesis was performed with reverse transcriptase using random nonamers as primers. In order to protect internal EcoRI sites in the cDNA from digestion at later steps in the procedure, methylated dCTP was used for first strand synthesis. After removal of RNA by an alkaline hydrolysis, the first strand of cDNA was precipitated using isopropanol in order to eliminate residual primers.

For both the chemical and the enzymatic methods, synthesis of the second strand of the cDNA is conducted as follows. After removal of RNA by alkaline hydrolysis, the first strand of cDNA is precipitated using isopropanol in order to eliminate residual primers. The second strand of the cDNA was synthesized with Klenow using a primer corresponding to the 5'end of the ligated oligonucleotide described in Example 12. Preferably, the primer is 20-25 bases in length. Methylated dCTP was also used for second strand synthesis in order to protect internal EcoRI sites in the cDNA from digestion during the cloning process.

Following cDNA synthesis, the cDNAs were cloned into pBlueScript as described in Example below.

EXAMPLE Insertion of cDNAs into BlueScript Following second strand synthesis, the ends of the cDNA were blunted with T4 DNA polymerase (Biolabs) and the cDNA was digested with EcoRI. Since methylated dCTP was used during cDNA synthesis, the EcoRI site present in the tag was the only site which was hemi-methylatcd. Consequently, only the EcoRI site in the oligonucleotide tag was susceptible to EcoRI digestion. The cDNA was then size fractionated using exclusion chromatography (AcA, Biosepra). Fractions corresponding to cDNAs of more than 150 bp were pooled and ethanol precipitated. The cDNA was directionally cloned into the Smal and EcoRI ends of the phagemid pBlueScript vector (Stratagene). The ligation mixture was electroporated into bacteria and propagated under appropriate antibiotic selection.

Clones containing the oligonucleotide tag attached were selected as described in Example 16 below.

WO 99/25825 PCT/IB98/01862 22 EXAMPLE 16 Selection of Clones Having the Oligonucleotide Tag Attached Thereto The plasmid DNAs containing 5' EST libraries made as described above were purified (Qiagen). A positive selection of the tagged clones was performed as follows. Briefly, in this selection procedure, the plasmid DNA was converted to single stranded DNA using gene 11 endonuclease of the phage Fl in combination with an exonuclease (Chang et al.. Gene 127:95-8, (1993)) such as exonuclease III or T7 gene 6 exomtclease. The resulting single stranded DNA was then purified using paramagnetic beads as described by Fry re al.. UBioeclhniques. 13: 124-131 (1992). In this procedure, thle single stranded DNA was hybridized with a biotinylated oligonucleotide having a sequence corresponding to the 3' end of the oligonucleotide described in Example 13. Preferably, the primer has a length of 20-25 bases. Clones including a sequence complementary to the biotinylated oligonucleotide were captured by incubation with streptavidin coated magnetic beads followed by magnetic selection. After capture of the positive clones, the plasmid DNA was released from the magnetic beads and converted into double stranded DNA using a DNA polymerase such as the ThermoSequenase obtained from Amersham Pharmacia Biotech. Alternatively, protocols such as the Gene Trapper kit (Gibco BRL) may be used. The double stranded DNA was then electroporated into bacteria. The percentage of positive clones having the 5' tag oligonucleotide was estimated to typically rank between 90 and 98% using dot blot analysis.

Following electroporation, the libraries were ordered in 384-microtiter plates (MTP). A copy of the MTP was stored for future needs. Then the libraries were transferred into 96 MTP and sequenced as described below.

EXAMPLE 17 Sequencing of Inserts in Selected Clones Plasmid inserts were first amplified by PCR on PE 9600 thermocyclers (Perkin-Elmer), using standard SETA-A and SETA-B primers (Genset SA), AmpliTaqGold (Perkin-Elmer). dNTPs (Bochringer), buffer and cycling conditions as recommended by the Perkin-Elmer Corporation.

PCR products were then sequenced using automatic ABI Prism 377 sequencers (Perkin Elmer, Applied Biosystems Division, Foster City, CA). Sequencing reactions were performed using PE 9600 thermocyclers (Perkin Elmer) with standard dye-primer chemistry and ThermoSequenase (Amersham Life Science). The primers used were either T7 or 21M 13 (available from Genset SA) as appropriate. The primers were labeled with the JOE, FAM, ROX and TAMRA dyes. The dNTPs and ddNTPs used in the sequencing reactions were purchased from Boehringer. Sequencing buffer, reagent concentrations and cycling conditions were as recommended by Amersham.

Following the sequencing reaction, the samples were precipitated with EtOH, resuspended in formamide loading buffer, and loaded on a standard 4% acrylamide gel. Electrophoresis was performed for hours at 3000V on an ABI 377 sequencer, and the sequence.data were collected and analyzed using the WO 99/25825 PCT/IB98/01862 23 ABI Prism DNA Sequencing Analysis Software, version 2.1.2.

The sequence data from the 44 cDNA libraries made as described above were transferred to a proprietary database, where quality control and validation steps were performed. A proprietary base-caller ("Trace"), working using a Unix system automatically flagged suspect peaks, taking into account the shape of the peaks, the inter-peak resolution, and the noise level. The proprietary base-caller also performed an automatic trimming. Any stretch of 25 or fewer bases having more than 4 suspect peaks was considered unreliable and was discarded. Sequences corresponding to cloning vector or ligation oligonucleotides were automatically removed from the EST sequences. However, the resulting ES'T sequences nmay contain I to bases belonging to the above mentioned sequences at their 5' end. If needed, these can easily be removed on a case by case basis.

Thereafter, the sequences were transferred to the proprietary NETGENETN' Database for further analysis as described below.

Following sequencing as described above, the sequences of the 5' ESTs were entered in a proprietary database called NETGENETM for storage and manipulation. It will be appreciated by those skilled in the art that the data could be stored and manipulated on any medium which can be read and accessed by a computer. Computer readable media include magnetically readable media, optically readable media, or electronically readable media. For example, the computer readable media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art.

In addition, the sequence data may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the sequence data may be stored as text in a word processing file, such as Microsoft WORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.

The computer readable media on which the sequence information is stored may be in a personal computer, a network, a server or other computer systems known to those skilled in the art. The computer or other system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data.

Once the sequence data has been stored it may be manipulated and searched to locate those stored sequences which contain a desired nucleic acid sequence or which encode a protein having a particular functional domain. For example, the stored sequence information may be compared to other known sequences to identify homologies, motifs implicated in biological function, or structural motifs.

Programs which may be used to search or compare the stored sequences include the MacPattern (EMBL), BLAST, and BLAST2 program series (NCBI), basic local alignment search tool programs for nucleotide (BLASTN) and peptide (BLASTX) comparisons (Altschul et al, J. Mol. Biol. 215:403 (1990)) and FASTA (Pearson and Lipman, Proc. Nail. Acad. Sci. USA, 85: 2444 (1988)). The BLAST programs then extend the alignments on the basis of defined match and mismatch criteria.

WO 99/25825 PCT/IB98/01862 24 Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets.

signal sequences encoding signal pcptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homcoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

Before searching the cDNAs in the NETGENETM database for sequence motifs of interest, cDNAs derived from mRNAs which were not of interest were identified and eliminated from further consideration as described in Example 18 below.

EXAMPLE 18 Elimination of Undesired Sequences from Further Consideration ESTs in the NETGENETM database which were derived from undesired sequences such as transfer RNAs. ribosomal RNAs, mitochondrial RNAs. procaryotic RNAs, fungal RNAs, Alu sequences, LI sequences, or repeat sequences were identified using the FASTA and BLASTN programs with the parameters listed in Table I.

To eliminate 5' ESTs encoding tRNAs from further consideration, the 5' EST sequences were compared to the sequences of 1190 known tRNAs obtained from EMBL release 38, of which 100 were human. The comparison was performed using FASTA on both strands of the 5' ESTs. Sequences having more than 80% homology over more than 60 nucleotides were identified as tRNA. Of the 144,341 sequences screened, 26 were identified as tRNAs and eliminated from further consideration.

To eliminate 5' ESTs encoding rRNAs from further consideration, the 5' EST sequences were compared to the sequences of 2497 known rRNAs obtained from EMBL release 38, of which 73 were human. The comparison was performed using BLASTN on both strands of the 5' ESTs with the parameter S=108. Sequences having more than 80% homology over stretches longer than 40 nucleotides were identified as rRNAs. Of the 144,341 sequences screened, 3,312 were identified as rRNAs and eliminated from further consideration.

To eliminate 5' ESTs encoding mtRNAs from further consideration, the 5' EST sequences were compared to the sequences of the two known mitochondrial genomes for which the entire genomic sequences are available and all sequences transcribed from these mitochondrial genomes including tRNAs, rRNAs, and mRNAs for a total of 38 sequences. The comparison was performed using BLASTN on both strands of the 5' ESTs with the parameter S=108. Sequences having more than 80% homology over stretches longer than 40 nucleotides were identified as mtRNAs. Of the 144,341 sequences screened, 6,1 were identified as mtRNAs and eliminated from further consideration.

Sequences which might have resulted from exogenous contaminants were eliminated from further consideration by comparing the 5' EST sequences to release 46 of the EMBL bacterial and fungal divisions using BLASTN with the parameter S=144. All sequences having more than 90% homology over at least WO 99/25825 PCT/IB98/01862 nucleotides were identified as exogenous contaminants. Of the 42 cDNA libraries examined, the average percentages of procaryotic and fungal sequences contained therein were 0.2% and 0.5% respectively.

Among these sequences, only one could be identified as a sequence specific to fungi. The others were either fungal or procaryotic sequences having homologies with vertebrate sequences or including repeat sequences which had not been masked during the electronic comparison.

In addition, the 5' ESTs were compared to 6093 Alu sequences and 1115 LI sequences to mask ESTs containing such repeat sequences from further consideration. 5' ESTs including THE and MER repeats. SSTR sequences or satellite, micro-satellite, or telomeric repeats were also eliminated from further consideration. On average, 11.5% of the sequences in the libraries contained repeat sequences. Of this 11.5%, 7% contained Alu repeats. 3.3% contained LI repeats and the remaining 1.2% were derived from the other types of repetitive sequences which were screened. These percentages are consistent with those found in cDNA libraries prepared by other groups. For example, the cDNA libraries of Adams et al.

contained between 0% and 7.4% Alu repeats depending on the source of the RNA which was used to prepare the cDNA library (Adams et al., Nature 377:174, 1996).

The sequences of those 5' ESTs remaining after the elimination of undesirable sequences were compared with the sequences of known human mRNAs to determine the accuracy of the sequencing procedures described above.

EXAMPLE 19 Measurement of Sequencine Accuracy by Comparison to Known Sequences To further determine the accuracy of the sequencing procedure described above, the sequences of ESTs derived from known sequences were identified and compared to the known sequences. First, a FASTA analysis with overhangs shorter than 5 bp on both ends was conducted on the 5' ESTs to identify those matching an entry in the public human mRNA database. The 6655 5' ESTs which matched a known human mRNA were then realigned with their cognate mRNA and dynamic programming was used to include substitutions, insertions, and deletions in the list of "errors" which would be recognized. Errors occurring in the last 10 bases of the 5' EST sequences were ignored to avoid the inclusion of spurious cloning sites in the analysis of sequencing accuracy.

This analysis revealed that the sequences incorporated in the NETGENETM database had an accuracy of more than 99.5%.

To determine the efficiency with which the above selection procedures select cDNAs which include the 5' ends of their corresponding mRNAs, the following analysis was performed.

EXAMPLE Determination of Efficiency of 5' EST Selection To determine the efficiency at which the above selection procedures isolated 5' ESTs which WO 99/25825 PCT/IB98/01862 26 included sequences close to the 5' end of the mRNAs from which they were derived, the sequences of the ends of the 5' ESTs which were derived from the elongation factor 1 subunit a and ferritin heavy chain genes were compared to the known cDNA sequences for these genes. Since the transcription start sites for the elongation factor 1 subunit a and ferritin heavy chain are well characterized, they may be used to determine the percentage of 5' ESTs derived from these genes which included the authentic transcription start sites.

For both genes, more than 95% of the cDNAs included sequences close to or upstream of the 5' end of the corresponding mRNAs.

To extend the analysis of the reliability of the procedures for isolating 5' ESTs from ESTs in the NETGENErI database, a similar analysis was conducted using a database composed of human mRNA sequences extracted from GenBank database release 97 for comparison. For those 5' ESTs derived from mRNAs included in the GeneBank database, more than 85% had their 5' ends close to the 5' ends of the known sequence. As some of the mRNA sequences available in the GenBank database are deduced from genomic sequences, a 5' end matching with these sequences will be counted as an internal match. Thus, the method used here underestimates the yield of ESTs including the authentic 5' ends of their corresponding mRNAs.

The EST libraries made above included multiple 5' ESTs derived from the same mRNA. The sequences of such 5' ESTs were compared to one another and the longest 5' ESTs for each mRNA were identified. Overlapping cDNAs were assembled into continuous sequences (contigs). The resulting continuous sequences were then compared to public databases to gauge their similarity to known sequences, as described in Example 21 below.

EXAMPLE 21 Clusterine of the 5' ESTs and Calculation of Novelty Indices for cDNA Libraries For each sequenced EST library, the sequences were clustered by the 5' end. Each sequence in the library was compared to the others with BLASTN2 (direct strand, parameters S=107). ESTs with High Scoring Segment Pairs (HSPs) at least 25 bp long, having 95% identical bases and beginning closer than bp from each EST 5' end were grouped. The longest sequence found in the cluster was used as representative of the cluster. A global clustering between libraries was then performed leading to the dcfinition of super-contigs.

To assess the yield of new sequences within the EST libraries, a novelty rate (NR) was defined as: NR= 100 X (Number of new unique sequences found in the library/Total number of sequences from the library). Typically, novelty rating range between 10% and 41% depending on the tissue from which the EST library was obtained. For most of the libraries, the random sequencing of 5' EST libraries was pursued until the novelty rate reached Following characterization as described above, the collection of 5' ESTs in NETGENETM was WO 99/25825 PCT/IB98/01862 27 screened to identify those 5' ESTs bearing potential signal sequences as described in Example 22 below.

EXAMPLE 22 Identification of Potential Signal Sequences in 5' ESTs The 5' ESTs in the NETGENE' database were screened to identify those having an uninterrupted open reading frame (ORF) longer than 45 nucleotides beginning with an ATG codon and extending to the end of the EST. Approximately half of the cDNA sequences in NETGENETM contained such an ORF. The ORFs of these 5' ESTs were searched to identify potential signal motifs using slight modifications of the procedures disclosed in Von Heijne, G. A New Method for Predicting Signal Sequence Cleavage Sites.

Nucleic Acids Res. 14:4683-4690 (1986). Those 5' EST sequences encoding a 15 amino acid long stretch with a score of at least 3.5 in the Von Heijne signal peptide identification matrix were considered to possess a signal sequence. Those 5' ESTs which matched a known human mRNA or EST sequence and had a end more than 20 nucleotides downstream of the known 5' end were excluded from further analysis. The remaining cDNAs having signal sequences therein were included in a database called SIGNALTAGM.

To confirm the accuracy of the above method for identifying signal sequences, the analysis of Example 23 was performed.

EXAMPLE 23 Confirmation of Accuracy of Identification of Potential Signal Sequences in 5' ESTs The accuracy of the above procedure for identifying signal sequences encoding signal peptides was evaluated by applying the method to the 43 amino terminal amino acids of all human SwissProt proteins.

The computed Von Heijne score for each protein was compared with the known characterization of the protein as being a secreted protein or a non-secreted protein. In this manner, the number of non-secreted proteins having a score higher than 3.5 (false positives) and the number of secreted proteins having a score lower than 3.5 (false negatives) could be calculated.

Using the results of the above analysis, the probability that a peptide encoded by the 5' region of the mRNA is in fact a genuine signal peptide based on its Von Heijne's score was calculated based on either the assumption that 10% of human proteins are secreted or the assumption that 20% of human proteins are secreted. The results of this analysis are shown in Figures 2 and 3.

Using the above method of identifying secretory proteins, 5' ESTs for human glucagon, gamma interferon induced monokine precursor, secreted cyclophilin-like protein, human pleiotropin, and human biotinidase precursor all of which are polypeptides which are known to be secreted, were obtained. Thus, the above method successfully identified those 5' ESTs which encode a signal peptide.

To confirm that the signal peptide encoded by the 5' ESTs actually functions as a signal peptide, the signal sequences from the 5' ESTs may be cloned into a vector designed for the identification of signal peptides. Some signal peptide identification vectors are designed to confer the ability to grow in selective WO 99/25825 PCT/IB98/01862 28 medium on host cells which have a signal sequence operably inserted into the vector. For example, to confirm that a 5' EST encodes a genuine signal peptide, the signal sequence of the 5' EST may be inserted upstream and in frame with a non-secreted form of the yeast invertase gene in signal peptide selection vectors such as those described in U.S. Patent No. 5.536.637. Growth of host cells containing signal sequence selection vectors having tile signal scquence from the 5' EST inserted therein confirms that the EST encodes a genuine signal peptide.

Alternatively. the presence of a signal peptide may be confirmed by cloning the extended cDNAs obtained using lhe ESTs into expression vectors such as pXTI (as described below), or by constructing promoter-signal sequence-reporter gene vectors which encode fusion proteins between the signal peptide and an assayable reporter protein. After introduction of these vectors into a suitable host cell, such as COS cells or NIH 3T3 cells, the growth medium numy be harvested and analyzed for the presence of the secreted protein. The medium from these cells is compared to the medium from cells containing vectors lacking the signal sequence or extended cDNA insert to identify vectors which encode a functional signal peptide or an authentic secreted protein.

Those 5' ESTs which encoded a signal peptide, as determined by the method of Example 22 above, were further grouped into four categories based on their homology to known sequences. The categorization of the 5' ESTs is described in Example 24 below.

EXAMPLE 24 Categorization of 5' ESTs Encoding a Sienal Peptide Those 5' ESTs having a sequence not matching any known vertebrate sequence nor any publicly available EST sequence were designated "new." Of the sequences in the SIGNALTAGTM database, 947 of the 5' ESTs having a Von Heijne's score of at least 3.5 fell into this category.

Those 5' ESTs having a sequence not matching any vertebrate sequence but matching a publicly known EST were designated "EST-ext", provided that the known EST sequence was extended by at least nucleotides in the 5' direction. Of the sequences in the SIGNALTAGTM database, 150 of the 5' ESTs having a Von Heijne's score of at least 3.5 fell into this category.

Those ESTs not matching any vertebrate sequence but matching a publicly known EST without extending the known EST by at least 40 nucleotides in the 5' direction were designated "EST." Of the sequences in the SIGNALTAGTM database, 599 of the 5' ESTs having a Von Heijne's score of at least fell into this category.

Those 5' ESTs matching a human mRNA sequence but extending the known sequence by at least nucleotides in the 5' direction were designated "VERT-ext." Of the sequences in the SIGNALTAG T M database, 23 of the 5' ESTs having a Von Heijne's score of at least 3.5 fell into this category. Included in this category was a 5' EST which extended the known sequence of the human translocase mRNA by more than 200 bases in the 5' direction. A 5' EST which extended the sequence of a human tumor suppressor WO 99/25825 PCT/IB98/01862 29 gene in the 5' direction was also identified.

Figure 4 shows the distribution of 5' ESTs in each category and the number of 5' ESTs in each category having a given minimum von Heijne's score.

Each of the 5' ESTs was categorized based on the tissue from which its corresponding mRNA was obtained, as described below in Example EXAMPLIE Carieorization of Expression Patterns Figure 5 shows the tissues from which the mRNAs corresponding to the 5' ESTs in each of the above described categories were obtained.

In addition to categorizing the 5' ESTs by the tissue from which the cDNA library in which they were first identified was obtained, the spatial and temporal expression patterns of the mRNAs corresponding to the 5' ESTs, as well as their expression levels, may be determined as described in Example 26 below. Characterization of the spatial and temporal expression patterns and expression levels of these mRNAs is useful for constructing expression vectors capable of producing a desired level of gene product in a desired spatial or temporal manner, as will be discussed in more detail below.

In addition, 5' ESTs whose corresponding mRNAs are associated with disease states may also be identified. For example, a particular disease may result from lack of expression, over expression, or under expression of an mRNA corresponding to a 5' EST. By comparing mRNA expression patterns and quantities in samples taken from healthy individuals with those from individuals suffering from a particular disease, 5' ESTs responsible for the disease may be identified.

It will be appreciated that the results of the above characterization procedures for 5' ESTs also apply to extended cDNAs (obtainable as described below) which contain sequences adjacent to the 5' ESTs.

It will also be appreciated that if it is desired to defer characterization until extended cDNAs have been obtained rather than characterizing the ESTs themselves, the above characterization procedures can be applied to characterize the extended cDNAs after their isolation.

EXAMPLE 26 Evaluation of Expression Levels and Patterns of mRNAs Corresponding to 5' ESTs or Extended cDNAs Expression levels and patterns of mRNAs corresponding to 5' ESTs or extended cDNAs (obtainable as described below) may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277. Briefly, a 5' EST, extended cDNA, or fragment thereof corresponding to the gene encoding the mRNA to be characterized is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA.

Preferably, the 5' EST or extended cDNA has 100 or more nucleotides. The plasmid is linearized and WO 99/25825 PCT/IB98/01862 transcribed in the presence of ribonucleotides comprising modified ribonucleotides biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest. The hybridizations are performed under standard stringent conditions (40-50oC for 16 hours in an 80% formamide, 0.4 M NaCI buffer, pH The unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA RNases CL3. T I Phy M. U2 or The presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin. The presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an ani-DIG antibody coupled to alkaline phosphatase.

The 5' ESTs, extended cDNAs, or fragments thereof may also be tagged with nucleotide sequences for the serial analysis of gene expression (SAGE) as disclosed in UK Patent Application No. 2,305.241 A.

In this method, cDNAs are prepared from a cell, tissue, organism or other source of nucleic acid for which it is desired to determine gene expression patterns. The resulting cDNAs are separated into two pools. The cDNAs in each pool are cleaved with a first restriction endonuclease, called an "anchoring enzyme," having a recognition site which is likely to be present at least once in most cDNAs. The fragments which contain the 5' or 3' most region of the cleaved cDNA are isolated by binding to a capture medium such as streptavidin coated beads. A first oligonucleotide linker having a first sequence for hybridization of an amplification primer and an internal restriction site for a "tagging endonuclease" is ligated to the digested cDNAs in the first pool. Digestion with the second endonuclease produces short "tag" fragments from the cDNAs.

A second oligonucleotide having a second sequence for hybridization of an amplification primer and an internal restriction site is ligated to the digested cDNAs in the second pool. The cDNA fragments in the second pool are also digested with the "tagging endonuclease" to generate short "tag" fragments derived from the cDNAs in the second pool. The "tags" resulting from digestion of the first and second pools with the anchoring enzyme and the tagging endonuclease are ligated to one another to produce "ditags." In some embodiments, the ditags are concatamerized to produce ligation products containing from 2 to 200 ditags.

The tag sequences are then determined and compared to the sequences of the 5' ESTs or extended cDNAs to determine which 5' ESTs or extended cDNAs are expressed in the cell, tissue, organism, or other source of nucleic acids from which the tags were derived. In this way, the expression pattern of the 5' ESTs or extended cDNAs in the cell, tissue, organism, or other source of nucleic acids is obtained.

Quantitative analysis of gene expression may also be performed using arrays. As used herein, the term array means a one dimensional, two dimensional, or multidimensional arrangement of full length cDNAs extended cDNAs which include the coding sequence for the signal peptide, the coding sequence for the mature protein, and a stop codon), extended cDNAs, 5' ESTs or fragments of the full length cDNAs, extended cDNAs, or 5' ESTs of sufficient length to permit specific detection of gene expression. Preferably, the fragments are at least 15 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. More preferably, the fragments are more than 100 nucleotides in

L

WO 99/25825 PCT/IB98/01862 31 length. In some embodiments the fragments may be more than 500 nucleotides in length.

For example, quantitative analysis of gene expression may be performed with full length cDNAs.

extended cDNAs, 5' ESTs, or fragments thereof in a complementary DNA microarray as described by Schena et al. Science 270:467-470, 1995; Proc. Natl. Acad. Sci. U.S.A. 93:10614-10619 (1996). Full length cDNAs. extended cDNAs, 5' ESTs or fragments thereof are amplified by PCR and arrayed from 96-well microtiter plates onto silylated microscope slides using high-speed robotics. Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution. The arrays are submerged in water for 2 min at 950C, transferred into 0.2% SDS for 1 min. rinsed twice with water, air dried and stored in the dark at 250C.

Cell or tissue mRNA is isolated or commercially obtained and probes are prepared by a single round of reverse transcription. Probes are hybridized to I cm 2 microarrays under a 14 x 14 mm glass coverslip for 6-12 hours at 600C. Arrays are washed for 5 min at 25°C in low stringency wash buffer (1 x SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1 x SSC/0.2% SDS). Arrays are scanned in 0.1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.

Quantitative analysis of the expression of genes may also be performed with full length cDNAs, extended cDNAs, 5' ESTs, or fragments thereof in complementary DNA arrays as described by Pietu et al.

Genome Research 6:492-503 (1996). The full length cDNAs, extended cDNAs, 5' ESTs or fragments thereof are PCR amplified and spotted on membranes. Then, mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.

Alternatively, expression analysis of the 5' ESTs or extended cDNAs can be done through high density nucleotide arrays as described by Lockhart et al. Nature Biotechnology 14: 1675-1680, 1996. and Sosnowsky et al. Proc. Nail. Acad. Sci. 94:1119-1123, 1997. Oligonucleotides of 15-50 nucleotides corresponding to sequences of the 5' ESTs or extended cDNAs are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra). Preferably, the oligonuclcotides are about 20 nucleotides in length.

cDNA probes labeled with an appropriate compound, such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., Proc. Natl. Acad. Sci.

94:1119-1123)., the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed. Comparative analysis of the intensity of the signal originating from cDNA probes on the same WO 99/25825 PCT/IB98/01862 32 target oligonucleotide in different cDNA samples indicates a differential expression of the mRNA corresponding to the 5' EST or extended cDNA from which the oligonucleotide sequence has been designed.

III. Use of 5' ESTs to Clone Extended cDNAs and to Clone the Corresponding Gcnomic DNAs Once 5' ESTs which include the 5' end of the corresponding mRNAs have been selected using the procedures described above, they can be utilized to isolate extended cDNAs which contain sequences adjacent to the 5' ESTs. The extended cDNAs may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the authentic translation start site, the signal sequence, and the sequence encoding the mature protein remaining after cleavage of the signal peptide. Such extended cDNAs are referred to herein as "full length cDNAs." Alternatively, the extended cDNAs may include only the sequence encoding the mature protein remaining after cleavage of the signal peptide, or only the sequence encoding the signal peptide.

Example 27 below describes a general method for obtaining extended cDNAs. Example 28 below describes the cloning and sequencing of several extended cDNAs, including extended cDNAs which include the entire coding sequence and authentic 5' end of the corresponding mRNA for several secreted proteins.

The methods of Examples 27, 28, and 29 can also be used to obtain extended cDNAs which encode less than the entire coding sequence of the secreted proteins encoded by the genes corresponding to the ESTs. In some embodiments, the extended cDNAs isolated using these methods encode at least 10 amino acids of one of the proteins encoded by the sequences of SEQ ID NOs: 134-180. In further embodiments, the extended cDNAs encode at least 20 amino acids of the proteins encoded by the sequences of SEQ ID NOs: 134-180. In further embodiments, the extended cDNAs encode at least 30 amino acids of the sequences of SEQ ID NOs: 134-180. In a preferred embodiment, the extended cDNAs encode a full length protein sequence, which includes the protein coding sequences of SEQ ID NOs: 134-180.

EXAMPLE 27 General Method for Using 5' ESTs to Clone and Sequence Extended cDNAs which Include the Entire Coding Region and the Authentic 5' End of the Corresponding mRNA The following general method has been used to quickly and efficiently isolate extended cDNAs including sequence adjacent to the sequences of the 5' ESTs used to obtain them. This method may be applied to obtain extended cDNAs for any 5' EST in the NetGene T database, including those 5' ESTs encoding secreted proteins. The method is summarized in figure 6.

I. Obtainine Extended cDNAs a) First strand synthesis The method takes advantage of the known 5' sequence of the mRNA. A reverse transcription reaction is conducted on purified mRNA with a poly 14dT primer containing a 49 nucleotide sequence at its end allowing the addition of a known sequence at the end of the cDNA which corresponds to the 3' end of WO 99/25825 PCT/IB98/01862 33 the mRNA. For example, the primer may have the following sequence: 5'-ATC G1T GAG ACT CGT ACC AGC AGA GTC ACG AGA GAG ACT ACA CGG TAC TGG TTT TTTT TTT T TTVN (SEQ ID NO: 14). Those skilled in the art will appreciate that other sequences may also be added to the poly dT sequence and used to prime the first strand synthesis. Using this primer and a reverse transcriptase such as the Superscript II (Gibco BRL) or Rnase H Minus M-MLV (Promega) enzyme. a reverse transcript anchored at the 3' polyA site of the RNAs is generated.

After removal of the mRNA hybridized to the first cDNA strand by alkaline hydrolysis, the products of the alkaline hydrolysis and the residual poly dT primer are eliminated with an exclusion column such as an AcA34 (Biosepra) matrix as explained in Example 11.

b) Second strand synthesis A pair of nested primers on each end is designed based on the known 5' sequence from the 5' EST and the known 3' end added by the poly dT primer used in the first strand synthesis. Softwares used to design primers are either based on GC content and melting temperatures of oligonucleotides, such as OSP (lllier and Green, PCR Meth. Appl. 1:124-128, 1991), or based on the octamer frequency disparity method (Griffais et al., Nucleic Acids Res. 19: 3SS7-3S91, 1991 such as PC-Rare (http://bioinformatics.weizmann.ac.il/software/PC-Rare/doc/manuel.html).

Preferably, the nested primers at the 5' end are separated from one another by four to nine bases.

The 5' primer sequences may be selected to have melting temperatures and specificities suitable for use in

PCR.

Preferably, the nested primers at the 3' end are separated from one another by four to nine bases.

For example, the nested 3' primers may have the following sequences: CCA GCA GAG TCA CGA GAG AGA CTA CAC GG -3'(SEQ ID NO: 15), and CAC GAG AGA GAC TAC ACG GTA CTG G -3' (SEQ ID NO: 16). These primers were selected because they have melting temperatures and specificities compatible with their use in PCR. However, those skilled in the art will appreciate that other sequences may also be used as primers.

The first PCR run of 25 cycles is performed using the Advantage Tth Polymerase Mix (Clontech) and the outer primer from each of the nested pairs. A second 20 cycle PCR using the same enzyme and the inner primer from each of the nested pairs is then performed on 1/2500 of the first PCR product. Thereafter, the primers and nucleotides are removed.

2. Sequencing of Full Length Extended cDNAs or Fragments Thereof Due to the lack of position constraints on the design of 5' nested primers compatible for PCR use using the OSP software, amplicons of two types are obtained. Preferably, the second 5' primer is located upstream of the translation initiation codon thus yielding a nested PCR product containing the whole coding sequence. Such a full length extended cDNA undergoes a direct cloning procedure as described in section a. However, in some cases, the second 5' primer is located downstream of the translation initiation codon, thereby yielding a PCR product containing only part of the ORF. Such incomplete PCR products are WO 99/25825 PCT/B98/01862 34 submitted to a modified procedure described in section b.

a) Nested PCR products containing complete ORFs When the resulting nested PCR product contains the complete coding sequence, as predicted from the 5'EST sequence, it is cloned in an appropriate vector such as pED6dpc2. as described in section 3.

b) Nested PCR products containing incomplete ORFs When the amplicon does not contain the complete coding sequence, intermediate steps are necessary to obtain both the complete coding sequence and a PCR product containing the full coding sequence. The complete coding sequence can be assembled from several partial sequences determined directly from different PCR products as described in the following section.

Once the full coding sequence has been completely determined, new primers compatible for PCR use are designed to obtain amplicons containing the whole coding region. However, in such cases, 3' primers compatible for PCR use are located inside the 3' UTR of the corresponding mRNA, thus yielding amplicons which lack part of this region, i.e. the polyA tract and sometimes the polyadenylation signal, as illustrated in figure 6. Such full length extended cDNAs are then cloned into an appropriate vector as described in section 3.

c) Sequencing extended cDNAs Sequencing of extended cDNAs is performed using a Die Terminator approach with the AmpliTaq DNA polymerase FS kit available from Perkin Elmer.

In order to sequence PCR fragments, primer walking is performed using software such as OSP to choose primers and automated computer software such as ASMG (Sutton et al., Genome Science Technol.

1: 9-19, 1995) to construct contigs of walking sequences including the initial 5' tag using minimum overlaps of 32 nucleotides. Preferably, primer walking is performed until the sequences of full length cDNAs are obtained.

Completion of the sequencing of a given extended cDNA fragment is assessed as follows. Since sequences located after a polyA tract are difficult to determine precisely in the case of uncloned products, sequencing and primer walking processes for PCR products are interrupted when a polyA tract is identified in extended cDNAs obtained as described in case b. The sequence length is compared to the size of the nested PCR product obtained as described above. Due to the limited accuracy of the determination of the PCR product size by gel electrophoresis, a sequence is considered complete if the size of the obtained sequence is at least 70 the size of-the first nested PCR product. If the length of the sequence determined from the computer analysis is not at least 70% of the length of the nested PCR product, these PCR products are cloned and the sequence of the insertion is determined. When Northern blot data are available, the size of the mRNA detected for a given PCR product is used to finally assess that the sequence is complete.

Sequences which do not fulfill the above criteria are discarded and will undergo a new isolation procedure.

Sequence data of all extended cDNAs are then transferred to a proprietary database, where quality controls and validation steps are carried out as described in example WO 99/25825 PCT/IB98/01862 3. Cloning of Full Length Extended cDNAs The PCR product containing the full coding sequence is then cloned in an appropriate vector. For example, the extended cDNAs can be cloned into the expression vector pED6dpc2 (DiscoverEase. Genetics Institute, Cambridge. MA) as follows. The structure of pED6dpc2 is shown in Figure 7. pED6dpc2 vector DNA is prepared with blunt ends by performing an EcoRI digestion followed by a fill in reaction. The blunt ended vector is dephosphorylated. After removal of PCR primers and ethanol precipitation, the PCR product containing the full coding sequence or the extended cDNA obtained as described above is phosphorylated with a kinase subsequently removed by phenol-Sevag extraction and precipitation. The double stranded extended cDNA is then ligated to the vector and the resulting expression plasmid introduced into appropriate host cells.

Since the PCR products obtained as described above are blunt ended molecules that can be cloned in either direction, the orientation of several clones for each PCR product is determined. Then, 4 to clones are ordered in microtiter plates and subjected to a PCR reaction using a first primer located in the vector close to the cloning site and a second primer located in the portion of the extended cDNA corresponding to the 3' end of the mRNA. This second primer may be the antisense primer used in anchored PCR in the case of direct cloning (case a) or the antisense primer located inside the 3'UTR in the case of indirect cloning (case Clones in which the start codon of the extended cDNA is operably linked to the promoter in the vector so as to permit expression of the protein encoded by the extended cDNA are conserved and sequenced. In addition to the ends of cDNA inserts, approximately 50 bp of vector DNA on each side of the cDNA insert are also sequenced.

The cloned PCR products are then entirely sequenced according to the aforementioned procedure.

In this case, contig assembly of long fragments is then performed on walking sequences that have already contigated for uncloned PCR products during primer walking. Sequencing of cloned amplicons is complete when the resulting contigs include the whole coding region as well as overlapping sequences with vector DNA on both ends.

4. Computer Analysis of Full Length Extended cDNA Sequences of all full length extended cDNAs are then submitted to further analysis as described below and using the parameters found in Table I with the following modifications. For screening of miscellaneous subdivisions of Genbank, FASTA was used instead of BLASTN and 15 nucleotide of homology was the limit instead of 17. For Alu detection, BLASTN was used with the following parameters: S=72; identity=70%; and length 40 nucleotides. Polyadenylation signal and polyA tail which were not search for the 5' ESTs were searched. For polyadenylation signal detection the signal (AATAAA) was searched with one permissible mismatch in the last ten nucleotides preceding the 5' end of the polyA.

For the polyA, a stretch of 8 amino acids in the last 20 nucleotides of the sequence was searched with BLAST2N in the sense strand with the following parameters S=10, E=1000, and WO 99/25825 PCT/IB98/01862 36 Finally, patented sequences and ORF homologies were searched using, respectively. BLASTN and BLASTP on GenSEQ (Derwent's database of patented nucleotide sequences) and SWISSPROT for ORFs with the following parameters (W=8 and B=10). Before examining the extended full length cDNAs for sequences of interest, extended cDNAs which are not of interest are searched as follows.

a) Elimination of undesired sequences Although 5'ESTs were checked to remove contaminants sequences as described in Example 18. a last verification was carried out to identify extended clNAs sequences derived from undesired sequences such as vector RNAs. transfer RNAs, ribosonal rRNAs. mitochondrial RNAs, prokaryotic RNAs and fungal RNAs using the FASTA and BLASTN programs on both strands of extended cDNAs as described below.

To identify the extended cDNAs encoding vector RNAs, extended cDNAs are compared to the known sequences of vector RNA using the FASTA program. Sequences of extended cDNAs with more than 90% homology over stretches of 15 nucleotides are identified as vector RNA.

To identify the extended cDNAs encoding tRNAs, extended cDNA sequences were compared to the sequences of 1190 known tRNAs obtained from EMBL release 38, of which 100 were human.

Sequences of extended cDNAs having more than 80% homology over 60 nucleotides using FASTA were identified as tRNA.

To identify the extended cDNAs encoding rRNAs, extended cDNA sequences were compared to the sequences of 2497 known rRNAs obtained from EMBL release 38. of which 73 were human.

Sequences of extended cDNAs having more than 80% homology over stretches longer than 40 nucleotides using BLASTN were identified as rRNAs.

To identify the extended cDNAs encoding mtRNAs, extended cDNA sequences were compared to the sequences of the two known mitochondrial genomes for which the entire genomic sequences are available and all sequences transcribed from these mitochondrial genomes including tRNAs, rRNAs, and mRNAs for a total of 38 sequences. Sequences of extended cDNAs having more than 80% homology over stretches longer than 40 nucleotides using BLASTN were identified as mtRNAs.

Sequences which might have resulted from other exogenous contaminants were identified by comparing extended cDNA sequences to release 105 of Genbank bacterial and fungal divisions. Sequences of extended cDNAs having more than 90% homology over 40 nucleotides using BLASTN were identified as exogenous prokaryotic or fungal contaminants.

In addition, extended cDNAs were searched for different repeat sequences, including Alu sequences, Ll sequences, THE and MER repeats, SSTR sequences or satellite, micro-satellite, or telomeric repeats. Sequences of extended cDNAs with more than 70% homology over 40 nucleotide stretches using BLASTN were identified as repeat sequences and masked in further identification procedures. In addition, clones showing extensive homology to repeats matches of either more than 50 nucleotides if the homology was at least 75% or more than 40 nucleotides if the homology was at least or more than 30 nucleotides if the homology was at least 90%, were flagged.

WO 99/25825 PCT/IB98/01862 37 b) Identification of structural features Structural features, e.g. polyA tail and polyadenylation signal, of the sequences of full length extended cDNAs are subsequently determined as follows.

A polyA tail is defined as a homopolymeric stretch of at least 11 A with at most one alternative base within it. The polyA tail scarch is restricted to the last 20 nt of the sequence and limited to stretches of 11 consecutive A's because sequencing reactions are often not readable after such a polyA stretch. Stretches with 100% homology over 6 nucleotides are identified as polyA tails.

To search for a polyadenylation signal, the polyA tail is clipped from the full-length sequence. The bp preceding the polyA tail are searched for the canonic polyadenylation AAUAAA signal allowing one mismatch to account for possible sequencing errors and known variation in the canonical sequence of the polyadenylation signal.

c) Identification of functional features Functional features, e.g. ORFs and signal sequences, of the sequences of full length extended cDNAs were subsequently determined as follows.

The 3 upper strand frames of extended cDNAs are searched for ORFs defined as the maximum length fragments beginning with a translation initiation codon and ending with a stop codon. ORFs encoding at least 20 amino acids are preferred.

Each found ORF is then scanned for the presence of a signal peptide in the first 50 amino-acids or, where appropriate, within shorter regions down to 20 amino acids or less in the ORF, using the matrix method of von Heijne (Nuc. Acids Res. 14: 4683-4690 (1986)), the disclosure of which is incorporated herein by reference and the modification described in Example 22.

d) Homology to either nuclcotidic or proteic sequences Sequences of full length extended cDNAs are then compared to known sequences on a nucleotidic or proteic basis.

Sequences of full length extended cDNAs are compared to the following known nucleic acid sequences: vertebrate sequences (Genbank release GB), EST sequences (Genbank release GB), patented sequences (Genseqn release GSEQ) and recently identified sequences (Genbank daily release) available at the time of filing. Full length cDNA sequences are also compared to the sequences of a private database (Genset internal sequences) in order to find sequences that have already been identified by applicants.

Sequences of full length extended cDNAs with more than 90% homology over 30 nucleotides using either BLASTN or BLAST2N as indicated in Table II are identified as sequences that have already been described. Matching vertebrate sequences are subsequently examined using FASTA; full length extended cDNAs with more than 70% homology over 30 nucleotides are identified as sequences that have already been described.

ORFs encoded by full length extended cDNAs as defined in section c) are subsequently compared to known amino acid sequences found in Swissprot release CHP, PIR release PIR# and Genpept release WO 99/25825 PCT/B98/01862 38 UPE-'E1 public databases using BLASTP with the parameter W=8 and allowing a maximum of 10 matches.

Sequences of full length extended cDNAs showing extensive homology to known protein sequences are recognized as already identified proteins.

In addition, the three-frame conceptual translation products of the top strand of full length extended cDNAs are compared to publicly known amino acid sequences of Swissprot using BLASTX with the parameter E=0.001. Sequences of full length extended cDNAs with more than 70% homology over amino acid stretches are detected as already identified proteins.

Selection of Cloned Full Lentlli Sequences of the Present Invention Cloned full length extended cDNA sequences that have already been characterized by the aforementioned computer analysis are then submitted to an automatic procedure in order to preselect full length extended cDNAs containing sequences of interest.

a) Automatic sequence preselection All complete cloned full length extended cDNAs clipped for vector on both ends are considered.

First, a negative selection is operated in order to eliminate unwanted cloned sequences resulting from either contaminants or PCR artifacts as follows. Sequences matching contaminant sequences such as vector RNA, tRNA, mtRNA, rRNA sequences are discarded as well as those encoding ORF sequences exhibiting extensive homology to repeats as defined in section 4 Sequences obtained by direct cloning using nested primers on 5' and 3' tags (section 1. case a) but lacking polyA tail are discarded. Only ORFs containing a signal peptide and ending either before the polyA tail (case a) or before the end of the cloned 3'UTR (case b) are kept. Then, ORFs containing unlikely mature proteins such as mature proteins which size is less than amino acids or less than 25% of the immature protein size are eliminated.

In the selection of the OFR, priority was given to the ORF and the frame corresponding to the polypeptides described in SignalTag Patents (United States Patent Application Serial Nos: 08/905,223; 08/905,135; 08/905,051; 08/905,144; 08/905,279; 08/904,468; 08/905,134; and 08/905.133). If the ORF was not found among the OFRs described in the SignalTag Patents, the ORF encoding the signal peptide with the highest score according to Von Heijne method as defined in Example 22 was chosen. If the scores were identical, then the longest ORF was chosen.

Sequences of full length extended cDNA clones are then compared pairwise with BLAST after masking of the repeat sequences. Sequences containing at least 90% homology over 30 nucleotides are clustered in the same class. Each cluster is then subjected to a cluster analysis that detects sequences resulting from internal priming or from alternative splicing, identical sequences or sequences with several frameshifts. This automatic analysis serves as a basis for manual selection of the sequences.

b) Manual sequence selection Manual selection is carried out using automatically generated reports for each sequenced full length extended cDNA clone. During this manual procedures, a selection is operated between clones belonging to the same class as follows. ORF sequences encoded by clones belonging to the same class are aligned and WO 99/25825 PCT/IB98/01862 39 compared. If the homology between nucleotidic sequences of clones belonging to the same class is more than 90% over 30 nucleotide stretches or if the homology between amino acid sequences of clones belonging to the same class is more than 80% over 20 amino acid stretches, than the clones are considered as being identical. The chosen ORF is the best one according to the criteria mentioned below. If the nucleotide and amino acid homologies are less than 90% and 80% respectively, the clones are said to encode distinct proteins which can be both selected if they contain sequences of interest.

Selection of full length extended cDNA clones encoding sequences of interest is performed using the following criteria. Structural parameters (initial tag. polyadenylation site and signal) are first checked.

Then, homologies with known nucleic acids and proteins are examined in order to determine whether the clone sequence match a known nucleic/proteic sequence and, in the latter case, its covering rate and the date at which the sequence became public. If there is no extensive match with sequences other than ESTs or genomic DNA, or if the clone sequence brings substantial new information, such as encoding a protein resulting from alternative slicing of an mRNA coding for an already known protein, the sequence is kept.

Examples of such cloned full length extended cDNAs containing sequences of interest are described in Example 28. Sequences resulting from chimera or double inserts as assessed by homology to other sequences are discarded during this procedure.

EXAMPLE 28 Cloning and Sequencing of Extended cDNAs The procedure described in Example 27 above was used to obtain the extended cDNAs of the present invention. Using this approach, the full length cDNA of SEQ ID NO: 17 was obtained. This cDNA falls into the "EST-ext" category described above and encodes the signal peptide MKKVLLLITAILAVAVG (SEQ ID NO: 18) having a von Hcijne score of 8.2.

The full length cDNA of SEQ ID NO:49 was also obtained using this procedure. This cDNA falls into the "EST-ext" category described above and encodes the signal peptide MWWFQQGLSFLPSALVIWTSA (SEQ ID NO:20) having a von Heijne score of Another full length cDNA obtained using the procedure described above has the sequence of SEQ ID NO:21. This cDNA, falls into the "EST-ext" category described above and encodes the signal peptide MVLTTLPSANSANSPVNMPTTGPNSLSYASSALSPCLT (SEQ ID NO:22) having a von Heijne score of 5.9.

The above procedure was also used to obtain a full length cDNA having the sequence of SEQ ID NO:23. This cDNA falls into the "EST-ext" category described above and encodes the signal peptide ILSTVTALTFAXA (SEQ ID NO:24) having a von Heijne score of The full length cDNA of SEQ ID NO:25 was also obtained using this procedure. This cDNA falls into the "new" category described above and encodes a signal peptide LVLTLCTLPLAVA (SEQ ID NO:26) having a von Heijne score of 10.1.

WO 99/25825 PCT/IB98/01862 The full length cDNA of SEQ ID NO:27 was also obtained using this procedure. This cDNA falls into the "new" category described above and encodes a signal peptide LWLLFFLVTADHA (SEQ ID NO:28) having a von Heijne score of 10.7.

The above procedures were also used to obtain the extended cDNAs of the present invention. ESTs expressed in a variety of tissues were obtained as described above. The appended sequence listing provides the tissues from which the extended cDNAs were obtained. It will be appreciated that the extended cDNAs may also be expressed in tissues other than the tissue listed in the sequence listing.

ESTs obtained as described above were used to obtain extended cDNAs having the sequences of SEQ ID NOs: 40-86. Table II provides the sequence identification numbers of the extended cDNAs of the present invention, the locations of the full coding sequences in SEQ ID NOs: 40-86 the nucleotides encoding both the signal peptide and the mature protein, listed under the heading FCS location in Table II).

the locations of the nucleotides in SEQ ID NOs: 40-86 which encode the signal peptides (listed under the heading SigPep Location in Table the locations of the nucleotides in SEQ ID NOs: 40-86 which encode the mature proteins generated by cleavage of the signal peptides (listed under the heading Mature Polypeptide Location in Table II), the locations in SEQ ID NOs: 40-86 of stop codons (listed under the heading Stop Codon Location in Table II), the locations in SEQ ID NOs: 40-86 of polyA signals (listed under the heading Poly A Signal Location in Table II) and the locations of polyA sites (listed under the heading Poly A Site Location in Table II).

The polypeptides encoded by the extended cDNAs were screened for the presence of known structural or functional motifs or for the presence of signatures, small amino acid sequences which are well conserved amongst the members of a protein family. The conserved regions have been used to derive consensus patterns or matrices included in the PROSITE data bank, in particular in the file prosite.dat (Release 13.0 of November 1995, located at http://expasy.hcuge.ch/sprot/prositc.html. Prosite_convert and prosite scan programs (http://ulrec3.unil.ch/ftpscrveur/prositescan) were used to find signatures on the extended cDNAs.

For each pattern obtained with the prosite.convert program from the prosite.dat file, the accuracy of the detection on a new protein sequence has been tested by evaluating the frequency of irrelevant hits on the population of human secreted proteins included in the data bank SWISSPROT. The ratio between the number of hits on shuffled proteins (with a window size of 20 amino acids) and the number of hits on native (unshuffled) proteins was used as an index. Every pattern for which the ration was greater than 20% (one hit on shuffled proteins for 5 hits on native proteins) was skipped during the search with prosite_scan. The program used to shuffle protein sequences (dbshuffled) and the program used to determine the statistics for each pattern in the protein data banks (prosite_statistics) are available on the ftp site http://ulrec3.unil.ch/ftpserveur/prositescan.

The results of the search are provided in Table Il. The first column provides the ID number of the sequence. The second column indicates the beginning and end positions of the signature. The Prosite WO 99/25825 PCT/IB98/01862 41 definition of the signature is indicated in the third column.

Table IV lists the sequence identification numbers of the polypeptides of SEQ ID NOs: 87-133. the locations of the amino acid residues of SEQ ID NOs: 87-133 in the full length polypeptide (second column).

the locations of the amino acid residues of SEQ ID NOs: 87-133 in the signal peptidcs (third column), and tie locations of the amino acid residues of SEQ ID NOs: 87-133 in the mature polypeptide created by cleaving the signal peptide from the full length polypeptide (fourth column). In Table IV. the first amino acid of the signal peptide is designated as amino acid number 1. In the appended sequence listing, the first amino acid of the mature protein resulting fronm cleavage of the signal peptide is designated as amino acid number 1 and the first amino acid of the signal peptide is designated with the appropriate negative number.

in accordance with the regulations governing sequence listings.

The extended cDNAs of the present invention were categorized based on their homology to known sequences. Genebank release #103, division ESTs. and Geneseq release #28 were used to scan the extended cDNAs using Blast. For each extended cDNA ID, the covering rate of the sequence by another sequence was determined as follows. The length in nucleotides of the matching segment was calculated (even when gaps were present) and divided by the length in nucleotides of the extended cDNA sequence. When more than one covering rate was obtained for a given extended cDNA, the higher covering rate was used to classify the extended cDNA. The Geneseq sequences have been categorized as either ESTs or vertebrate, with ESTs being those sequences obtained by random sequencing of cDNA libraries and vertebrate sequences being those sequences containing sequences resembling known functional motifs.

The results of this categorization are provided in Table V. The first column lists the sequence identification number of the sequence being categorized. The second column indicates those sequences having no matches with the database scanned. The third column indicates those sequences having a covering rate of less than 30%. The fourth column indicates those sequences having a covering rate greater than 30%. The fifth column indicates sequences partially or totally covered by vertebrate sequences as described above.

The nucleotide sequences of the sequences of SEQ ID NOs: 40-86 and 134-180, and the amino acid sequences encoded by SEQ ID NOs: 40-86 and 134-180 amino acid sequences of SEQ ID NOs: 87-133 and 181-227) are provided in the appended sequence listing. In some instances, the sequences are preliminary and may include some incorrect or ambiguous sequences or amino acids. The sequences of SEQ ID NOs: 40-86 and 134-180 can readily be screened for any errors therein and any sequence ambiguities can be resolved by resequencing a fragment containing such errors or ambiguities on both strands. Nucleic acid fragments for resolving sequencing errors or ambiguities may be obtained from the deposited clones or can be isolated using the techniques described herein. Resolution of any such ambiguities or errors may be facilitated by using primers which hybridize to sequences located close to the ambiguous or erroneous sequences. For example, the primers may hybridize to sequences within 50-75 bases of the ambiguity or error. Upon resolution of an error or ambiguity, the corresponding corrections can be made in the protein WO 99/25825 PCT/IB98/01862 42 sequences encoded by the DNA containing the error or ambiguity. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell.

collecting the protein, and determining its sequence.

For each amino acid sequence, Applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing. Some of the amino acid sequences may contain "Xaa" designators. These "Xaa" designators indicate cither a residue which cannot be identified because of nucleotide sequence ambiguity or a stop codon in the determined sequence where Applicants believe one should not exist (if the sequence were determined more accurately).

Cells containing the 47 extended cDNAs (SEQ ID NOs: 134-180) of the present invention in the vector pED6dpc2, are maintained in permanent deposit by the inventors at Genset, 24 Rue Royale, 75008 Paris, France.

A pool of the cells containing the 47 extended cDNAs (SEQ ID NOs: 134-180), from which the cells containing a particular polynucleotide is obtainable, will be deposited with the American Type Culture Collection. Each extended cDNA clone will be transfected into separate bacterial cells (E-coli) in this composite deposit. A pool of cells containing the 43 extended cDNAs (SEQ ID NOs: 134, 136-143, 145- 162, 164-174, and 176-180), from which the cells containing a particular polynucleotide is obtainable, were deposited with the American Type Culture Collection on December 16, 1997, under the name SignalTag 1- 43. and ATCC accession No. 98619. A pool of cells comprising the 2 extended cDNAs (SEQ ID NOs: 144 and 163), from which the cells containing a particular polynucleotide is obtainable, were deposited with the American Type Culture Collection on October 15, 1998. under the name SignalTag 44-66, and ATCC accession No. 98923. Each extended cDNA can be removed from the pED6dpc2 vector in which it was deposited by performing a Notl, Pstl double digestion to produce the appropriate fragment for each clone.

The proteins encoded by the extended cDNAs may also be expressed from the promoter in pED6dpc2.

Bacterial cells containing a particular clone can be obtained from the composite deposit as follows: An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The design of the oligonucleotide probe should preferably follow these parameters: It should be designed to an area of the sequence which has the fewest ambiguous bases if any; Preferably, the probe is designed to have a Tm of approx. 80 0 C (assuming 2 degrees for each A or T and 4 degrees for each G or However, probes having melting temperatures between 40 °C and 80 OC may also be used provided that specificity is not lost.

The oligonucleotide should preferably be labeled with g- 3 PATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe WO 99/25825 PCT/IB98/01862 43 should be quantified by measurement in a scintillation counter. Preferably. specific activity of the resulting probe should be approximately 4X 106 dpn/pmole.

The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ptl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 tpg/ml. The culture should preferably be grown to saturation at 370C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 1(X) pg/ml and agar at in a 150 mm petri dish when grown overnight at 370C. Other known methods of obtaining distinct, well-separated colonies can also be employed.

Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.

The filter is then preferably incubatedat 65 0 C for 1 hour with gentle agitation in 6X SSC stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 pg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to IX 06 dpm/mL. The filter is then preferably incubated at 65CC with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.IX SSC/0.5% SDS at 65CC for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.

The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.

The plasmid DNA obtained using these procedures may then be manipulated using standard cloning techniques familiar to those skilled in the art. Alternatively, a PCR can be done with primers designed at both ends of the extended cDNA insertion. For example, a PCR reaction may be conducted using a primer having the sequence GGCCATACACTTGAGTGAC (SEQ ID NO:38) and a primer having the sequence ATATAGACAAACGCACACC (SEQ. ID. NO:39). The PCR product which corresponds to the extended cDNA can then be manipulated using standard cloning techniques familiar to those skilled in the art.

In addition to PCR based methods for obtaining extended cDNAs, traditional hybridization based methods may also be employed. These methods may also be used to obtain the genomic DNAs which encode the mRNAs from which the 5' ESTs were derived, mRNAs corresponding to the extended cDNAs, or nucleic acids which are homologous to extended cDNAs or 5' ESTs. Example 29 below provides an example of such methods.

WO 99/25825 PCT/IB98/01862 44 EXAMPLE 29 Methods for Obtainine Extended cDNAs or Nucleic Acids Homologous to Extended cDNAs or 5' ESTs A full length cDNA library can be made using the strategies described in Examples 13. 14. 15. and 16 above by replacing the random nonamer used in Example 14 with an oligo-dT primer. For instance, the oligoniucleoide of SEQ ID NO: 14 may be used.

Alternatively, a cDNA library or genomic DNA library may be obtained from a commercial source or made using techniques familiar to those skilled in the art. The library includes cDNAs which are derived from the mRNA corresponding to a 5' EST or which have homology to an extended cDNA or 5' EST. The cDNA library or genomic DNA library is hybridized to a detectable probe comprising at least consecutive nucleotides from the 5' EST or extended cDNA using conventional techniques. Preferably, the probe comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST or extended cDNA. More preferably, the probe comprises at least 20-30 consecutive nucleotides from the 5' EST or extended cDNA.

In some embodiments, the probe comprises more than 30 nucleotides from the 5' EST or extended cDNA.

Techniques for identifying cDNA clones in a cDNA library which hybridize to a given probe sequence are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press. (1989). The same techniques may be used to isolate genomic DNAs.

Briefly, cDNA or genomic DNA clones which hybridize to the detectable probe are identified and isolated for further manipulation as follows. A probe comprising at least 10 consecutive nucleotides from the 5' EST or extended cDNA is labeled with a detectable label such as a radioisotope or a fluorescent molecule. Preferably, the probe comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST or extended cDNA. More preferably, the probe comprises 20-30 consecutive nucleotides from the 5' EST or extended cDNA. In some embodiments, the probe comprises more than 30 nuclcotides from the 5' EST or extended cDNA.

Techniques for labeling the probe are well known and include phosphorylation with polynucleotide kinase, nick translation, in vitro transcription, and non-radioactive techniques. The cDNAs or genomic DNAs in the library are transferred to a nitrocellulose or nylon filter and denatured. After incubation of the filter with a blocking solution, the filter is contacted with the labeled probe and incubated for a sufficient amount of time for the probe to hybridize to cDNAs or genomic DNAs containing a sequence capable of hybridizing to the probe.

By varying the stringency of the hybridization conditions used to identify extended cDNAs or genomic DNAs which hybridize to the detectable probe, extended cDNAs having different levels of homology to the probe can be identified and isolated. To identify extended cDNAs or genomic DNAs having a high degree of homology to the probe sequence, the melting temperature of the probe may be calculated using the following formulas: WO 99/25825 PCT/IB98/01862 For probes between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula: Tm=81.5+16.6(log I(fraction G+C)-(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation Tm=S1.5+16.6(log 1(fraction formamide)- (600/N) where N is the length of the probe.

Prehybridization may be carried out in 6X SSC, 5X Dcnhardt's reagent. 0.5% SDS, 100tg denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS. l00pg denatured fragmented salmon sperm DNA, 50% formamide. The formulas for SSC and Denhardt's solutions are listed in Sambrook et al., supra.

Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to extended cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in_length, the hybridization may be carried out at 15-250C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 15-25CC below the Tm. Preferably, for hybridizations in 6X SSC, the hybridization is conducted at approximately 680C. Preferably, for hybridizations in 50% formamide containing solutions, the hybridization is conducted at approximately 420C.

All of the foregoing hybridizations would be considered to be under "stringent" conditions.

Following hybridization, the filter is washed in 2X SSC, 0.1% SDS at room temperature for minutes. The filter is then washed with 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour.

Thereafter, the solution is washed at the hybridization temperature in 0.1X SSC, 0.5% SDS. A final wash is conducted in 0. IX SSC at room temperature.

Extended cDNAs, nucleic acids homologous to extended cDNAs or 5' ESTs, or genomic DNAs which have hybridized to the probe are identified by autoradiography or other conventional techniques.

The above procedure may be modified to identify extended cDNAs, nucleic acids homologous to extended cDNAs, or genomic DNAs having decreasing levels of homology to the probe sequence. For example, to obtain extended cDNAs, nucleic acids homologous to extended cDNAs, or genomic DNAs of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 50C from 680C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate" conditions above 50 0 C and "low" conditions below Alternatively, the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 420C. In this case, the concentration of formamide in the hybridization WO 99/25825 PCT/IB98/01862 46 buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6X SSC, 0.5% SDS at 500C. These conditions are considered to be "moderate" conditions above 25% formamide and "low" conditions below 25% formamide.

Extended cDNAs, nucleic acids homologous to extended cDNAs. or genomic DNAs which have hybridized to the probe are identified by autoradiography.

If it is desired to obtain nucleic acids homologous to extended cDNAs. such as allelic variants thereof or nucleic acids encoding proteins related to the proteins encoded by the extended cDNAs, the level of homology between the hybridized nucleic acid and the extended cDNA or 5' EST used as the probe may readily be determined. To determine the level of homology between the hybridized nucleic acid and the extended cDNA or 5'EST from which the probe was derived, the nucleotide sequences of the hybridized nucleic acid and the extended cDNA or 5'EST from which the probe was derived are compared. For example, using the above methods, nucleic acids having at least 95% nucleic acid homology to the extended cDNA or 5'EST from which the probe was derived may be obtained and identified. Similarly, by using progressively less stringent hybridization conditions one can obtain and identify nucleic acids having at least at least 85%, at least 80% or at least 75% homology to the extended cDNA or 5'EST from which the probe was derived.

To determine whether a clone encodes a protein having a given amount of homology to the protein encoded by the extended cDNA or 5' EST, the amino acid sequence encoded by the extended cDNA or EST is compared to the amino acid sequence encoded by the hybridizing nucleic acid. Homology is determined to exist when an amino acid sequence in the extended cDNA or 5' EST is closely related to an amino acid sequence in the hybridizing nucleic acid. A sequence is closely related when it is identical to that of the extended cDNA or 5' EST or when it contains one or more amino acid substitutions therein in which amino acids having similar characteristics have been substituted for one another. Using the above methods, one can obtain nucleic acids encoding proteins having at least 95%, at least 90%, at least 85%, at least 80% or at least 75% homology to the proteins encoded by the extended cDNA or 5'EST from which the probe was derived.

Alternatively, extended cDNAs may be prepared by obtaining mRNA from the tissue, cell, or organism of interest using mRNA preparation procedures utilizing poly A selection procedures or other techniques known to those skilled in the art. A first primer capable of hybridizing to the poly A tail of the mRNA is hybridized to the mRNA and a reverse transcription reaction is performed to generate a first cDNA strand.

The first cDNA strand is hybridized to a second primer containing at least 10 consecutive nucleotides of the sequences of the 5' EST for which an extended cDNA is desired. Preferably, the primer comprises at least 12, 15, or 17 consecutive nucleotides from the sequences of the 5' EST. More preferably, the primer comprises 20-30 consecutive nucleotides from the sequences of the 5' EST. In some WO 99/25825 PCT/IB98/01862 47 embodiments, the primer comprises more than 30 nucleotides from the sequences of the 5' EST. If it is desired to obtain extended cDNAs containing the full protein coding sequence, including the authentic translation initiation site, the second primer used contains sequences located upstream of the translation initiation site. The second primer is extended to generate a second cDNA strand complementary to the first cDNA strand. Alternatively, RTPCR may be performed as described above using primers from both ends of the cDNA to be obtained.

Extended cDNAs containing 5' fragments of the mRNA may be prepared by contacting an mRNA comprising the sequence of the 5' EST for which an extended cDNA is desired with a primer comprising at least 10 consecutive nucleotides of the sequences complementary to the 5' EST, hybridizing the primer to the mRNAs, and reverse transcribing the hybridized primer to make a first cDNA strand from the mRNAs.

Preferably, the primer comprises at least 12, 15. or 17 consecutive nucleotides from the 5' EST. More preferably, the primer comprises 20-30 consecutive nucleotides from the 5' EST.

Thereafter, a second cDNA strand complementary to the first cDNA strand is synthesized. The second cDNA strand may be made by hybridizing a primer complementary to sequences in the first cDNA strand to the first cDNA strand and extending the primer to generate the second cDNA strand.

The double stranded extended cDNAs made using the methods described above are isolated and cloned. The extended cDNAs may be cloned into vectors such as plasmids or viral vectors capable of replicating in an appropriate host cell. For example, the host cell may be a bacterial, mammalian, avian, or insect cell.

Techniques for isolating mRNA, reverse transcribing a primer hybridized to mRNA to generate a first cDNA strand, extending a primer to make a second cDNA strand complementary to the first cDNA strand, isolating the double stranded cDNA and cloning the double stranded cDNA are well known to those skilled in the art and are described in Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc.

(1997); and Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, (1989).

Alternatively, kits for obtaining full length cDNAs, such as the GeneTrapper (Cat. No. 10356-020, Gibco, BRL), may be used for obtaining full length cDNAs or extended cDNAs. In this approach, full length or extended cDNAs are prepared from mRNA and cloned into double stranded phagemids. The cDNA library in the double stranded phagemids is then rendered single stranded by treatment with an endonuclease, such as the Gene II product of the phage Fl, and Exonuclease III as described in the manual accompanying the GeneTrapper kit. A biotinylated oligonucleotide comprising the sequence of a 5' EST, or a fragment containing at least 10 nucleotides thereof, is hybridized to the single stranded phagemids.

Preferably, the fragment comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST. More preferably, the fragment comprises 20-30 consecutive nucleotides from the 5' EST. In some procedures, the fragment may comprise more than 30 consecutive nucleotides from the 5' EST.

Hybrids between the biotinylated oligonucleotide and phagemids having inserts containing the WO 99/25825 PCT/IB98/01862 48 EST sequence are isolated by incubating the hybrids with streptavidin coated paramagnetic beads and retrieving the beads with a magnet. Thereafter, the resulting phagemids containing the 5' EST sequence are released from tile beads and converted into double stranded DNA using a primer specific for the 5' EST sequence. The resulting double stranded DNA is transformed into bacteria. Extended cDNAs containing the 5' EST sequence are identified by colony PCR or colony hybridization.

A plurality of extended cDNAs containing full length protein coding sequences or sequences encoding only the mature protein remaining after the signal peptide is cleaved may be provided as cDNA libraries for subsequent evaluation of the encoded proteins or use in diagnostic assays as described below.

IV. Expression of Proteins Encoded by Extended cDNAs Isolated Using 5' ESTs Extended cDNAs containing the full protein coding sequences of their corresponding mRNAs or portions thereof, such as cDNAs encoding the mature protein, may be used to express the secreted proteins or portions thereof which they encode as described in Example 30 below. If desired, the extended cDNAs may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein. It will be appreciated that a plurality of extended cDNAs containing the full protein coding sequences or portions thereof may be simultaneously cloned into expression vectors to create an expression library for analysis of the encoded proteins as described below.

EXAMPLE Expression of the Proteins Encoded by Extended cDNAs or Portions Thereof To express the proteins encoded by the extended cDNAs or portions thereof, nucleic acids containing the coding sequence for the proteins or portions thereof to be expressed are obtained as described in Examples 27-29 and cloned into a suitable expression vector. If desired, the nucleic acids may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein. For example, the nucleic acid may comprise the sequence of one of SEQ ID NOs: 134-180 listed in Table VII and in the accompanying sequence listing. Alternatively, the nucleic acid may comprise those nucleotides which make up the full coding sequence of one of the sequences of SEQ ID NOs: 134-180 as defined in Table VII above.

It will be appreciated that should the extent of the full coding sequence the sequence encoding the signal peptide and the mature protein resulting from cleavage of the signal peptide) differ from that listed in Table VII as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be readily able to identify the extent of the full coding sequences in the sequences of SEQ ID NOs. 134-180. Accordingly. the scope of any claims herein relating to nucleic acids containing the full coding sequence of one of SEQ ID NOs. 134-180 is not to be construed as excluding any readily identifiable variations from or equivalents to the full coding sequences listed in Table VII. Similarly, should the extent of the full length polypeptides differ from those indicated in Table VIII as a result of any of the preceding factors, the scope of claims relating to polypeptides WO 99/25825 PCT/IB98/01862 49 comprising the amino acid sequence of the full length polypeptides is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table VII.

Alternatively, the nucleic acid used to express the protein or portion thereof may comprise those nucleotides which encode the mature protein the protein created by cleaving the signal peptide off) encoded by one of the sequences of SEQ ID NOs: 134-180 as defined in Table VII.

It will be appreciated that should the extent of the sequence encoding the mature protein differ from that listed in Table VII as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein.

or other biological factors, one skilled in the art would be readily able to identify the extent of the sequence encoding the mature protein in the sequences of SEQ ID NOs: 134-180. -Accordingly, the scope of any claims herein relating to nucleic acids containing the sequence encoding the mature protein encoded by one of SEQ ID NOs: 134-180 is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table VII. Thus, claims relating to nucleic acids containing the sequence encoding the mature protein encompass equivalents to the sequences listed in Table VII, such as sequences encoding biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the proteins in addition to cleavage of the signal peptide. Similarly, should the extent of the mature polypeptides differ from those indicated in Table VIII as a result of any of the preceding factors, the scope of claims relating to polypeptides comprising the sequence of a mature protein included in the sequence of one of SEQ ID NOs. 181-227 is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table VIII. Thus, claims relating to polypeptides comprising the sequence of the mature protein encompass equivalents to the sequences listed in Table VIII, such as biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the proteins in addition to cleavage of the signal peptide. It will also be appreciated that should the biologically active form of the polypeptides included in the sequence of one of SEQ ID NOs.

181-227 or the nucleic acids encoding the biologically active form of the polypeptides differ from those identified as the mature polypeptide in Table VIII or the nucleotides encoding the mature polypeptide in Table VII as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be 1radily able to identify the amino acids in the biologically active form of the polypeptides and the nucleic acids encoding the biologically active form of the polypeptides. In such instances, the claims relating to polypeptides comprising the mature protein included in one of SEQ ID NOs. 181-227 or nucleic acids comprising the nucleotides of one of SEQ ID NOs. 134- 180 encoding the mature protein shall not be construed to exclude any readily identifiable variations from the sequences listed in Table VII and Table VIII.

In some embodiments, the nucleic acid used to express the protein or portion thereof may comprise WO 99/25825 PCT/IB98/01862 those nucleotides which encode the signal peptide encoded by one of the sequences of SEQ ID NOs: 134- 180 as defined in Table VII above.

It will be appreciated that should the extent of the sequence encoding the signal peptide differ from that listed in Table VII as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be readily able to identify the extent of the sequence encoding the signal peptide in the sequences of SEQ ID NOs. 134-180. Accordingly. the scope of any claims herein relating to nucleic acids containing the sequence encoding the signal peptide encoded by one of SEQ ID NOs.134-180 is not to be construed as excluding any readily identifiable variations from the sequences listed in Table VII. Similarly, should the extent of the signal peptides differ from those indicated in Table V11I as a result of any of the preceding factors, the scope of claims relating to polypeptides comprising the sequence of a signal peptide included in the sequence of one of SEQ ID NOs. 181-227 is not to be construed as excluding any readily identifiable variations from the sequences listed in Table VIII.

Alternatively, the nucleic acid may encode a polypeptide comprising at least 10 consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227. In some embodiments, the nucleic acid may encode a polypeptide comprising at least 15 consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227. In other embodiments, the nucleic acid may encode a polypeptide comprising at least consecutive amino acids of one of the sequences of SEQ ID NOs: 181-227.

The nucleic acids inserted into the expression vectors may also contain sequences upstream of the sequences encoding the signal peptide, such as sequences which regulate expression levels or sequences which confer tissue specific expression.

The nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The expression vector may be any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767.

The following is provided as one exemplary method to express the proteins encoded by the extended cDNAs corresponding to the 5' ESTs or the nucleic acids described above. First, the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the extended cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and WO 99/25825 PCT/IB98/01862 51 incorporating it into the mammalian expression vector pXTI (Stratagene). pXTI contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The extended cDNA or portion thereof encoding the polypeptide to be expressed is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the extended cDNA or portion thereof and containing restriction cndonuclease sequences for Pst I incorporated into the 5'primer and Bglll at the 5' end of the corresponding cDNA 3' primer, taking care to ensure that the extended cDNA is positioned in frame with the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with Pstl. blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXTI, now containing a poly A signal and digested with Bgll.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml G418 (Sigma, St. Louis, Missouri). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.

Alternatively, the extended cDNAs may be cloned into pED6dpc2 as described above. The resulting pED6dpc2 constructs may be transfected into a suitable host cell, such as COS I cells.

Methotrexate resistant cells are selected and expanded. Preferably, the protein expressed from the extended cDNA is released into the culture medium thereby facilitating purification.

Proteins in the culture medium are separated by gel electrophoresis. If desired, the proteins may be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis.

As a control, the expression vector lacking a cDNA insert is introduced into host cells or organisms and the proteins in the medium are harvested. The secreted proteins present in the medium are detected using techniques such as Coomassic or silver staining or using antibodies against the protein encoded by the extended cDNA. Coomassie and silver staining techniques are familiar to those skilled in the art.

Antibodies capable of specifically recognizing the protein of interest may be generated using synthetic 15-mer peptides having a sequence encoded by the appropriate 5' EST, extended cDNA, or portion thereof. The synthetic peptides are injected into mice to generate antibody to the polypeptide encoded by the 5' EST, extended cDNA, or portion thereof.

Secreted proteins from the host cells or organisms containing an expression vector which contains the extended cDNA derived from a 5' EST or a portion thereof are compared to those from the control cells or organism. The presence of a band in the medium from the cells containing the expression vector which is absent in the medium from the control cells indicates that the extended cDNA encodes a secreted protein.

Generally, the band corresponding to the protein encoded by the extended cDNA will have a mobility near that expected based on the number of amino acids in the open reading frame of the extended cDNA.

However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

WO 99/25825 PCT/IB98/01862 52 Alternatively, if the protein expressed from the above expression vectors does not contain sequences directing its secretion, the proteins expressed from host cells containing an expression vector containing an insert encoding a secreted protein or portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the desired protein or portion thereof is being expressed. Generally, tie band will have the mobility expected for the secreted protein or portion thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

The protein encoded by the extended cDNA may be purified using standard inmmunochromatography techniques. In such procedures, a solution containing the secreted protein, such as the culture medium or a cell extract, is applied to a column having antibodies against the secreted protein attached to the chromatography matrix. The secreted protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound secreted protein is then released from the column and recovered using standard techniques.

If antibody production is not possible, the extended cDNA sequence or portion thereof may be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the coding sequence of the extended cDNA or portion thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera may be p-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to p-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites may be engineered between the p-globin gene or the nickel binding polypeptide and the extended cDNA or portion thereof. Thus, the two polypeptides of the chimera may be separated from one another by protease digestion.

One useful expression vector for generating p-globin chimerics is pSG5 (Stratagene), which encodes rabbit p-globin. Intron II of the rabbit 0-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (Basic Methods in Molecular Biology, L.G. Davis, M.D.

Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express T Translation Kit (Stratagene).

Following expression and purification of the secreted proteins encoded by the 5' ESTs, extended cDNAs, or fragments thereof, the purified proteins may be tested for the ability to bind to the surface of various cell types as described in Example 31 below. It will be appreciated that a plurality of proteins expressed from these cDNAs may be included in a panel of proteins to be simultaneously evaluated for the WO 99/25825 PCT/IB98/01862 53 activities specifically described below, as well as other biological roles for which assays for determining activity are available.

EXAMPLE 31 Analysis of Secreted Proteins to Determine Whether they find to the Cell Surface The proteins encoded by the 5' ESTs. extended cDNAs. or fragments thereof are cloned into expression vectors such as those described in Example 30. The proteins are purified by size. charge.

immunochromatography or other techniques familiar to those skilled in the art. Following purification, the proteins are labeled using techniques known to those skilled in the art. The labeled proteins are incubated with cells or cell lines derived from a variety of organs or tissues to allow the proteins to bind to any receptor present on the cell surface. Following the incubation, the cells are washed to remove nonspecifically bound protein. The labeled proteins are detected by autoradiography. Alternatively, unlabeled proteins may be incubated with the cells and detected with antibodies having a detectable label, such as a fluorescent molecule, attached thereto.

Specificity of cell surface binding may be analyzed by conducting a competition analysis in which various amounts of unlabeled protein are incubated along with the labeled protein. The amount of labeled protein bound to the cell surface decreases as the amount of competitive unlabeled protein increases. As a control, various amounts of an unlabeled protein unrelated to the labeled protein is included in some binding reactions. The amount of labeled protein bound to the cell surface does not decrease in binding reactions containing increasing amounts of unrelated unlabeled protein, indicating that the protein encoded by the cDNA binds specifically to the cell surface.

As discussed above, secreted proteins have been shown to have a number of important physiological effects and, consequently, represent a valuable therapeutic resource. The secreted proteins encoded by the extended cDNAs or portions thereof made according to Examples 27-29 may be evaluated to determine their physiological activities as described below.

EXAMPLE 32 Assaving the Proteins Expressed from Extended cDNAs or Portions Thereof for Cvtokine. Cell Proliferation or Cell Differentiation Activity As discussed above, secreted proteins may act as cytokines or may affect cellular proliferation or differentiation. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAIG, T10, B9, B9/11, BaF3, MC9/G, M+ (preB 2E8, RB5, DAI, 123, TI 165, HT2, CTLL2, TF-1, Mo7c and CMK. The proteins encoded by the above extended cDNAs or portions thereof WO 99/25825 PCT/IB98/01862 54 may be evaluated for their ability to regulate T cell or thymocyte proliferation in assays such as those described above or in the following references: Current Protocols in Immunology, Ed. by J.E. Coligan et al..

Greene Publishing Associates and Wiley-Interscience; Takai et al. J. Inmunol. 137:3494-3500 (1986); Bertagnolli et al. J. Innol. 145:1706-1712 (1990): Bertagnolli et al.. Cellular hnnunology 133:327-341 (1991); Bertagnolli. et al. J. hnmiunol. 149:3778-3783 (1992); and Bowman et al., J. Imnmunol. 152:1756- 1761 (1994).

In addition, numerous assays for cytokine production and/or the proliferation of spleen cells, lymph node cells and thymocytes are known. These include the techniques disclosed in Current Protocols in Imunnology. J.E. Coligan et al. Eds., Vol 1 pp. 3.12.1-3.12.14 John Wiley and Sons. Toronto. (1994); and Schreiber. R.D. Current Protocols in Imnunology., supra Vol I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. (1994).

The proteins encoded by the cDNAs may also be assayed for the ability to regulate the proliferation and differentiation of hematopoictic or lymphopoietic cells. Many assays for such activity are familiar to those skilled in the art, including the assays in the following references: Bottomly, Davis, L.S. and Lipsky, Measurement of Human and Murine Interleukin 2 and Interleukin 4, Current Protocols in Immunology., J.E. Coligan et al. Eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. (1991); deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al.. Nature 36:690-692, (1988); Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, (1983); Nordan, Measurement of Mouse and Human Interleukin 6. Current Protocols in Inmunology. J.E. Coligan ct al. Eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. (1991); Smith et al.. Proc. Nall. Acad. Sci. U.S.A. 83:1857-1861, 1986; Bennett, F., Giannotti, Clark, S.C. and Turner, Measurement of Human Interleukin 11. Current Protocols in Imnunology. J.E. Coligan et al. Eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. (1991); and Ciarletta, Giannotti, Clark, S.C. and Turner, Measurement of Mouse and Human Interleukin 9. Current Protocols in Immunuology. J.E. Coligan et al., Eds. Vol 1 pp. 6.13.1, John Wilcy and Sons, Toronto. (1991).

The proteins encoded by the cDNAs may also be assayed for their ability to regulate T-cell responses to antigens. Many assays for such activity are familiar to those skilled in the art, including the assays described in the following references: Chapter 3 (In Vitro Assays for Mouse Lymphocyte Function), Chapter 6 (Cytokines and Their Cellular Receptors) and Chapter 7, (Immunologic Studies in Humans) Current Protocols in Immunology, J.E. Coligan et al. Eds. Greene Publishing Associates and Wiley- Interscience; Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095 (1980); Weinberger et al., Eur. J.

Iummun. 11:405-411 (1981); Takai et al., J. Inmunol. 137:3494-3500 (1986); and Takai et al., J. Immunol.

140:508-512 (1988).

Those proteins which exhibit cytokine, cell proliferation, or cell differentiation activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which induction of cell proliferation or differentiation is beneficial. Alternatively, as described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host WO 99/25825 PCT/IB98/01862 cells to increase or decrease tile expression of the proteins as desired.

EXAMPLE 33 Assaying t he-Proteins Expressed from Extended cDNAs or Portions Thereof for Activity as Irnmuni S -Ystcm Regulators Tile proteins encoded by tile cDNAs may also be evaluated for their effects as immune regulators.

l'or example. the proteins may be evaluated for their activity to influence thymocyte or splenocyte cytotoxienty. Numerous assays for such activity are familiar to those skilled in the art including thle assays described in thle following references: Chapter 3 (in Vitro Assays for Mouse Lymphocyte Function 3. 1 3.19) and Chapter 7 (Immunologic Studies in Humans) Currenit Protocols in Imiminology', J.E. Coligan et al.

Eds. Greene Publishing Associates and Wiley-Interscietice;, Herrmann et al., Proc. Nall. AcadI. Sci. USA 78:2488-24921 (198 Herrmann et lInuinol. 128:1968-1974 (1982); Handa et al., himmol.

135:1564-1572 (1985). Takai et al., J. hnumnol. 137:3494-3500 (1986); Takai et al.. hinunol. 140:50S- 512 (1988); Herrmann et Proc. Nail. Acad. Sci. US.A 78:2488-2492 (1981)-. Herrmann et al J. hnnwinol.

128:1968-1974 (1982); Handa et hinuuol. 135:1564-1572 (1985); Takai et al., J. hinunol.

137:3494-3500 (1986); Bowman et Virology 61:1992-1998; Takai et lmnmunol. 140:508-5 12 (19I88); Bertagnolli et al., Cellular Immutnology 133:327-341 (199 and Brown et al., J. Iminnmol.

153:3079-3092 (1994).

The proteins cncoded by the cDNAs may also be cvaluated for their effects on T-ccll dependent immunoglobulin responses and isotype switching. Numerous assays for such activity are familiar to those skilled in the art, including thle assays disclosed in thle following references: Maliszewski, J. Inmnunol.

144:3028-3033 (1990); and Mond, J.J. and Brunswick, M. Assays for B Cell Function: i vitro Antibody Production, Vol I pp. 3.8.1-3.8.16 Current Protocols in lIninunology. J.E-Coligan et al Eds., John Wiley and Sons, Toronto. (1994).

The proteins encoded by the cDNAs may also be evaluated for their effect on immune effector cells, including their effect on Th I cells and cytotoxic lymphocytes. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Chapter 3 (in Vitro Assays for Mouse Lymphocyte Function 3.1-3. 19) and Chapter 7 (Immunologic Studies in Humans) Current Protocols inz Innuiology, J.E. Coligan et al. Eds., Greene Publishing Associates and Wiley- Interscience; Takai et al., J. lmnzunol. 137:3494-3500 (1986); Takai et al.; J. lInnunol. 140:508-512 (1988); and Bertagnolli et limmunol. 149:3778-3783 (1992).

The proteins encoded by the cDNAs may also be evaluated for their effect on dendritic cell mediated activation of naive T-cells. Numerous assays for such activity are familiar to those skilled in thle art, including the assays disclosed in the following references: Guery et al., J. inintunol. 134:536-544 (1995); Inaba et al., Journzal of Experimental Medicine 173:549-559 (199 Macatonia et al., humiunol.

154:5071-5079 (1995); Porgador et al., Journal of Experimental Medicine 182:255-260 (1995); Nair et al., WO 99/25825 PCT/B98/01862 56 Journal of Virology 67:4062-4069 (1993); Huang et al.. Science 264:961-965 (1994): Macatonia et al., Journal of Experimental Medicine 169:1255-1264 (1989); Bhardwaj et al.. Journal of Clinical Investigation 94:797-807 (1994); and Inaba et al.. Journal of Eperimental'Medicine 172:631-640 (1990).

The proteins encoded by the cDNAs may also be evaluated for their influence on the lifetime of lymphocytes. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: D)arzynkiewicz et al.. Cvyometry 13:795-808 (1992); Gorczyca ct al..

Leukemia 7:659-670 (1993); Gorcyca et al.. Ca'ncer Research 53:1945-1951 (1993); Itoh et al.. Cell 66:233-243 (1991); Zacharchuk et al., J. Inunnol. 145:4037-4045 (1990); Zanmi et al., Cvyometrv 14:891- 897 (1993): and Gorczyca et al., Internaiional Journal of Oncology 1:639-648 (1992).

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117 (1994); Fine et al., Cellular immunology 155:111-122 (1994); Galy et al., Blood 85:2770-2778 (1995); and Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551 (1991).

Those proteins which exhibit activity as immune system regulators activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of immune activity is beneficial.

For example, the protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), in regulating (up or down) growth and proliferation ofT and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases caused by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses. mycobactcria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.

Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.

Using the proteins of the invention it may also be possible to regulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T- WO 99/25825 PCT/IB98/01862 57 cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.

Immunosuppression ofT cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing nonresponsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolcrizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (including without limitation 1 lymphocyte antigen functions (such as, for example, 137)), preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versushost disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen B7-1, 137-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject. it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.

Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, (1989), pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.

Many autoimmune disorders are the result of inappropriate activation ofT cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.

WO 99/25825 PCT/IB98/01862 58 Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally. blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus crythmatosis in MIUJpr/pr mice or NZB hybrid mice. murine autoimmuno collagen arthritis, diabetes mellitus in OD mice and BB rats, and murine experimental nyasthenia gravis (see Paul ed., Fwultmwntal Innumology, Raven Press, New York, (19S9), pp. 8-10-856).

Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory form of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to T cells in vivo. thereby activating the T cells.

In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acids encoding all or a portion of a WO 99/25825 PCT/IB98/01862 59 cytoplasmic-dornain truncated portion) of an MHC class I ca chain protein and 03: macroglobulin protein or an MHC class 11 a chain protein and an MHC class 1 chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class II or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen B7-1. B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MI-C class II associated protein, such as the invariant chain, can also be cotransfecied with a DNA encoding a peptide having the activity of aB lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. Alternatively, us described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.

EXAMPLE 34 Assaying the Proteins Expressed from Exended cDNAs or Portions Thereof for Hematopoiesis Regulating Activity The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their hematopoiesis regulating activity. For example, the effect of the proteins on embryonic stem cell differentiation may be evaluated. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Johansson et al. Cellular Biology 15:141-151 (1995); Keller et al.. Molecular and Cellular Biology 13:473-486 (1993); and McClanahan et al.. Blood 81:2903-2915 (1993).

The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their influence on the lifetime of stem cells and stem cell differentiation. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Freshney, M.G. Methylcellulose Colony Forming Assays, Culture of Hematopoietic Cells. R.I. Freshney, et al. Eds.

pp. 265-268, Wiley-Liss, Inc., New York, NY. (1994); Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911 (1992); McNiece, I.K. and Briddell, R.A. Primitive Hematopoietic Colony Forming Cells with High Proliferative Potential, Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. (1994); Neben et al., Experimental Hematology 22:353-359 (1994); Ploemacher, R.E. Cobblestone Area Forming Cell Assay, Culture of Hematopoietic Cells. R.I. Freshncy, et al. Eds. pp. 1-21, Wiley-Liss, Inc., New York, NY. (1994); Spooncer, Dexter, M. and Allen, T. Long Term Bone Marrow Cultures in the Presence of Stromal Cells, Culture of Hematopoietic Cells. R.I.

Freshney, et al. Eds. pp. 163-179, Wiley-Liss, Inc., New York, NY. (1994); and Sutherland, H.J. Long Term Culture Initiating Cell Assay, Culture of Hematopoietic Cells. R.I. Freshney, et al. Eds. pp. 139-162, Wiley-Liss, Inc., New York, NY. (1994).

WO 99/25825 PCT/IB98/01862 Those proteins which exhibit hematopoiesis regulatory activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of hematopoeisis is beneficial. For example, a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoicsis.

e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines. thereby indicating utility, for example. in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantion, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria). as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. Alternatively, as described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.

EXAMPLE Assaying the Proteins Expressed from Extended cDNAs or Portions Thereof for Regulation of Tissue Growth The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their effect on tissue growth. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in International Patent Publication No. W095/16035, International Patent Publication No. W095/05846 and International Patent Publication No. W091/07491.

Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mcrtz, J. Invest. Dermatol. 71:382-84 (1978).

Those proteins which are involved in the regulation of tissue growth may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of tissue growth is beneficial. For example, a protein of the present invention also may have utility in compositions used for bone, cartilage, WO 99/25825 PCT/IB98/01862 61 tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of bums, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, o oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.

Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligamentlike tissue or other tissue formation in circumstances where sucif tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.

De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendonor ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and WO 99/25825 PCT/IB98/01862 62 localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders. head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium) muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to generate. A protein of the invention may also exhibit angiogenic activity.

A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokinc damage.

A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

Alternatively, as described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.

EXAMPLE 36 Assaying the Proteins Expressed from Extended cDNAs or Portions Thereof for Regulation of Reproductive Hormones or Cell Movement The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their ability to regulate reproductive hormones, such as follicle stimulating hormone. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Vale et al., Endocrinology 91:562-572 (1972); Ling et al., Nature 321:779-782 (1986); Vale et al., Nature 321:776-779 (1986); Mason ct al., Nature 318:659-663 (1985); Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095 (1986). Chapter 6.12 (Measurement of Alpha and Beta Chemokines) Current Protocols in Immunology, J.E. Coligan et al. Eds. Greene Publishing Associates and Wiley-Intersciece Taub et al. J.

Clin. Invest. 95:1370-1376 (1995); Lind et al. APMIS 103:140-146 (1995); Muller et al. Eur. J. Immunol.

WO 99/25825 PCT/IB98/01862 63 25:1744-1748; Gruber et al. J. of Inunol. 152:5860-5867 (1994): and Johnston et al. J. of liunuol.

153:1762-1768 (1994).

Those proteins which exhibit activity as reproductive hormones or regulators of cell movement may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of reproductive hormones or cell movement are beneficial. For example, a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSI while activins are characterized by their ability to stimulate the release of folic stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heerodimers with a member of the inhibin ac family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.

Administration of sufficient amounts of other inhibins can induce infertility in these mammals.

Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-B group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798.885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.

Alternatively, as described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.

EXAMPLE 36A Assavine the Proteins Expressed from Extended cDNAs or Portions Thereof for Chemotactic/Chemokinctic Activity The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for chemotacti/chemokinetic activity. For example, a protein of the present invention may have chemotactic or chemokinetic activity act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, cosinophils, epithelial and/or endothelial cells. Chemotactic and chmokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.

Chemotactic or chcmokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has WO 99/25825 PCT/IB98/01862 64 chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chcmotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhension of one cell population to another cell population.

Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Iimmuiology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies. E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokincs 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376 (1995); Lind et al. APMIS 103:140- 146 (1995); Mueller et al. Eur. J. Inmmunol. 25:174-174748; Gruber et al. J. of hImunol. 152:5860-5867 (1994); and Johnston et al. J. of hnmunnol. 153:1762-1768 (1994).

EXAMPLE 37 Assaying the Proteins Expressed from Extended cDNAs or Portions Thereof for Regulation of Blood Clottine The proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their effects on blood clotting. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Linet et al., J. Clin. Pharmacol. 26:131-140 (1986); Burdick et al., Thrombosis Res. 45:413419 (1987); Humphrey et al., Fibrinolysis 5:71-79 (1991); and Schaub, Prostaglandins 35:467-474 (1988).

Those proteins which are involved in the regulation of blood clotting may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of blood clotting is beneficial. For example, a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulations disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels stroke). Alternatively, as described in more detail below, genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.

WO 99/25825 PCT/IB98/01862 EXAMPLE 38 Assavine the Proteins Expressed from Extended cDNAs or Portions Thereof for Involvement in Receptor/Ligand Interactions The proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for their involvement in receptor/ligand interactions. Numerous assays for such involvement are familiar to those skilled in the art. including the assays disclosed in the following references: Chapter 7.28 (Measurement of Cellular Adhesion under Static Conditions 7.28.1-7.28.22) Current Protocols in Inununmlogy, J.E. Coligan et al. Eds. Greene Publishing Associates and Wiley-lnterscience; Takai et al., Proc. Nal. Acad. Sci. USA 84:6864-6868 (1987); Bierer et al., J. Eqp. Med, 168:1145-1156 (1988); Rosenstein et al.. J. Exp. Mel.

169:149-160 (1989); Stoltenborg et al.. J. Inmmunol. Methods 175:59-68 (1994); Stit et al., Cell 80:661- 670 (1995); and Gyuris et al., Cell 75:791-803 (1993).

For example, the proteins of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands. receptor kinases and their ligands.

receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as sclectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

EXAMPLE 38A Assaying the Proteins Expressed from Extended cDNAs or Portions Thereof for Anti-Inflammatory Activity The proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for antiinflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the WO 99/25825 PCT/IB98/01862 66 invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.

EXAMPLE 38B Assavine the Proteins Expressed from Extended cDNAs or Portions Thereof for Tumor Inhibition Activity The proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for tumor inhibition activity. In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.

A protein of the invention may also exhibit one or more ofthe following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or climination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics. including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoictic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

EXAMPLE 39 Identification of Proteins which Interact with Polvpeptides Encoded by Extended cDNAs Proteins which interact with the polypeptides encoded by extended cDNAs or portions thereof, such WO 99/25825 PCT/IB98/01862 67 as receptor proteins, may be identified using two hybrid systems such as the Matchmaker Two Hybrid System 2 (Catalog No. K 1604-1. Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K 1604-1. Clontech). the extended cDNAs or portions thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GALA. cDNAs in a cDNA library which encode proteins which might interact with the polypeptides encoded by the extended cDNAs or portions thereof are inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GAL4 dependent expression of the HIS3 gene. Transformants capable of growing on medium lacking histidine are-screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain plasmids encoding proteins which interact with the polypeptide encoded by the extended cDNAs or portions thereof.

Alternatively, the system described in Lustig et al., Methods in Enzvymology 283: 83-99 (1997). may be used for identifying molecules which interact with the polypeptides encoded by extended cDNAs. In such systems, in vitro transcription reactions are performed on a pool of vectors containing extended cDNA inserts cloned downstream of a promoter which drives in vitro transcription. The resulting pools of mRNAs are introduced into Xenopuls laevis oocytes. The oocytes are then assayed for a desired activity.

Alternatively, the pooled in vitro transcription products produced as described above may be translated in vitro. The pooled in vitro translation products can be assayed for a desired activity or for interaction with a known polypeptide.

Proteins or other molecules interacting with polypeptides encoded by extended cDNAs can be found by a variety of additional techniques. In one method, affinity columns containing the polypeptide encoded by the extended cDNA or a portion thereof can be constructed. In some versions, of this method the affinity column contains chimeric proteins in which the protein encoded by the extended cDNA or a portion thereof is fused to glutathione S-transferase. A mixture of cellular proteins or pool of expressed proteins as described above and is applied to the affinity column. Proteins interacting with the polypeptide attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al. Electrophoresis 18:588-598 (1997). Alternatively, the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.

Proteins interacting with polypeptides encoded by extended cDNAs or portions thereof can also be screened by using an Optical Biosensor as described in Edwards Leatherbarrow, Analytical Biochemistry, 246:1-6 (1997). The main advantage of the method is that it allows the determination of the association rate between the protein and other interacting molecules. Thus, it is possible to WO 99/25825 PCT/IB98/01862 68 specifically select interacting molecules with a high or low association rate. Typically a target molecule is linked to the sensor surface (through a carboxymethl dextran matrix) and a sample of test molecules is placed in contact with the target molecules. The binding of a test molecule to the target molecule causes a change in the refractive index and/or thickness. This change is detected by the Biosensor provided it occurs in the evanescent field (which extend a few hundred manometers from the sensor surface). In these screening assays, the target molecule can be one of the polypeptides encoded by extended cDNAs or a portion thereof and the test sample can be a collection of proteins extracted from tissues or cells, a pool of expressed proteins, combinatorial peptide and/ or chemical libraries, or phage displayed peptides.

The tissues or cells from which the test proteins are extracted can originate from any species.

In other methods, a target protein is immobilized and the test population is a collection of unique polypeptides encoded by the extended cDNAs or portions thereof.

To study the interaction of the proteins encoded by the extended cDNAs or portions thereof with drugs, the microdialysis coupled to HPLC method described by Wang et al., Chromatographia 44:205- 208(1997) or the affinity capillary electrophoresis method described by Busch et al., J. Chronmaogr.

777:311-328 (1997).

The system described in U.S. Patent No. 5.654,150, may also be used to identify molecules which interact with the polypeptides encoded by the extended cDNAs. In this system. pools of extended cDNAs are transcribed and translated in vitro and the reaction products are assayed for interaction with a known polypeptide or antibody.

It will be appreciated by those skilled in the art that the proteins expressed from the extended cDNAs or portions may be assayed for numerous activities in addition to those specifically enumerated above. For example, the expressed proteins may be evaluated for applications involving control and regulation of inflammation, tumor proliferation or metastasis, infection, or other clinical conditions. In addition, the proteins expressed from the extended cDNAs or portions thereof may be useful as nutritional agents or cosmetic agents.

The proteins expressed from the extended cDNAs or portions thereof may be used to generate antibodies capable of specifically binding to the expressed protein or fragments thereof as described in Example 40 below. The antibodies may capable of binding a full length protein encoded by one of the sequences of SEQ ID NOs. 134-180, a mature protein encoded by one of the sequences of SEQ ID NOs.

134-180, or a signal peptide encoded by one of the sequences of SEQ ID Nos. 134-180. Alternatively, the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 10 amino acids of the sequences of SEQ ID NOs: 181-227. In some embodiments, the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 15 amino acids of the sequences of SEQ ID NOs: 181-227. In other embodiments, the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 25 amino acids of the sequences of SEQ ID NOs: 181-227. In further embodiments, the WO 99/25825 PCT/IB98/01862 69 antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 40 amino acids of the sequences of SEQ ID NOs: 1S 1-227.

EXAMPLE Production of an Antibody to a 1Human Protein Substantially pure protein or polypeptide is isolated from the transfected or transformed cells as described in Example 30. The concentration of protein in the final preparation is adjusted. for example. by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows: A. Monoclonal Antibody Production by Hybridonma Fusion Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, Nature 256:495 (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfuscd cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa. as originally described by Engvall. Meth. Enzymol.

70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

B. Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in: WO 99/25825 PCT/IB98/01862 Handbook ofEvperimental Inmunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 jIM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher. Chap. 42 in: Manual of Clinical Immunology. 2d Ed. (Rose and Friedman. Eds.) Amer. Soc. For Microbiol., Washington.

D.C.(1980).

Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

V. Use of Extended cDNAs or Portions Thereof as Reagents The extended cDNAs of the present invention may be used as reagents in isolation procedures, diagnostic assays, and forensic procedures. For example, sequences from the extended cDNAs (or genomic DNAs obtainable therefrom) may be detectably labeled and used as probes to isolate other sequences capable of hybridizing to them. In addition, sequences from the extended cDNAs (or genomic DNAs obtainable therefrom) may be used to design PCR primers to be used in isolation, diagnostic, or forensic procedures.

EXAMPLE 41 Preparation of PCR Primers and Amplification of DNA The extended cDNAs (or genomic DNAs obtainable therefrom) may be used to prepare PCR primers for a variety of applications, including isolation procedures for cloning nucleic acids capable of hybridizing to such sequences, diagnostic techniques and forensic techniques. The PCR primers are at least bases, and preferably at least 12, 15, or 17 bases in length. More preferably, the PCR primers are at least 20-30 bases in length. In some embodiments, the PCR primers may be more than 30 bases in length. It is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerasc such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.

WO 99/25825 PCT/IB98/01862 71 EXAMPLE 42 Use of Extended cDNAs as Probes Probes derived from extended cDNAs or portions thereof (or genomic DNAs obtainable therefrom) may be labeled with detectable labels familiar to those skilled in the art, including radioisotopes and nonradioactive labels, to provide a detectable probe. The detectable probe may be single stranded or double stranded and may be made using techniques known in the art. including in vitro transcription, nick translation, or kinase reactions. A nucleic acid sample containing a sequence capable of hybridizing to the labeled probe is contacted with the labeled probe. If the nucleic acid in the sample is double stranded, it may be denatured prior to contacting the probe. In some applications, the nucleic acid sample may be immobilized on a surface such as a nitrocellulose or nylon membrane. The nucleic acid sample may comprise nucleic acids obtained from a variety of sources, including genomic DNA, cDNA libraries, RNA, or tissue samples.

Procedures used to detect the presence of nucleic acids capable of hybridizing to the detectable probe include well known techniques such as Southern blotting, Northern blotting, dot blotting, colony hybridization, and plaque hybridization. In some applications, the nucleic acid capable of hybridizing to the labeled probe may be cloned into vectors such as expression vectors, sequencing vectors, or in vitro transcription vectors to facilitate the characterization and expression of the hybridizing nucleic acids in the sample. For example, such techniques may be used to isolate and clone sequences in a genomic library or cDNA library which are capable of hybridizing to the detectable probe as described in Example 30 above.

PCR primers made as described in Example 41 above may be used in forensic analyses, such as the DNA fingerprinting techniques described in Examples 43-47 below. Such analyses may utilize detectable probes or primers based on the sequences of the extended cDNAs isolated using the 5' ESTs (or genomic DNAs obtainable therefrom).

EXAMPLE 43 Forensic Matching by DNA Seuencing In one exemplary method, DNA samples are isolated from forensic specimens of, for example, hair, semen, blood or skin cells by conventional methods. A panel of PCR primers based on a number of the extended cDNAs (or genomic DNAs obtainable therefrom), is then utilized in accordance with Example 41 to amplify DNA of approximately 100-200 bases in length from the forensic specimen. Corresponding sequences are obtained from a test subject. Each of these identification DNAs is then sequenced using standard techniques, and a simple database comparison determines the differences, if any, between the sequences from the subject and those from the sample. Statistically significant differences between the suspect's DNA sequences and those from the sample conclusively prove a lack of identity. This lack of identity can be proven, for example, with only one sequence. Identity, on the other hand, should be demonstrated with a large number of sequences, all matching. Preferably, a minimum of 50 statistically WO 99/25825 PCT/IB98/01862 72 identical sequences of 100 bases in length are used to prove identity between the suspect and the sample.

EXAMPLE 44 Positive Identification by DNA Sequcncin The technique outlined in the previous example may also be used on a larger scale to provide a unique fingerprint-type identification of any individual. In this technique, primers are prepared from a large number of sequences from Table II and the appended sequence listing. Preferably. 20 to 50 different primers are used. These primers are used to obtain a corresponding number of PCR-generated

DNA

segments from the individual in question in accordance with Example 41. Each of these DNA segments is sequenced, using the methods set forth in Example 43. The database of sequences generated through this procedure uniquely identifies the individual from whom the sequences were obtained. The same panel of primers may then be used at any later time to absolutely correlate tissue or other biological specimen with that individual.

EXAMPLE Southern Blot Forensic Identification The procedure of Example 44 is repeated to obtain a panel of at least 10 amplified sequences from an individual and a specimen. Preferably, the panel contains at least 50 amplified sequences. More preferably, the panel contains 100 amplified sequences. In some embodiments, the panel contains 200 amplified sequences. This PCR-generated DNA is then digested with one or a combination of, preferably, four base specific restriction enzymes. Such enzymes are commercially available and known to those of skill in the art. After digestion, the resultant gene fragments are size separated in multiple duplicate wells on an agarose gel and transferred to nitrocellulose using Southern blotting techniques well known to those with skill in the art. For a review of Southern blotting see Davis et al. Basic Methods in Molecular Biology, (1986), Elsevier Press. pp 62-65).

A panel of probes based on the sequences of the extended cDNAs (or genomic DNAs obtainable therefrom), or fragments thereof of at least 10 bases, are radioactively or colorimetrically labeled using methods known in the art, such as nick translation or end labeling, and hybridized to the Southern blot using techniques known in the art (Davis et al., sura). Preferably, the probe comprises at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the probe comprises at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In some embodiments, the probe comprises more than nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom).

Preferably, at least 5 to 10 of these labeled probes are used, and more preferably at least about 20 or 30 are used to provide a unique pattern. The resultant bands appearing from the hybridization of a large sample of extended cDNAs (or genomic DNAs obtainable therefrom) will be a unique identifier. Since the WO 99/25825 PCT/IB98/01862 73 restriction enzyme cleavage will be different for every individual, the band pattern on the Southern blot will also be unique. Increasing the number of extended cDNA probes will provide a statistically higher level of confidence in the identification since there will be an increased number of sets of bands used for identification.

EXAMPLE 46 Dot illot Idenilicat ion Proc lure Another technique for identifying individuals using the extended cDNA sequences disclosed herein utilizes a dot blot hybridization technique.

Genomic DNA is isolated from nuclei of subject to be identified. Oligonucleotide probes of approximately 30 bp in length are synthesized that correspond to at least 10, preferably 50 sequences from the extended cDNAs or genomic DNAs obtainable therefrom. The probes are used to hybridize to the genomic DNA through conditions known to those in the art. The oligonucleotides are end labeled with P32 using polynucleotide kinase (Pharmacia). Dot Blots are created by spotting the genomic DNA onto nitrocellulose or the like using a vacuum dot blot manifold (BioRadRichmond California). The nitrocellulose filter containing the genomic sequences is baked or UV linked to the filter, prehybridized and hybridized with labeled probe using techniques known in the art (Davis et al. sunra). The "P labeled DNA fragments are sequentially hybridized with successively stringent conditions to detect minimal differences between the 30 bp sequence and the DNA. Tetramethylammonium chloride is useful for identifying clones containing small numbers of nucleotide mismatches (Wood et al., Proc. Natl. Acad. Sci. USA 82(6):1585- 1588 (1985)). A unique pattern of dots distinguishes one individual from another individual.

Extended cDNAs or oligonucleotides containing at least 10 consecutive bases from these sequences can be used as probes in the following alternative fingerprinting technique. Preferably, the probe comprises at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the probe comprises at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In some embodiments, the probe comprises more than nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom).

Preferably, a plurality of probes having sequences from different genes are used in the alternative fingerprinting technique. Example 47 below provides a representative alternative fingerprinting procedure in which the probes are derived from extended cDNAs.

EXAMPLE 47 Alternative "Fingerprint" Identification Technique oligonucleotides are prepared from a large number, e.g. 50, 100, or 200, of extended cDNA sequences (or genomic DNAs obtainable therefrom) using commercially available oligonucleotide services such as Genset, Paris, France. Cell samples from the test subject are processed for DNA using WO 99/25825 PCT/IB98/01862 74 techniques well known to those with skill in the art. The nucleic acid is digested with restriction enzymes such as EcoRI and XbaI. Following digestion, samples are applied to wells for electrophoresis. The procedure, as known in the art, may be modified to accommodate polyacrylamide electrophoresis, however in this example. samples containing 5 ug of DNA are loaded into wells and separated on 0.8% agarose gels.

The gels are transferred onto nitrocellulose using standard Southern blotting techniques.

ng of each of the oligonucleotides are pooled and end-labeled with The nitrocellulose is prehybridized with blocking solution and hybridized with the labeled probes. Following hybridization and washing, the nitrocellulose filler is exposed to X-Omat AR X-ray film. The resulting hybridization pattern will be unique for each individual.

It is additionally contemplated within this example that the number of probe sequences used can be varied for additional accuracy or clarity.

The antibodies generated in Examples 30 and 40 above may be used to identify the tissue type or cell species from which a sample is derived as described above.

EXAMPLE 48 Identification of Tissue Types or Cell Species by Means of Labeled Tissue Specific Antibodies Identification of specific tissues is accomplished by the visualization of tissue specific antigens by means of antibody preparations according to Examples 30 and 40 which are conjugated, directly or indirectly to a detectable marker. Selected labeled antibody species bind to their specific antigen binding partner in tissue sections, cell suspensions, or in extracts of soluble proteins from a tissue sample to provide a pattern for qualitative or semi-qualitative interpretation.

Antisera for these procedures must have a potency exceeding that of the native preparation, and for that reason, antibodies are concentrated to a mg/mI level by isolation of the gamma globulin fraction, for example, by ion-exchange chromatography or by ammonium sulfate fractionation. Also, to provide the most specific antisera, unwanted antibodies, for example to common proteins, must be removed from the gamma globulin fraction, for example by means of insoluble immunoabsorbents, before the antibodies are labeled with the marker. Either monoclonal or heterologous antisera is suitable for either procedure.

A. Immunohistochemical Techniques Purified, high-titer antibodies, prepared as described above, are conjugated to a detectable marker, as described, for example, by Fudenberg, Chap. 26 in: Basic 503 Clinical Immunology, 3rd Ed. Lange, Los Altos, California (1980) or Rose, N. et al., Chap. 12 in: Methods in Inmmunodiagnosis, 2d Ed. John Wiley 503 Sons, New York (1980).

A fluorescent marker, either fluorescein or rhodamine, is preferred, but antibodies can also be labeled with an enzyme that supports a color producing reaction with a substrate, such as horseradish peroxidase. Markers can be added to tissue-bound antibody in a second step, as described below.

WO 99/25825 PCT/IB98/01862 Alternatively, the specific antitissue antibodies can be labeled with ferritin or other electron dense particles, and localization of the ferritin coupled antigen-antibody complexes achieved by means of an electron microscope. In yet another approach, the antibodies are radiolabeled. with, for example and detected by overlaying the antibody treated preparation with photographic emulsion.

Preparations to carry out the procedures can comprise monoclonal or polyclonal antibodies to a single protein or peptide identified as specific to a tissue type, for example, brain tissue, or antibody preparations to several antigenically distinct tissue specific antigens can be used in panels, independently or in mixtures, as required.

Tissue sections and cell suspensions are prepared for inmmunohistochemical examination according to common histological techniques. Multiple cryostat sections (about 4 p.m, unfixed) of the unknown tissue and known control, are mounted and each slide covered with different dilutions of the antibody preparation.

Sections of known and unknown tissues should also be treated with preparations to provide a positive control, a negative control, for example, pre-immune sera. and a control for non-specific staining, for example, buffer.

Treated sections are incubated in a humid chamber for 30 min at room temperature, rinsed, then washed in buffer for 3045 min. Excess fluid is blotted away, and the marker developed.

If the tissue specific antibody was not labeled in the first incubation, it can be labeled at this time in a second antibody-antibody reaction, for example, by adding fluorescein- or enzyme-conjugated antibody against the immunoglobulin class of the antiserum-producing species, for example, fluorescein labeled antibody to mouse IgG. Such labeled scra arc commercially available.

The antigen found in the tissues by the above procedure can be quantified by measuring the intensity of color or fluorescence on the tissue section, and calibrating that signal using appropriate standards.

B. Identification of Tissue Specific Soluble Proteins The visualization of tissue specific proteins and identification of unknown tissues from that procedure is carried out using the labeled antibody reagents and detection strategy as described for immunohistochemistry; however the sample is prepared according to an electrophoretic technique to distribute the proteins extracted from the tissue in an orderly array on the basis of molecular weight for detection.

A tissue sample is homogenized using a Virtis apparatus; cell suspensions are disrupted by Dounce homogenization or osmotic lysis, using detergents in either case as required to disrupt cell membranes, as is the practice in the art. Insoluble cell components such as nuclei, microsomes, and membrane fragments are removed by ultracentrifugation, and the soluble protein-containing fraction concentrated if necessary and reserved for analysis.

A sample of the soluble protein solution is resolved into individual protein species by conventional SDS polyacrylamide electrophoresis as described, for example, by Davis, L. et al., Section 19-2 in: Basic WO 99/25825 PCT/IB98/01862 76 Methods in Molecular Biology Leder, ed). Elsevier. New York (1986). using a range of amounts of polyacrylamide in a set of gels to resolve the entire molecular weight range of proteins to be detected in the sample. A size marker is run in parallel for purposes of estimating molecular weights of the constituent proteins. Sample size for analysis is a convenient volume of from 5 to55 .tl. and containing from about 1 to 100 pg protein. An aliquot of each of the resolved proteins is transferred by blotting to a nitrocellulose filter paper, a process that maintains the pattern of resolution. Multiple copies are prepared. The procedure.

known as Western Blot Analysis, is well described in Davis. L. et al.. (above) Section 19-3. One set of nitrocellulose blots is stained with Coomassie Blue dye to visualize the entire set of proteins for comparison with the antibody bound proteins. The remaining nitrocellulose filters are then incubated with a solution of one or more specific antisera to tissue specific proteins prepared as described in Examples 30 and 40. In this procedure, as in procedure A above, appropriate positive and negative sample and reagent controls are run.

In either procedure A or B, a detectable label can be attached to the primary tissue antigen-primary antibody complex according to various strategies and permutations thereof. In a straightforward approach, the primary specific antibody can be labeled; alternatively, the unlabeled complex can be bound by a labeled secondary anti-IgG antibody. In other approaches, either the primary or secondary antibody is conjugated to a biotin molecule, which can, in a subsequent step, bind an avidin conjugated marker. According to yet another strategy, enzyme labeled or radioactive protein A, which has the property of binding to any IgG, is bound in a final step to either the primary or secondary antibody.

The visualization of tissue specific antigen binding at levels above those seen in control tissues to one or more tissue specific antibodies, prepared from the gene sequences identified from extended cDNA sequences, can identify tissues of unknown origin, for example, forensic samples, or differentiated tumor tissue that has metastasized to foreign bodily sites.

In addition to their applications in forensics and identification, extended cDNAs (or genomic DNAs obtainable therefrom) may be mapped to their chromosomal locations. Example 49 below describes radiation hybrid (RH) mapping of human chromosomal regions using extended cDNAs. Example 50 below describes a representative procedure for mapping an extended cDNA (or a genomic DNA obtainable therefrom) to its location on a human chromosome. Example 51 below describes mapping of extended cDNAs (or genomic DNAs obtainable therefrom) on metaphase chromosomes by Fluorescence In Situ Hybridization (FISH).

EXAMPLE 49 Radiation hybrid manping of Extended cDNAs to the human genome Radiation hybrid (RH) mapping is a somatic cell genetic approach that can be used for high resolution mapping of the human genome. In this approach, cell lines containing one or more human chromosomes are lethally irradiated, breaking each chromosome into fragments whose size depends on the radiation dose. These fragments are rescued by fusion with cultured rodent cells, yielding subclones WO 99/25825 PCT/IB98/01862 77 containing different portions of the human genome. This technique is described by Benham et al. Genomics 4:509-517 (1989) and Cox et al., Science 250:245-250 (1990). The random and independent nature of the subclones permits efficient mapping of any human genome marker. Human DNA isolated from a panel of 80-100 cell lines provides a mapping reagent for ordering extended cDNAs (or genomic DNAs obtainable therefrom). In this approach, the frequency of breakage between markers is used to measure distance.

allowing construction of fine resolution maps as has been done using conventional ESTs Schuler et al., Science 274:540-546 (1996).

RHI mapping has been used to generate a high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for growth hormone (GH) and thymidine kinase (TK) Foster et al., Genomics 33:185-192 (1996), the region surrounding the Gorlin syndrome gene (Obermayr et al., Eur. J. Hunt. Genet. 4:242-245, 1996), 60 loci covering the entire short arm of chromosome 12 (Raeymaekers et al.. Genomics 29:170-178, (1995)). the region of human chromosome 22 containing the neurofibromatosis type 2 locus (Frazer et al., Genomics 14:574-584 (1992)) and 13 loci on the long arm of chromosome 5 (Warrington et al., Genomics 11:701-708 (1991)).

EXAMPLE Manning of Extended cDNAs to Human Chromosomes using PCR techniques Extended cDNAs (or genomic DNAs obtainable therefrom) may be assigned to human chromosomes using PCR based methodologies. In such approaches, oligonuclcotide primer pairs are designed from the extended cDNA sequence (or the sequence of a genomic DNA obtainable therefrom) to minimize the chance of amplifying through an intron. Preferably, the oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification. The creation of PCR primers from known sequences is well known to those with skill in the art. For a review of PCR technology sec Erlich, PCR Technology; Principles and Applications for DNA Amplification. (1992). W.H. Freeman and Co., New York.

The primers are used in polymerase chain reactions (PCR) to amplify templates from total human genomic DNA. PCR conditions are as follows: 60 ng of genomic DNA is used as a template for PCR with ng of each oligonucleotide primer, 0.6 unit of Taq polymerase, and 1 gCu of a "P-labeled deoxycytidine triphosphate. The PCR is performed in a microplate thermocycler (Techne) under the following conditions: cycles of 94 0 C, 1.4 min; 55 0 C, 2 min; and 72 0 C, 2 min; with a final extension at 720C for 10 min. The amplified products are analyzed on a 6% polyacrylamide sequencing gel and visualized by autoradiography.

If the length of the resulting PCR product is identical to the distance between the ends of the primer sequences in the extended cDNA from which the primers are derived, then the PCR reaction is repeated with DNA templates from two panels of human-rodent somatic cell hybrids, BIOS PCRable DNA (BIOS Corporation) and NIGMS Human-Rodent Somatic Cell Hybrid Mapping Panel Number 1 (NIGMS, WO 99/25825 PCT/B98/01862 78 Camden, NJ).

PCR is used to screen a series of somatic cell hybrid cell lines containing defined sets of human chromosomes for the presence of a given extended cDNA (or genomic DNA obtainable therefrom). DNA is isolated from the somatic hybrids and used as starting templates for PCR reactions using the primer pairs from the extended cDNAs (or genomic DNAs obtainable therefrom). Only those sonatic cell hybrids with chromosomes containing the human gene corresponding to the extended cDNA (or genomic DNA obtainable therefrom) will yield an amplified fragment. The extended cDNAs (or genomic DNAs obtainable therefrom) are assigned to a chromosome by analysis of the segregation pattern of PCR products from the somatic hybrid DNA templates. The single human chromosome present in all cell hybrids that give rise to an amplified fragment is the chromosome containing that extended cDNA (or genomic DNA obtainable therefrom). For a review of techniques and analysis of results from somatic cell gene mapping experiments. (See Ledbetter et al., Genomics 6:475-481 (1990).) Alternatively, the extended cDNAs (or genomic DNAs obtainable therefrom) may be mapped to individual chromosomes using FISH as described in Example 51 below.

EXAMPLE 51 Manning of Extended 5' ESTs to Chromosomes Using Fluorescence in situ Hybridization Fluorescence in situ hybridization allows the extended cDNA (or genomic DNA obtainable therefrom) to be mapped to a particular location on a given chromosome. The chromosomes to be used for fluorescence in situ hybridization techniques may be obtained from a variety of sources including cell cultures, tissues, or whole blood.

In a preferred embodiment, chromosomal localization of an extended cDNA (or genomic DNA obtainable therefrom) is obtained by FISH as described by Cherifet al. Proc. Natl. Acad. Sci. U.S.A., 87:6639-6643 (1990). Metaphase chromosomes are prepared from phytohemagglutinin (PHA)-stimulated blood cell donors. PHA-stimulated lymphocytes from healthy males are cultured for 72 h in RPMI-1640 medium. For synchronization, methotrexate (10 uM) is added for 17 h, followed by addition of bromodeoxyuridine (5-BudR, 0.1 mM) for 6 h. Colcemid (1 gg/ml) is added for the last 15 min before harvesting the cells. Cells are collected, washed in RPMI, incubated with a hypotonic solution of KCI mM) at 370C for 15 min and fixed in three changes of methanol:acetic acid The cell suspension is dropped onto a glass slide and air dried. The extended cDNA (or genomic DNA obtainable therefrom) is labeled with biotin-16 dUTP by nick translation according to the manufacturer's instructions (Bethesda Research Laboratories, Bethesda, MD), purified using a Sephadex G-50 column (Pharmacia, Upssala, Sweden) and precipitated. Just prior to hybridization, the DNA pellet is dissolved in hybridization buffer (50% formamide, 2 X SSC, 10% dextran sulfate, 1 mg/ml sonicated salmon sperm DNA, pH 7) and the probe is denatured at 70 0 C for 5-10 min.

WO 99/25825 PCT/IB98/01862 79 Slides kept at -20°C are treated for 1 h at 370C with RNase A (100 tg/ml), rinsed three times in 2 X SSC and dehydrated in an ethanol series. Chromosome preparations are denatured in 70% formamide, 2 X SSC for 2 min at 70 0 C. then dehydrated at 40C. The slides are treated with proteinase K (10 pg/100 ml in mM Tris-HCI. 2 mM CaCl 2 at 37°C for 8 min and dehydrated. The hybridization mixture containing the probe is placed on the slide, covered with a coverslip. sealed with rubber cement and incubated overnight in a humid chamber at 370C. After hybridization and post-hybridization washes, the biotinylated probe is detected by avidin-FITC and amplified with additional layers of biotinylated goat anti-avidin and avidin- FITC. For chromosomal localization, fluorescent R-bands are obtained as previously described (Cherifet al., supra.). The slides are observed under a LEICA fluorescence microscope (DMRXA). Chromosomes are counterstained with propidium iodide and the fluorescent signal of the probe appears as two symmetrical yellow-green spots on both chromatids of the fluorescent R-band chromosome (red). Thus, a particular extended cDNA (or genomic DNA obtainable therefrom) may be localized to a particular cytogenetic Rband on a given chromosome.

Once the extended cDNAs (or genomic DNAs obtainable therefrom) have been assigned to particular chromosomes using the techniques described in Examples 49-51 above, they may be utilized to construct a high resolution map of the chromosomes on which they are located or to identify the chromosomes in a sample.

EXAMPLE 52 Use of Extended cDNAs to Construct or Expand Chromosome Maps Chromosome mapping involves assigning a given unique sequence to a particular chromosome as described above. Once the unique sequence has been mapped to a given chromosome, it is ordered relative to other unique sequences located on the same chromosome. One approach to chromosome mapping utilizes a series of yeast artificial chromosomes (YACs) bearing several thousand long inserts derived from the chromosomes of the organism from which the extended cDNAs (or genomic DNAs obtainable therefrom) are obtained. This approach is described in Ramaiah Nagaraja et al. Genome Research 7:210- 222, (March, 1997). Briefly, in this approach each chromosome is broken into overlapping pieces which are inserted into the YAC vector. The YAC inserts are screened using PCR or other methods to determine whether they include the extended cDNA (or genomic DNA obtainable therefrom) whose position is to be determined. Once an insert has been found which includes the extended cDNA (or genomic DNA obtainable therefrom), the insert can be analyzed by PCR or other methods to determine whether the insert also contains other sequences known to be on the chromosome or in the region from which the extended cDNA (or genomic DNA obtainable therefrom) was derived. This process can be repeated for each insert in the YAC library to determine the location of each of the extended cDNAs (or genomic DNAs obtainable therefrom) relative to one another and to other known chromosomal markers. In this way, a high resolution map of the distribution of numerous unique markers along each of the organisms chromosomes may be WO 99/25825 PCT/IB98/01862 obtained.

As described in Example 53 below extended cDNAs (or genomic DNAs obtainable therefrom) may also be used to identify genes associated with a particular phenotype, such as hereditary disease or drug response.

EXAMPLE 53 Identification of Cenes associated with hereditary diseases or druI response This example illustrates an approach useful for the association of extended cDNAs (or genomic DNAs obtainable therefrom) with particular phenotypic characteristics. In this example, a particular extended cDNA (or genomic DNA obtainable therefrom) is used as a test probe to associate that extended cDNA (or genomic DNA obtainable therefrom) with a particular phenotypic characteristic.

Extended cDNAs (or genomic DNAs obtainable therefrom) are mapped to a particular location on a human chromosome using techniques such as those described in Examples 49 and 50 or other techniques known in the art. A search of Mendelian Inheritance in Man McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) reveals the region of the human chromosome which contains the extended cDNA (or genomic DNA obtainable therefrom) to be a very gene rich region containing several known genes and several diseases or phenotypes for which genes have not been identified. The gene corresponding to this extended cDNA (or genomic DNA obtainable therefrom) thus becomes an immediate candidate for each of these genetic diseases.

Cells from patients with these diseases or phenotypes are isolated and expanded in culture. PCR primers from the extended cDNA (or genomic DNA obtainable therefrom) are used to screen genomic DNA, mRNA or cDNA obtained from the patients. Extended cDNAs (or genomic DNAs obtainable therefrom) that are not amplified in the patients can be positively associated with a particular disease by further analysis. Alternatively, the PCR analysis may yield fragments of different lengths when the samples are derived from an individual having the phenotype associated with the disease than when the sample is derived from a healthy individual, indicating that the gene containing the extended cDNA may be responsible for the genetic disease.

VI. Use of Extended cDNAs (or genomic DNAs obtainable therefrom) to Construct Vectors The present extended cDNAs (or genomic DNAs obtainable therefrom) may also be used to construct secretion vectors capable of directing the secretion of the proteins encoded by genes inserted in the vectors. Such secretion vectors may facilitate the purification or enrichment of the proteins encoded by genes inserted therein by reducing the number of background proteins from which the desired protein must be purified or enriched. Exemplary secretion vectors are described in Example 54 below.

WO 99/25825 PCT/IB98/01862 81 EXAMPLE 54 Construction of Secretion Vectors The secretion vectors of the present invention include a promoter capable of directing gene expression in the host cell, tissue, or organism of interest. Such promoters include the Rous Sarcoma Virus promoter, the SV40 promoter. the human cytomegalovirus promoter. and other promoters familiar to those skilled in the art.

A signal sequence from an extended cDNA (or genomic DNA obtainable therefrom), such as one of the signal sequences in SEQ ID NOs: 134-180 as defined in Table VII above, is operably linked to the promoter such that the mRNA transcribed from the promoter will direct the translation of the signal peptide.

The host cell, tissue, or organism may be any cell, tissue, or organism which recognizes the signal peptide encoded by the signal sequence in the extended cDNA (or genomic DNA obtainable therefrom). Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues, or organisms, insect cells, tissues or organisms, or yeast.

In addition, the secretion vector contains cloning sites for inserting genes encoding the proteins which are to be secreted. The cloning sites facilitate the cloning of the insert gene in frame with the signal sequence such that a fusion protein in which the signal peptide is fused to the protein encoded by the inserted gene is expressed from the mRNA transcribed from the promoter. The signal peptide directs the extracellular secretion of the fusion protein.

The secretion vector may be DNA or RNA and may integrate into the chromosome of the host, be stably maintained as an extrachromosomal replicon in the host, be an artificial chromosome, or be transiently present in the host. Many nucleic acid backbones suitable for use as secretion vectors are known to those skilled in the art, including retroviral vectors, SV40 vectors, Bovine Papilloma Virus vectors, yeast integrating plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P element vectors, baculovirus vectors, or bacterial plasmids capable of being transiently introduced into the host.

The secretion vector may also contain a polyA signal such that the polyA signal is located downstream of the gene inserted into the secretion vector.

After the gene encoding the protein for which secretion is desired is inserted into the secretion vector, the secretion vector is introduced into the host cell, tissue, or organism using calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection, viral particles or as naked DNA. The protein encoded by the inserted gene is then purified or enriched from the supernatant using conventional techniques such as ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, and hplc.

Alternatively, the secreted protein may be in a sufficiently enriched or pure state in the supernatant or growth media of the host to permit it to be used for its intended purpose without further enrichment.

The signal sequences may also be inserted into vectors designed for gene therapy. In such vectors, WO 99/25825 PCT/B98/01862 82 the signal sequence is operably linked to a promoter such that mRNA transcribed from the promoter encodes the signal peptide. A cloning site is located downstream of the signal sequence such that a gene encoding a protein whose secretion is desired may readily be inserted into the vector and fused to the signal sequence. The vector is introduced into an appropriate host cell. The protein expressed from the promoter is secreted extracellularly, thereby producing a therapeutic effect.

The extended cDNAs or 5' ESTs may also be used to clone sequences located upstream of the extended cDNAs or 5' ESTs which are capable of regulating gene expression, including promoter sequences, enhancer sequences, and other upstream sequences which influence transcription or translation levels. Once identified and cloned, these upstream regulatory sequences may be used in expression vectors designed to direct the expression of an inserted gene in a desired spatial, temporal, developmental, or quantitative fashion. Example 55 describes a method for cloning sequences upstream of the extended cDNAs or 5' ESTs.

EXAMPLE Use of Extended cDNAs or 5' ESTs to Clone Upstream Seuences from Genomic DNA Sequences derived from extended cDNAs or 5' ESTs may be used to isolate the promoters of the corresponding genes using chromosome walking techniques. In one-chromosome walking technique, which utilizes the GenomeWalkerM kit available from Clontech, five complete genomic DNA samples are each digested with a different restriction enzyme which has a 6 base recognition site and leaves a blunt end.

Following digestion, oligonucleotide adapters are ligated to each end of the resulting genomic DNA fragments.

For each of the five genomic DNA libraries, a first PCR reaction is performed according to the manufacturer's instructions using an outer adaptor primer provided in the kit and an outer gene specific primer. The gene specific primer should be selected to be specific for the extended cDNA or 5' EST of interest and should have a melting temperature, length, and location in the extended cDNA or' EST which is consistent with its use in PCR reactions. Each first PCR reaction contains 5ng of genomic DNA, 5 pl of Tth reaction buffer, 0.2 mM of each dNTP, 0.2 pM each of outer adaptor primer and outer gene specific primer, 1.1 mM of Mg(OAc) 2 and 1 pl of the Tth polymerase 50X mix in a total volume of 50 pl.

The reaction cycle for the first PCR reaction is as follows: 1 min 94 0 C 2 sec 94°C, 3 min 72°C (7 cycles) 2 sec 94oC, 3 min 67°C (32 cycles) 5 min 67°C.

The product of the first PCR reaction is diluted and used as a template for a second PCR reaction according to the manufacturer's instructions using a pair of nested primers which are located internally on the amplicon resulting from the first PCR reaction. For example, 5 pl of the reaction product of the first PCR reaction mixture may be diluted 180 times. Reactions are made in a 50 pi volume having a composition identical to that of the first PCR reaction except the nested primers are used. The first nested WO 99/25825 PCT/IB98/01862 83 primer is specific for the adaptor, and is provided with the GenomeWalkerTm kit. The second nested primer is specific for the particular extended cDNA or 5' EST for which the promoter is to be cloned and should have a melting temperature, length, and location in the extended cDNA or 5' EST which is consistent with its use in PCR reactions. The reaction parameters of the second PCR reaction are as follows: 1 min 94°C/ 2 sec 940C. 3 min 72°C (6 cycles) 2 sec 94°C. 3 min 670C (25 cycles) 5 min 67 0

C

The product of the second PCR reaction is purified, cloned, and sequenced using standard techniques. Alternatively, tow or more human genomic DNA libraries can be constructed by using two or more restriction enzymes. The digested genomic DNA is cloned into vectors which can be converted into single stranded, circular, or linear DNA. A biotinylated oligonucleotide comprising at least 15 nucleotides from the extended cDNA or 5' EST sequence is hybridized to the single stranded DNA. Hybrids between the biotinylated oligonucleotide and the single stranded DNA containing the extended cDNA or EST sequence are isolated as described in Example 29 above. Thereafter, the single stranded DNA containing the extended cDNA or EST sequence is released from the beads and convened into double stranded DNA using a primer specific for the extended cDNA or 5' EST sequence or a primer corresponding to a sequence included in the cloning vector. The resulting double stranded DNA is transformed into bacteria. DNAs containing the 5' EST or extended cDNA sequences are identified by colony PCR or colony hybridization.

Once the upstream genomic sequences have been cloned and sequenced as described above, prospective promoters and transcription start sites within the upstream sequences may be identified by comparing the sequences upstream of the extended cDNAs or 5' ESTs with databases containing known transcription start sites, transcription factor binding sites, or promoter sequences.

In addition, promoters in the upstream sequences may be identified using promoter reporter vectors as described in Example 56.

EXAMPLE 56 Identification of Promoters in Cloned Upstream Sequences The genomic sequences upstream of the extended cDNAs or 5' ESTs are cloned into a suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, ppgal-Basic, ppgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech. Briefly, each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, 3 galactosidase, or green fluorescent protein. The sequences upstream of the extended cDNAs or 5' ESTs are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert. If necessary, the upstream sequences can be cloned into vectors which contain an enhancer for augmenting transcription levels from weak promoter sequences. A WO 99/25825 PCT/IB98/01862 84 significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.

Appropriate host cells for the promoter reporter vectors may be chosen based on the results of the above described determination of expression patterns of the extended cDNAs and ESTs. For example, if the expression pattern analysis indicates that the mRNA corresponding to a particular extended cDNA or EST is expressed in fibroblasts, the promoter reporter vector may be introduced into a human fibroblast cell line.

Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion.

The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into the cloning sites in the promoter reporter vectors.

EXAMPLE 57 Clonine and Identification of Promoters Using the method described in Example 55 above with 5' ESTs, sequences upstream of several genes were obtained. Using the primer pairs GGG AAG ATG GAG ATA GTA TTG CCT G (SEQ ID NO:29) and CTG CCA TGT ACA TGA TAG AGA GAT TC (SEQ ID NO:30), the promoter having the internal designation P13H2 (SEQ ID NO:31) was obtained.

Using the primer pairs GTA CCA GGGG ACT GTG ACC ATT GC (SEQ ID NO:32) and CTG TGA CCA TTG CTC CCA AGA GAG (SEQ ID NO:33), the promoter having the internal designation P15B4 (SEQ ID NO:34) was obtained.

Using the primer pairs CTG GGA TGG AAG GCA CGG TA (SEQ ID NO:35) and GAG ACC ACA CAG CTA GAC AA (SEQ ID NO:36), the promoter having the internal designation P29B6 (SEQ ID NO:37) was obtained.

Figure 7 provides a schematic description of the promoters isolated and the way they are assembled with the corresponding 5' tags. The upstream sequences were screened for the presence of motifs resembling transcription factor binding sites or known transcription start sites using the computer program Matlnspector release 2.0, August 1996.

Figure 8 describes the transcription factor binding sites present in each of these promoters. The columns labeled matrices provides the name of the Matlnspector matrix used. The column labeled position provides the 5' position of the promoter site. Numeration of the sequence starts from the transcription site as determined by matching the genomic sequence with the 5' EST sequence. The column labeled WO 99/25825 PCT/IB98/01862 "orientation" indicates the DNA strand on which the site is found, with the strand being the coding strand as determined by matching the genomic sequence with the sequence of the 5' EST. The column labeled "score" provides the MatInspector score found for this site. The column labeled "length" provides the length of the site in nucleotides. The column labeled "sequence" provides the sequence of the site found.

The promoters and other regulatory sequences located upstream of the extended cDNAs or 5' ESTs may be used to design expression vectors capable of directing the expression of an inserted gene in a desired spatial. temporal. developmental, or quantitative manner. A promoter capable of directing the desired spatial, temporal, developmental, and quantitative patterns may be selected using the results of the expression analysis described in Example 26 above. For example, if a promoter which confers a high level of expression in muscle is desired, the promoter sequence upstream of an extended cDNA or 5' EST derived from an mRNA which is expressed at a high level in muscle, as determined by the method of Example 26, may be used in the expression vector.

Preferably, the desired promoter is placed near multiple restriction sites to facilitate the cloning of the desired insert downstream of the promoter, such that the promoter is able to drive expression of the inserted gene."Tliepromoter may be inserted in conventional nucleic acid backbones designed for extrachromosomal replication, integration into the host chromosomes or transient expression. Suitable backbones for the present expression vectors include retroviral backbones, backbones from eukaryotic episomes such as SV40 or Bovine Papilloma Virus, backbones from bacterial episomes, or artificial chromosomes.

Preferably, the expression vectors also include a polyA signal downstream of the multiple restriction sites for directing the polyadenylation of mRNA transcribed from the gene inserted into the expression vector.

Following the identification of promoter sequences using the procedures of Examples 55-57, proteins which interact with the promoter may be identified as described in Example 58 below.

EXAMPLE 58 Identification of Proteins Which Interact with Promoter Sequences.

Unstream Regulatory Sequences, or mRNA Sequences within the promoter region which are likely to bind transcription factors may be identified by homology to known transcription factor binding sites or through conventional mutagenesis or deletion analyses of reporter plasmids containing the promoter sequence. For example, deletions may be made in a reporter plasmid containing the promoter sequence of interest operably linked to an assayable reporter gene. The reporter plasmids carrying various deletions within the promoter region are transfected into an appropriate host cell and the effects of the deletions on expression levels is assessed. Transcription factor binding sites within the regions in which deletions reduce expression levels may be further localized using site directed mutagenesis, linker scanning analysis, or other techniques familiar to those skilled in the WO 99/25825 PCT/IB98/01862 86 art. Nucleic acids encodingproteins which interact with sequences in the promoter may be identified using one-hybrid systems such as those described in the manual accompanying the Matchmaker One-Hybrid System kit available from Clontech (Catalog No. K 1603-1). Briefly, the Matchmaker One-hybrid system is used as follows. The target sequence for which it is desired to identify binding proteins is cloned upstream of a selectable reporter gene and integrated into the yeast genome. Preferably. multiple copies of the target sequences are inserted into tihe reporter plasmid in tandem.

A library comprised of fusions between cDNAs to be evaluated for the ability to bind to the promoter and tie activation domain of a yeast transcription factor, such as GAL4, is transformed into the yeast strain containing tihe integrated reporter sequence. The yeast are plated on selective media to select cells expressing the selectable marker linked to the promoter sequence. The colonies which grow on the selective media contain genes encoding proteins which bind the target sequence. The inserts in the genes encoding the fusion proteins are further characterized by sequencing. In addition, the inserts may be inserted into expression vectors or in vitro transcription vectors. Binding of the polypeptides encoded by the inserts to the promoter DNA may be confirmed by techniques familiar to those skilled in the art, such as gel shift analysis or DNAse protection analysis.

VII. Use of Extended cDNAs (or Genomic DNAs Obtainable Therefrom) in Gene Therapy The present invention also comprises the use of extended cDNAs (or genomic DNAs obtainable therefrom) in gene therapy strategies, including antisense and triple helix strategies as described in Examples 57 and 58 below. In antisense approaches, nucleic acid sequences complementary to an mRNA are hybridized to the mRNA intracellularly. thereby blocking the expression of the protein encoded by the mRNA. The antisense sequences may prevent gene expression through a variety of mechanisms. For example, the antisense sequences may inhibit the ability of ribosomes to translate the mRNA. Alternatively, the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation. Another mechanism through which antisense sequences may inhibit gene expression is by interfering with mRNA splicing. In yet another strategy, the antisense nucleic acid may be incorporated in a ribozyme capable of specifically cleaving the target mRNA.

EXAMPLE 59 Preparation and Use of Antisense Oligonucleotides The antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They may comprise a sequence complementary to the sequence of the extended cDNA (or genomic DNA obtainable therefrom). The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., Anm. Rev. Biochem. 55:569-597 (1986) and Izant and Weintraub, Cell 36:1007-1015 (1984).

WO 99/25825 PCT/IB98/01862 87 In some strategies, antisense molecules are obtained from a nucleotide sequence encoding a protein by reversing the orientation of the coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. The antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript. Another approach involves transcription of the antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in an expression vector.

Alternatively, oligonucleotides which are complementary to the strand normally transcribed in the cell may be synthesized in vitro. Thus, the antisense nucleic acids are complementary to the corresponding mRNA and are capable of hybridizing to the mRNA to create a duplex. In some embodiments, the antisense sequences may contain modified sugar phosphate backbones to increase stability and make them less sensitive to RNase activity. Examples of modifications suitable for use in antisense strategies are described by Rossi et al., Pharmacol. Ther. 50(2):245-254 (1991).

Various types of antisense oligonucleotides complementary to the sequence of the extended cDNA (or genomic DNA obtainable therefrom) may be used. In one preferred embodiment, stable and semi-stable antisense oligonucleotides described in International Application No. PCT W094/23026 are used. In these molecules, the 3' end or both the 3' and 5' ends are engaged in intramolecular hydrogen bonding between complementary base pairs. These molecules are better able to withstand exonuclease attacks and exhibit increased stability compared to conventional antisense oligonucleotides.

In another preferred embodiment, the antisense oligodeoxynucleotides against herpes simplex virus types 1 and 2 described in International Application No. WO 95/04141, are used.

In yet another preferred embodiment, the covalently cross-linked antisense oligonucleotides described in International Application No. WO 96/31523, are used. These double- or single-stranded oligonucleotides comprise one or more, respectively, inter- or intra-oligonucleotide covalent cross-linkages, wherein the linkage consists of an amide bond between a primary amine group of one strand and a carboxyl group of the other strand or of the same strand, respectively, the primary amine group being directly substituted in the 2' position of the strand nucleotide monosaccharide ring, and the carboxyl group being carried by an aliphatic spacer group substituted on a nucleotide or nucleotide analog of the other strand or the same strand, respectively.

The antisense oligodeoxynucleotides and oligonucleotides disclosed in International Application No. WO 92/18522, may also be used. These molecules are stable to degradation and contain at least one transcription control recognition sequence which binds to control proteins and are effective as decoys therefor. These molecules may contain "hairpin" structures, "dumbbell" structures, "modified dumbbell" structures, "cross-linked" decoy structures and "loop" structures.

In another preferred embodiment, the cyclic double-stranded oligonucleotides described in European Patent Application No. 0 572 287 A2 are used. These ligated oligonucleotide "dumbbells" contain the binding site for a transcription factor and inhibit expression of the gene under control of the WO 99/25825 PCT/IB98/01862 88 transcription factor by sequestering the factor.

Use of the closed antisense oligonucleotides disclosed in International Application No. WO 92/19732, is also contemplated. Because these molecules have no free ends. they are more resistant to degradation by exonucleases than are conventional oligonucleotides. These oligonucleotides may be multifunctional, interacting with several regions which are not adjacent to the target mRNA.

The appropriate level of antiscnse nucleic acids required to inhibit gene expression may be determined using in vitro expression analysis. The antisense molecule may be introduced into the cells by diffusion, injection, infection or transfection using procedures known in the art. For example, the antisense nucleic acids can be introduced into the body as a bare or naked oligonucleotide, oligonucleotide encapsulated in lipid, oligonucleotide sequence encapsidated by viral protein, or as an oligonucleotide operably linked to a promoter contained in an expression vector. The expression vector may be any of a variety of expression vectors known in the art, including retroviral or viral vectors, vectors capable of extrachromosomal replication, or integrating vectors. The vectors may be DNA or RNA.

The antisense molecules are introduced onto cell samples at a number of different concentrations preferably between lx10 M to IxlO0'M. Once the minimum concentration that can adequately control gene expression is identified, the optimized dose is translated into a dosage suitable for use in vivo. For example, an inhibiting concentration in culture of Ixl10 7 translates into a dose of approximately 0.6 mg/kg bodyweight. Levels of oligonucleotide approaching 100 mg/kg bodyweight or higher may be possible after testing the toxicity of the oligonucleotide in laboratory animals. It is additionally contemplated that cells from the vertebrate are removed, treated with the antisense oligonucleotide, and reintroduced into the vertebrate.

It is further contemplated that the antisense oligonucleotide sequence is incorporated into a ribozyme sequence to enable the antisense to specifically bind and cleave its target mRNA. For technical applications of ribozyme and antisense oligonucleotides see Rossi et al., supra.

In a preferred application of this invention, the polypeptide encoded by the gene is first identified, so that the effectiveness of antisense inhibition on translation can be monitored using techniques that include but are not limited to antibody-mediated tests such as RIAs and ELISA, functional assays, or radiolabeling.

The extended cDNAs of the present invention (or genomic DNAs obtainable therefrom) may also be used in gene therapy approaches based on intracellular triple helix formation. Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity as it is associated with a particular gene. The extended cDNAs (or genomic DNAs obtainable therefrom) of the present invention or, more preferably, a portion of those sequences, can be used to inhibit gene expression in individuals having diseases associated with expression of a particular gene. Similarly, a portion of the extended cDNA (or genomic DNA obtainable therefrom) can be used to study the effect of inhibiting transcription of a particular gene within a cell. Traditionally, homopurine WO 99/25825 PCT/IB98/01862 89 sequences were considered the most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences. Thus, both types of sequences from the extended cDNA or from the gene corresponding to the extended cDNA are contemplated within the scope of this invention.

EXAMPLE Preparation and use of Triple Helix Proles The sequences of the extended cDNAs (or genomic DNAs obtainable therefrom) are scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting gene expression. Following identification of candidate homopyrimidine or homopurine stretches, their efficiency in inhibiting gene expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which normally express the target gene. The oligonucleotides may be prepared on an oligonucleotide synthesizer or they may be purchased commercially from a company specializing in custom oligonucleotide synthesis, such as GENSET, Paris, France.

The oligonucleotides may be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.

Treated cells are monitored for altered cell function or reduced gene expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the target gene in cells which have been treated with the oligonucleotide The cell functions to be monitored are predicted based upon the homologies of the target gene corresponding to the extended cDNA from which the oligonucleotide was derived with known gene sequences that have been associated with a particular function. The cell functions can also be predicted based on the presence of abnormal physiologies within cells derived from individuals with a particular inherited disease, particularly when the extended cDNA is associated with the disease using techniques described in Example 53.

The oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above and in Example 59 at a dosage calculated based on the in vitro results, as described in Example 59.

In some embodiments, the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3' end of the alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation see Griffin et al. Science 245:967-971 (1989).

WO 99/25825 PCT/IB98/01862 EXAMPLE 61 Use of Extended cDNAs to Express an Encoded Protein in a Host Organism The extended cDNAs of the present invention may also be used to express an encoded protein in a host organism to produce a beneficial effect. In such procedures, tie encoded protein may be transiently expressed in the host organism or stably expressed in the host organism. The encoded protein may have any of the activities described above. The encoded protein may be a protein which the host organism lacks or, alternatively, the encoded protein may augment the existing levels of the protein in the host organism.

A full length extended cDNA encoding the signal peptide and the mature protein, or an extended cDNA encoding only the mature protein is introduced into the host organism. The extended cDNA may be introduced into the host organism using a variety of techniques known to those of skill in the art. For example, the extended cDNA may be injected into the host organism as naked DNA such that the encoded protein is expressed in the host organism, thereby producing a beneficial effect.

Alternatively, the extended cDNA may be cloned into an expression vector downstream of a promoter which is active in the host organism. The expression vector may be any of the expression vectors designed for use in gene therapy, including viral or retroviral vectors.

The expression vector may be directly introduced into the host organism such that the encoded protein is expressed in the host organism to produce a beneficial effect. In another approach, the expression vector may be introduced into cells in vitro. Cells containing the expression vector are thereafter selected and introduced into the host organism, where they express the encoded protein to produce a beneficial effect.

EXAMPLE 62 Use Of Signal Peptides Encoded By 5' Ests Or Sequences Obtained Therefrom To Import Proteins Into Cells The short core hydrophobic region of signal peptides encoded by the 5'ESTS or extended cDNAs derived from the 5'ESTs of the present invention may also be used as a carrier to import a peptide or a protein of interest, so-called cargo, into tissue culture cells (Lin et al., J. Biol. Chent., 270: 14225-14258 (1995); Du et al., J. Peptide Res., 51: 235-243 (1998); Rojas et al., Nature Biotech., 16: 370-375 (1998)).

When cell permeable peptides of limited size (approximately up to 25 amino acids) are to be translocated across cell membrane, chemical synthesis may be used in order to add the h region to either the C-terminus or the N-terminus to the cargo peptide of interest. Alternatively, when longer peptides or proteins are to be imported into cells, nucleic acids can be genetically engineered, using techniques familiar to those skilled in the art, in order to link the extended cDNA sequence encoding the h region to the5' or the 3' end of a DNA sequence coding for a cargo polypeptide. Such genetically engineered nucleic acids are then translated either in vitro or in vivo after transfection into appropriate cells, using conventional WO 99/25825 PCT/IB98/01862 91 techniques to produce the resulting cell permeable polypeptide. Suitable hosts cells are then simply incubated with the cell permeable polypeptide which is then translocated across the membrane.

This method may be applied to study diverse intracellular functions and cellular processes. For instance, it has been used to probe functionally relevant domains of intracellular proteins and to examine protein-protein interactions involved in signal transduction pathways (Lin et al., supra; Lin et al.. J. Biol.

Clhem., 271: 5305-5308 (1996); Rojas et al.. J. Biol. Chem., 271: 27456-27461 (1996): Liu et al., Proc. Natl.

Acadt. Sci. USA, 93: 11819-11824 (1996); Rojas et al., Bioch. Biophiys. Res. Coinunun., 234: 675-680 (1997)).

Such techniques may be used in cellular therapy to import proteins producing therapeutic effects.

For instance, cells isolated from a patient may be treated with imported therapeutic proteins and then reintroduced into the host organism.

Alternatively, the h region of signal peptides of the present invention could be used in combination with a nuclear localization signal to deliver nucleic acids into cell nucleus. Such oligonucleotides may be antisense oligonucleotides or oligonucleotides designed to form triple helixes, as described in examples 59 and 60 respectively, in order to inhibit processing and maturation of a target cellular RNA.

EXAMPLE 63 Reassembling Resequencing of Clones Further study of the clones reported in SEQ ID NOs: 40 to 86 revealed a series of abnormalities.

As a result, the clones were rescquenced twice, reanalyzed and the open reading frames were reassigned.

The corrected nucleotide sequences have been disclosed in SEQ ID NOs: 134 to 180 and the predicted amino acid sequences for the corresponding polypeptides have also been corrected and disclosed in SEQ ID NOs: 181 to 227. The corrected sequences have been placed in the Sequence Listing in the same order as the original sequences from which they were derived.

After this reanalysis process a few apparent abnormalities persisted. The sequences presented in SEQ ID NOs: 134, 149, 151, and 164 are apparently unlikely to be genuine full length cDNAs. These clones are missing a stop codon and are thus more probably 3' truncated cDNA sequences. Similarly, the sequences presented in SEQ ID NOs: 145, 155, and 166 may also not be genuine full length cDNAs based on homolgy studies with existing protein sequences. Although both of these sequences encode a potential start methionine each could represent of 5' truncated cDNA.

In addition, after the reassignment of open reading frames for the clones, new open reading frames were chosen in some instances. In case of SEQ ID NOs: 135, 149, 155, 160, 166, 171, and 175 the new open reading frames were no longer predicted to contain a signal peptide.

Table VII provides the sequence identification numbers of the extended cDNAs of the present invention, the locations of the full coding sequences in SEQ ID NOs: 134-180 the nucleotides encoding both the signal peptide and the mature protein, listed under the heading FCS location in Table VII), the WO 99/25825 PCT/IB98/01862 92 locations of the nucleotides in SEQ ID NOs: 134-180 which encode the signal peptides (listed under the heading SigPep Location in Table VII). the locations of the nucleotides in SEQ ID NOs: 134-180 which encode the mature proteins generated by cleavage of the signal peptides (listed under the heading Mature Polypcptide Location in Table VII), the locations in SEQ ID NOs: 134-180 of stop codons (listed under the heading Stop Codon Location in Table VII). the locations in SEQ ID NOs: 134-180 of polyA signals (listed tunder the heading PolyA Signal Location in Table VII) and the locations of polyA sites (listed under the heading PolyA Site Location in Table VII).

Table VIll lists the sequence identification numbers of the polypeptides of SEQ ID NOs: 181- 227, the locations of the amino acid residues of SEQ ID NOs: 181-227 in the full length polypeptide (second column), the locations of the amino acid residues of SEQ ID NOs: 181-227 in the signal peptides (third column), and the locations of the amino acid residues of SEQ ID NOs: 181-227 in the mature polypeptide created by cleaving the signal peptide from the full length polypeptide (fourth column). In Table VIII, and in the appended sequence listing, the first amino acid of the mature protein resulting from cleavage of the signal peptide is designated as amino acid number I and the first amino acid of the signal peptide is designated with the appropriate negative number, in accordance with the regulations governing sequence listings.

EXAMPLE 64 Functional Anavsis of Predicted Protein Sequences Following double-sequencing, new contigs were assembled for each of the extended cDNAs of the present invention and each was compared to known sequences available at the time of filing. These sequences originate from the following databases Genbank (release 108 and daily releases up to October, 15, 1998), Gcnseq (release 32) PIR (release 53) and Swissprot (release 35). The predicted proteins of the present invention matching known proteins were further classified into 3 categories depending on the level of homology.

It should be noted that the numbering of amino acids in the protein sequences discussed in Figures 9 to 16, and Table VI, the first methionine encountered is designated as amino acid number 1. In the appended sequence listing, the first amino acid of the mature protein resulting from cleavage of the signal peptide is designated as amino acid number I and the first amino acid of the signal peptide is designated with the appropriate negative number, in accordance with the regulations governing sequence listings.

The first category contains proteins of the present invention exhibiting more than 90% identical amino acid residues on the whole length of the matched protein. They are clearly close homologucs which most probably have the same function or a very similar function as the matched protein.

The second category contains proteins of the present invention exhibiting more remote homologies (40 to 90% over the whole protein) indicating that the protein of the present invention is WO 99/25825 PCT/IB98/01862 93 susceptible to have functions similar to those of matched protein.

The third category contains proteins exhibiting high homology (90 to 100%) to a domain of a known protein indicating that the matched protein and the protein of the invention may share similar features.

In addition all of the corrected amino acid sequences (SEQ ID NOs: 181 to 227) were scanned for the presence of known protein signatures and motifs. This search was performed against the Prosite 34.0 database. using the Proscan software from the GCG package. Functional signatures and their locations are indicated in Table VI.

A) Proteins which are closely related to known proteins Protein of SEO ID NO: 214: The protein of SEQ ID NO: 214 encoded by the extended cDNA SEQ ID NO: 167 isolated from brain shows extensive homology to a human SH3 binding domain glutamic acid-rich like protein or SH3BGRL (Egeo et al. Biochen. Diophys. Res. Conunn., 247:302-306 (1998)) with Genbank accession number is AF042081. As shown by the alignments of Figure 9, the amino acid residues are identical except for positions 63 and 101 in the 114 amino acid long matched sequence. This SH3BRGL protein is itself homologous to the middle proline-rich region of a protein containing an SH3 binding domain, the SH3BGR protein (Scartezzini et al., Hum. Genet., 99:387-392 (1997)). This proline-rich region is also highly conserved in mice. Both SH3BGR and SH3BGRL proteins are thought to be involved in the Down syndrome pathogenesis. The protein SEQ ID NO: 214 also contains the proline-rich SH3 binding domain (bold) and a potential RGD cell attachment sequence (underlined).

SH3 domains are small important functional modules found in several proteins from all cukaryotic organisms that are involved in a whole range of regulation of protein-protein interaction, e.g.

in regulating enzymatic activities, recruiting specific substrates to the enzyme in signal transduction pathways, in interacting with viral proteins and they are also thought to play a role in determining the localization of proteins to the plasma membrane or the cytoskeleton (for a review, see Cohen et al, Cell, 80:237-248 (1995)).

The Arg-Gly-Asp (RGD) attachment site promote cell adhesion of a large number of adhesive extracellular matrix, blood and cell surface proteins to their integrin receptors which have been shown to regulate cell migration, growth, differentiation and apoptosis. This cell adhesion activity is also maintained in short RGD containing synthetic peptides which were shown to exhibit anti-thrombolytic and anti-metastatic activities and to inhibit bone degradation in vivo (for review, see Ruoslahti, Annu.

Rev. Cell Dev. Biol., 12:697-715 (1996)).

Taken together, these data suggest that the protein of SEQ ID NO: 214 may be important in regulating protein-protein interaction in signal transduction pathways, and/or may play a role of localization of proteins to the plasma membrane or cytoskeleton, and/or may play a role in cell adhesion.

Moreover, this protein or part therein, especially peptides containing the RGD motif, may be useful in WO 99/25825 PCT/IB98/01862 94 diagnosing and treating cancer, thrombosis, osteoporosis and/or in diagnosing and treating disorders associated with the Down syndrome.

Proteins of SEQ ID NOs: 185 and 215: The nearly homologous proteins of SEQ ID NOs: 185 and 215 encoded by the extended cDNA SEQ ID NOs: 138 and 168, respectively, exhibit an extensive homology with a murine protein named MP'l for MEK binding partner 1 (Genbank accession number AF082526). Tlhe amino acid residues are identical to the murine protein except for positions 39. 118 and 119 of the Genbank MPI sequence for SEQ ID NO: 215 and except for positions 33, 39. 118 and 119 of the Genbank MPI sequence for SEQ ID NO: 185. The Genbank MP1 sequence is the 124 amino acid long matched protein region. See the amino acid sequence alignment in Figure 10. MPI was shown to enhance enzymatic activation of mitogen-activated protein (MAP) kinase cascade. The MAP kinase pathway is one of the important enzymatic cascade that is conserved among all eukaryotes from yeast to human. This kind of pathway is involved in vital functions such as the regulation of growth, differentiation and apoptosis. MPI probably acts by facilitating the interaction of the two sequentially acting kinases MEKI and ERKI (Schaffer et al., Science, 281:1668-1671 (1998)).

Taken together, these data suggest that the proteins of SEQ ID NO: 185 and 215 may be involved in regulating protein-protein interaction in the signal transduction pathways. Thus, these proteins may be useful in diagnosing and/or treating several types of disorders including, but not limited to, cancer, neurodegenerative diseases, cardiovascular disorders, hypertension, renal injury and repair and septic shock.

Protein of SEQ ID NO: 186 The protein of SEQ ID NO: 186 encoded by the extended cDNA SEQ ID NO: 139 exhibits an extensive homology with a murine protein named claudin-2 (Genbank accession number AF072128).

The amino acid residues are identical except for the conservative substitutions observed at positions: 6, 22, 23, 29, 31, 90, 110, 120, 130, 171, 176, 179, 187, 192, 1 9 7 21 1 ,21 2 214, and 217 of the 230 amino acids long matched protein claudin-2. One drastic substitution from glycine to arginine was observed at position 189. See the amino acid sequence alignment in Figure 11. The murine homologue claudin-2 is a integral membrane proteins with 4 putative transmembrane domains belonging to a family of proteins thought to be involved in the formation of tight junctions between cells in epithelial or endothelial cell sheets (Furuse et al., J. Cell. Biol., 141:1539-1550, (1998)).

In addition, the protein of SEQ ID NO: 186 shows more remote homology to a family of transmembrane proteins among which are receptors for Clostridium perfringens enterotoxin (CPE) with either high or low affinity for CPE (Katahira et al., J. Biol. Chem., 452:26652-26658 (1997)). The matched region include the 4 putative transmembrane regions.

Taken together, these data suggest that the protein of SEQ ID NO: 186 may be involved in the formation and/or regulation of tight junction, and more generally in cell-cell adhesion. This protein may WO 99/25825 PCT/IB98/01862 also function as a receptor for a yet unknown ligand that may show homology to CPE. This protein may thus be useful in diagnosing and/or treating disorders associated with changes in epithelium permeability such as infectious diseases caused by Clostridium parasites.

Protein of SEO ID NO: 213 The protein of SEQ ID NO: 213 encoded by the extended cDNA SEQ ID NO: 166 and expressed in lymphocytes exhibits an extensive homology to a stretch of 121 amino acid of a human hematopoictic maturation factor named glia maturation factor gamma or GMF-y (Genbank accession number AU001993) and also to other glia maturation factors found in human, bovine and rodent species. The amino acid residues are identical as shown below except for conservative substitutions at positions and 77 of the 142 amino acids long matched protein GMF-y which is itself highly homologous to GMF-p (Asai et al., Biochem. Biophys. Acta, 1396:242-244 (1998)). See the amino acid sequence alignment in Figure 12. GMF-p was shown to act as a growth and differentiation factor for neurons and glial cells in human brain (Lim et al., Proc Natl Acad Sci U S A 86:3901-3905 (1989); and Harman et al., Brain Res.

56:332-335 (1991)) and is also thought to regulate ERK proteins of the evolutionarily conserved mitogen-activated protein (MAP) kinase cascade which is important in the regulation of growth, differentiation and apoptosis (Zaheer and Lim. J. Biol. Chem., 272:5183-5186 (1997)).

Taken together, these data suggest that the protein of SEQ ID NO: 213 may be involved in cell growth and differentiation and/or in apoptosis and/or in intracellular signaling. Thus, this protein may be useful in diagnosing and/or treating several types of disorders including, but not limiting to, cancer, neurodegencrative diseases, cardiovascular disorders, hypertension, renal injury and repair and septic shock.

Protein of SEO ID NO: 191 The protein of SEQ ID NO: 191 encoded by the extended cDNA SEQ ID NO: 144 and expressed in lymphocytes exhibits an extensive homology to a stretch of 91 amino acid of a human secreted protein expressed in peripheral blood mononucleocytes (Genpep accession number W36955 and Genseq accession number VOO433). The amino acid residues are identical except for the substitution of asparagine to isoleucine at positions 94, and the conservative substitutions at positions 108, 109 and 110 of the 110 amino acids long matched protein. See the amino acid sequence alignment in Figure 13.

Protein of SEO ID NO: 200 The protein of SEQ ID NO: 200 encoded by the extended cDNA SEQ ID NO: 153 exhibits extensive homologies to proteins encoding RING zinc finger proteins of the human ,chicken and rodent species, as well as an EGF-like domain. Two stretches of 341 and of 13 amino acids of the human RING zinc finger protein which might bind DNA (Genbank accession number AF037204). The amino acid residues are identical except for conservative substitutions at positions 18, 29, 156 and 282 of the 381 amino acid long human RING zinc finger. See the amino acid sequence alignment in Figure 14. Such RING zinc finger proteins are thought to be involved in protein-protein interaction and are especially WO 99/25825 PCT/IB98/01862 96 found in nucleic acid binding proteins. Secreted proteins may have nucleic acid binding domain as shown by a nematode protein thought to regulate gene expression which exhibits zinc fingers as well as a functional signal peptide (Hoist and Zipfel. J. Biol. Chem., 271:16275-16733 (1996)).

Taken together, these data suggest that the protein of SEQ ID NO: 200 may play a role in protein-protein interaction or be a nucleic acid binding protein.

Protein of SEO ID NO: 192 The protein of SEQ ID NO: 192 encoded by the extended cDNA SEQ ID NO: 145 exhibits extensive homologies to stretches of proteins encoding vacuolar proton-ATPase subunits M9.2 of either human (Genbank accession number Y15286) or bovine species (Genbank accession number Y15285).

These two highly conserved proteins are extremely hydrophobic membrane proteins with two membranespanning helices and a potential metal-binding domain conserved in mammalian protein homologues (Ludwig el al., J. Biol. Chew., 273:10939-10947 (1998)). The amino acid residues are completely identical as shown in the alignment in Figure 15. However, the protein of SEQ ID NO: 192 is missing amino acids 1 to 92 from the Genbank sequences. The protein of SEQ ID NO: 192 contains the second putative transmembrane domain as well as the potential metal-binding site.

Taken together, these data suggest that the protein of SEQ ID NO: 192 may play a role in energy conservation, secondary active transport, acidification of intracellular compartments and/or cellular pH homeostasis.

B) Proteins which are remotely related to proteins with known functions Proteins of SEO ID NOs: 201 and 227 The proteins of SEQ ID NOs: 201 and 227 encoded by the extended cDNA SEQ ID NOs: 154 and 180, respectively, belong to the stomatin or band 7 family. The human stomatin is an integral membrane phosphoprotein thought to be involved to regulate the cation conductance by interacting with other proteins of the junctional complex of the membrane skeleton (Gallagher and Forget, J. Biol. Chem., 270:26358-26363 (1995)). The proteins of SEQ ID NOs: 201 and 227 exhibit the PROSITE signature typical for the band 7 family signature. See the amino acid sequence alignment in Figure 16.

Taken together, these data suggest that the proteins of SEQ ID NOs: 201 and 227 play a role in the regulation of ion transport, hence in the control of cellular volume. These proteins may then be useful in diagnosing and/or treating stomatocytosis and/or cryohydrocytosis.

Protein of SEO ID NO: 198 The protein of SEQ ID NO: 198 encoded by the extended cDNA SEQ ID NO: 151 shows homologies with different DNA or RNA binding proteins such as the human Staf50 transcription factor (Genbank accession number X82200), the human Ro/SS-A ribonucleoprotein autoantigen (Swissprot accession number P19474) or the murine RPTI transcription factor (Swissprot accession number P15533). The protein of SEQ ID NO: 198 exhibits a putative signal peptide and also a PROSITE signature for a RING type zinc finger domain located from positions 15 to 59. Secreted proteins may WO 99/25825 PCT/IB98/01862 97 have nucleic acid binding domain as shown by a nematode protein thought to regulate gene expression which exhibits zinc fingers as well as a functional signal peptide (Hoist and Zipfel. J. Biol. Chem., 271:16275-16733 (1996)).

Taken together, these data suggest that the protein of SEQ ID NO: 198 may play a role in protein-protein interaction in intracellular signaling and eventually may directly or indirectly bind to DNA and/or RNA. hence regulating gene expression.

Protein of SEO I1) NO: 216 The protein of SEQ ID NO: 216 found in testis encoded by the extended cDNA SEQ ID NO: 169 shows homologies to protein domains with a 4-disulfide core signature found in either an extracellular proteinase inhibitor named chelonianin (Swissprot accession number P00993) or in rabbit and human proteins specifically expressed in epididymes (Genbank accession numbers U26725 and R13329). The matched domain in red sea turtle chelonianin is known to inhibit subtilisin, a serine protease (Kato and Tominaga, Fed. Proc., 38:832 (1979)). All cysteines of the 4 disulfide core signature thought to be crucial for biological activity are present in the protein of SEQ ID NO: 216. The 4 disulfide core signature is present except for a conservative substitution of asparagine to glutamine.

Taken together, these data suggest that the protein of SEQ ID NO: 216 may play a role in protein-protein interaction, act as a protease inhibitor and/or may also be related to male fertility.

Protein of SEQ ID NO: 197 The protein of SEQ ID NO: 197 encoded by the extended cDNA SEQ ID NO: 150 shows extensive homology to the connexin family conserved in the rodent, chicken, human, frog, sheep species.

Connexins are a family of integral membrane proteins that oligomerize into clusters of intercellular channels called gap junctions, which join cells in virtually all metazoans. These channels permit exchange of ions between neurons and between neurons and excitable cells-such as myocardiocytes (for review, see Goodenough et al., Ann. Rev. Biochem., 65:475-502 (1996)).

Taken together, these data suggest that the protein of SEQ ID NO: 197 may play a role in cell growth, differentiation and developmental signaling. Moreover, the protein of SEQ ID NO: 197 may be useful in diagnosing and/or treating cancer, neurodegenerative diseases and cardiovascular disorders.

C) Proteins homologous to a domain of a protein with known function Protein of SEQ ID NO: 183 The protein of SEQ ID NO: 183 encoded by the extended cDNA SEQ ID NO: 136 shows homology to a rabbit soluble protein called PiUS (Genbank accession number U74297) which is a stimulator of inorganic phosphate uptake and is thought to be involved in cellular phosphate metabolism and/or binding (Norbis et al., J. Memb. Biol., 156:19-24 (1997)).

Taken together, these data suggest that the protein of SEQ ID NO: 183 may play a role in WO 99/25825 PCT/IB98/01862 98 phosphate metabolism.

Protein of SEQ ID NO: 223 The protein of SEQ ID NO: 223 encoded by the extended cDNA SEQ ID NO: 176 shows homology to short stretches of a human protein called Tspan-1 (Genbank accession number AF054S38) which belongs to the 4 transmembrane superfamily of molecular facilitators called tetraspanin (Meakers et al.. FASEB 11:428-442 (1997)).

Taken together, these data suggest that the protein of SEQ ID NO: 223 may play a role in cell activation and proliferation, and/or adhesion and motility and/or differentiation and cancer.

Protein of SEQ ID NO: 193 The protein of SEQ ID NO: 193 encoded by the extended cDNA SEQ ID NO: 146 shows homology to short stretches of Drosophila, C. elegans and chloroplast proteins similar to E. coli ribosomal protein L16.

Taken together, these data suggest that the protein of SEQ ID NO: 193 may be a ribosomal protein.

As discussed above, the extended cDNAs of the present invention or portions thereof can be used for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination for expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The proteins or polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative WO 99/25825 PCT/IB98/01862 99 receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without litmittion Molecular Cloning: A Laboratory Manual, 2d ed.. Cole Spring Harbor Laboratory Press, Sambrook, E.F. Fritsch and T. Maniatis eds., (1989). and Methods in Enzymology; Guide to Molecular Cloning Techniques. Academic Press, Berger. S.L. and A.R. Kimmel eds., (1987).

Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.

In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

Although this invention has been described in terms of certain preferred embodiments, other embodiments which will be apparent to those of ordinary skill in the art in view of the disclosure herein are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by reference to the appended claims.

I

Search Char Step Program Strai Miscellaneous blastn both LRNA *Tasta both rRNA blastu both ntRNA blastn both Procaryotic blastn both Fungal blastn both AlI both acteristics Selection Characteristics d Parameters Identity Length (bp) S=61 X=16 90 17 .80 S=108 80 -S=108 80 S=144 90 S=144 90 70 S=72 70 S=72 70 S=54 X=16 90 S=108 90 S=108X=16 90 E=0.001 used for each step of EST analysis 1 1blastn both Repeats blastn both Promoters blastn top Vertebrate fasta* both ESTs blasta both

A

Proteins blasLO Table 1: Parameters USC "Quick Past" Database Scanner t aligncincni furthcr constraincd to begin closer Ohan lObp to EST 5' nd 0 using BLOSUM62 substitudoo matrix WO 99/25825 PCT/IB98/01862

FCS

Location 173-565 267-455 174-662 460-615 79-450 160-849 106-321 359-631 191-508 346-861 214-381 372-509 132-884 199-429 293-535 130-507 191-1009 141-614 212-364 147-1223 112-984 239-439 157-537 194-484 148-405 156-368 272-451 381-734 140-367 183-467 140-385 129-395 285-374 136-480 200-514 68-346 274-600 421.573 198-365 167-652 SigPep Location 173-211 267-371 174-266 460-555 79-369 160-231 106-201 359-466 191-286 346-408 214-339 372-437 132-215 199-288 293-385 130-189 191-325 141-251 212-268 147-248 112-237 239-316 157-345 194-253 S148-207 156-230 272-397 381-629 140-205 183-338 140-205 129-176 285-341 136-444 200-427 68-133 274-399 421-465 198-278 167-229 Mature Polypcptide Location 212-565 372-455 267-662 556-615 370-450 232-849 202-321 467-631 287-508 409-861 340-381 438-509 216-884 289-429 386-535 190-507 326-1009 252-614 269-364 249-1223 238-984 317-439 346-537 254-484 208-405 231-368 398-451 630-734 206-367 339-467 206-385 177-395 342-374 445-480 428-514 134-346 400-600 466-573 279-365 230-652 Stop Codon Location 566 456 663 616 451 850 322 632 509 862 382 510 885 430 536 508 1010 615 365 1224 985 440 538 485 406 369 452 735 368 468 386 396 375 481 515 347 601 574 366 653 PolyA Signal Location 1063-1068 817-822 1144- 149 614-619 1217-1222 1510-1515 577-582 1334-1339 755-760 1400-1405 1133-1138 812-817 1069-1074 464-469 733-738 546-551 1348-1353 1354-1359 1465-1470 1538-1543 976-981 586-591 771-776 768-773 789-794 706-7.11 503-508 736-741 965-970 620-625 383-388 513-518 575-580 835-840 1001-1006 472-477 943-948 553-558 364-369 1133-1138 PolyA Site Location 10S7-109S 842-855 1165-1176 635-648 1240-1251 1506-1519 598-610 1357-1370 780-791 1420-1433 1146-1158 838-850 1094-1107 4S9-500 752-765 572-584 1374-1387 1375-1385 1489-1497 1558-1570 1010-1022 603-615 791-804 780-792 820-832 709-721 518-531 770-783 984-996 644-657 405-416 530-543 592-605 851-864 1022-1033 490-499 966-978 575-587 387-400 1154-1166 WO 99/25825 PCTIIB98/O1 862 102 Mature Stop PolyA PolyA FCS SigPcp Polypcptide Codon Signal Site Id Location Location Location Location Location Location 180-557 180-383 384-557 558 722-727 743-754 SI 179-598 179-298 299-598 599 6S0-685 697-708 82 100-22S 100-171 172-228 229 211-216 230-243 83 346-552 346-40S 409-552 553 792-797 817-829 84 177-410 177-233 234-410 411 644-649 663-674 179-41S 179-3 19 320-41S 419 461-466 465-478 86 112-270 112-237 238-270 271 910-915 940-952 TAflLE~:t It11397 WO 99/25825 PCT/IB98/OI 862 103 TABLE rUT Motif Location 160-226 683-734 231-261 M Votif Zinc finger, C2H2 type, domain Conncxjns signatures Zinc finger, C3HC4 rype, signature TAOLS-3-is 1113~97 WO 99/25825 WO 9925825PCTIIB98/OI 862 104 !ABLE rV Id 87 88 89 91 92 93 94 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110

III

112 113 114 115 116 117 118 119 120 121 122 123 Full Length Polypcptide Location 1-131 1-63 1-163 1-52 -124 1-230 1-72 1-91 1-106 1-172 1-56 1-46 1-251 1-77 1-81 1-126 1-273 1-158 1-51 1-3 59 1-291 1-67 1-127 1-97 1-86 1-71 1-60 1-118 1-76 1-95 1-82 1-89 1-30 1-115 1-105 1-93 1-109 Signal Peptide Location 1-13 1-35 1-31 1-32 1-97 1-24 1-32 1-36 1-32 1-21 1-42 1-22 1-28 1-30 1-31 1-20 1-45 1-37 1-19 1-34 1-42 1-26 1-63 1-20 1-20 1-25 1-42 1-83 1-22 1-52 1-22 1-16 1-19 1-103 1-76 1-22 1-42 Mature Po lypep tide Location 14-131 36-63 32-163 33-52 9S- 124 25-230 3 3-72 37-91 3 3-106 22-172 43-56 23-46 29-251 3 1-77 32-81 2 1-126 46-273 3 8-158 20-5 1 3 5-359 43-29 1 27-67 64-127 2 1-97 21-86 26-7 1 43-60 84- 118 23-76 53-95 23-82 17-89 20-30 104-115 77-105 23-93 43- 109 WO 99/25825 PTI9/16 PCT/IB98/01862 Id 124 125 126 127 1 2S 129 130 131 132 133 Full Lcngth Polypcptidc Location 1-5 1 1-56 1-162 1-126 1-140 1-43 1-69 1-78 1-80 1-53 Signal Peptide Location 1-15 1-27 1-21 1-68 1-40 1-24 1-21 1-19 1-47 1-42 Mature Polypcptide Location 16-51 28-56 22- 162 69- 126 41-140 25-43 22-69 20-78 48-80 43-53 TAD LE4:ss 111397 WO 99/25825 PCTIIB98/OI 862

LALEV

No-matches Est <30% Est x WO 99/25825 WO 9925825PCT/IB98/01862 No-matches Est <3o% Est >30% Vrt TABLES~ss 11I1397 WO 99/25825 PCT/IB98/01862 108 PROTEIN SIGNATURE SEQ ID LOCATION 214 76 78 cell att 401TIF achment site 32 53 201 f289 291 164 192 46

=.L

227 239 241 114 142 205 179 182 226 78 81 181 99 -101 200 264 -278 240 -282 Microbodies C-terminal targeting signal Band 7 protein family Microbodies C-terminal targeting signal Band 7 protein family Endoplasmic reticulum targeting signal Microbodies C-terminal targeting signal cell attachment site EGF like domain C3HC4 zinc fincer (RINGz fi 196 10 -32 C2H2 zinc finger 198 15 -59 C3HC4 zinc finger (RING finger) 218 21 -42 Leucine zipper 197 164 -180 connexins TABLE VI WO 99/25825 PCT/IB98/01862 109 Table VII SEQ FCS SigPep Mnature Polypcptidc Stop Codon PolyA Signal PolyA Site ID Location Location Location Location Location Location 134 131/1042 131/169 170/1042 10421053 135 100/2-7 6 .107"^r '1042/1053 135 100/276 100/276 277 638/643 662/675 136 111/401 111/194 195/401 402 1080/10S5 1101/1112 137 359/514 359/454 455/514 515 536/547 13S 26/397 26/316 317/397 39S 1164/1169 1187/119 139 36/725 36/107 io08s/725 726 1302/1307 13S9/1400 140 35/250 35/130 131/250 251 505/51 0 526/538 141 169/4,JM 169/267 268/432 433 1132/1137 1155/1167 142 143/460 143/238 239/460 461 697/702 721/730 143 108/90S 108/170 171/908 909 1141/1146 1161/1174 144 209/532 209/532 533 1133/1138 1146/1158 145 5/211 5/142 143/2 11 212 716/721 742/754 146 98/850 98/181 182/850 851 1035/1040 1060/1073 147 46/342 46/189 190/342 343 377/382 402/413 148 139/381 139/231 232/381 382 579/584 598/609 149 72/512 72/512 512/522 150 126/944 126/260 261/944 945 123'/128 1309/1322 151 50/1279 50/160 161/1279 1280/1290 152 83/1261 83/139 140/1261 1262 135280/135290 153 57/1199 57/95 96/1199 1200 1438/1443 1458/1470 154 72/944 72/197 198/944 945 970/982 155 4/279 4/279 280 425/430 443/455 156 90/470 90/278 279/470 471 704/709 724n/38 157 88/339 88/147 148/339 340 619/624 637/649 158 33/578 33/92 93/578 579 703/7 14 159 33/245 33/107 108/245 246 546/551 584/596 160 125/343 125/343 344 375/380 390/403 161 126/632 126/575 576/632 633 670/675 721/727 162 90/317 90/155 156/317 318 913/918 932/944 163 126/410 126/287 288/410 411 561/566 587/598 164 85/348 85/150 151/348 -349/360 165 77/343 77/124 125/343 344 461/466 477/490 166 38/364 38/364 365 458/463 4751488 167 48/389 48/356 3571389 390 742/747 760/771 168 69/440 69/359 360/440 441 927/932 947/959 169 33/311 33198 99/311 312 437/442 455/464 170 110/730 110/235 236/730 731 764/769 787/799 171 38/214 38/2s14 215 308/320 172 129/296 129/209 210/296 297 318/331 173 78/563 78/359 360/563 564 1042/1047 1063/1075 174 62/523 62/265 266/523 524 602/607 621/632 175 24/320 24320 321 402/407 4 19/430 176 42/170 42/113 114/170 171 172/IS5 177 108/314 108/170 171/314 315 550/555 574/585 178 118/351 118/171 172/351 352 583/588 602/613 179 128/367 128/268 269/367 368 410/415 424/427 180 149/871 149/457 458/871 872 -893/912 WO 99/25825 WO 9925825PCTJIB98/01862 SEQ Full Length Polypeptide ID Locution 13-4 [3$ [36 137 138 139 140 141 142 143 144 [.46 147 [48 [-49 151 1$2 153 [54 155 156 157 158 159 160 161 162 163 16.4 165 166 167 168 169 170 171 172 [73 174 175 176 177 [78 [7 9 180 180 -13/291 1/59 *28/69 -32120 -97/27 -2-12-06 -32/-40 -3 3/55 -32/74 -21/2-46 1/108 -46/23 -281223 -48/51 -3 1/50 1/147 -45r228 37/373 19/374 -13/368 -421249 1/92 -63/64 .20/64 162 -25/46 -150/19 -12/54 1 -22,/66 -16/73 1/109 -103/11 -97/27 -22/71- n .42/ 165 1/59 -27/29 -94/68 -68/86 1/99 -24/19 -2 1/48 -18/60 -47/3 3 103/138 103/138 110 Table VIII signal Peptidle Location 3/-I -23S/-I -32/-I -47/-I .24/-I -45/-I -37/-1 -42/-1 -28/-I -480/-I -3 2/-I .54/-1 -22/-1 -13/-I -97/-l .420/-I -27/-l -95/-I -680/-1 -24/-I -47/-1 -163/-1 -103/-1 Mature Polypeptide Locution 11291 1/59 1/69- 1/20 1/27 1/206 1/40 1155 1/74 1/246 1/108 1/23 1/223 1151 1/147 1/228 1/37 3 1/374 1/3 63 11249 1/92 1/64 1/64 1/162 1/46 1/7 1/19 1/54 1/41 1/66 1/7 1/109 1/1 1 1/M 1/7 1/165 1/59 1/29 1/68 1/86 1/99 1/19 1/48 1/60 1/33 1/138 1/138 Editorial Note 10491/99 The following sequence listing, pages 1-177, are part of the description.

WO 99/25825 SEQUENCE LISTING <110> Genset SA <120> Extended cDNAS for Secreted Proteins <160> 227 <170> Patent-pm <210> 1 <211> 47 <212> RNA <213> <220> <221> modified-base <222> 1 <223> m7Gppp added to 1 <300> <400> 1 ggcauccuac ucccauccaa uuccacccua acuccuccca ucuccac <210> 2 <211> 46 <212> RNA <213> <22 0> <300> <400> 2 gcauccuacu cccauccaau uccacccuaa cuccucccau cuccac <210> 3 <211> <212> DNA <213> <220> <300> <400> 3 atcaagaatt cgcacgagac catta <210> 4 <211> <212> DNA <213> <220> <300> <400> 4 taatggtctc gtgcgaattc ttgat <210> <211> <212> DNA <213> <220> <300> <400> ccgacaagac caacgtcaag gccgc <210> 6 <211> <212> DNA <213> <22 0> <300> <400> 6 tcaccagcag gcagtggctt aggag <210> 7 <211> <212> DNA <213> <220> <300> <400> *7 agtgattcct gctactttgg atggc PCTIIB98/01862 WO 99/25825 PCT/IB98/01862 2 <210> 8 <211> <212> DNA <213> <220> <300> <400> 8 gcttggtct gttctggagt ttaga <210> 9 <211> <212> DNA <213> <220> <300> <400> 9 tCCdgagg gagacaagcc aat <210> 10 <211> <212> DNA <213> <220> <300> <400> agggaggagg aaacagcgtg agtcc <210> 11 <211> <212> DNA <213> <220> <300> <400> 11 atgggaaagg aaaagactca tatca <210> 12 <211> <212> DNA <213> <220> <300> <400> 12 agcagcaaca atcaggacag cacag <210> 13 <211> <212> DNA <213> <220> <300> <400> 13 atcaagaatt cgcacgagac catta <210> 14 <211> 67 <212> DNA <213> <220> <300> <400> 14 atcgttgaga ctcgtaccag cagagtcacg agagagacta cacggtactg gtttt ttttvn 67 <210> 15 <211> 29 <212> DNA <213> <22 0> <300> WO 99/25825 WO 9925825PCT/IB98/01 862 3 <400> 15 ccagcagagt cacgagagag actacacgg <210> 16 <211> <212> DNA <213> <220> <300> <400> 16 cacgagagag actacacggt aictgg <210> 17 <211> 526 <212> DNA <213> Homo Sapiens <220> <221> misc-feature <222> complement (261. .376) <223> blastn <221> misc...feacure <222> complement(380. .486) <223> blastn <221> misc-feacure <222> complement(11O. .145) <223> blastn <221> misc-feature <222> complement(196. .229) <223> blastn <221> sig..peptide <222> 90. .140 <223> Von Heijne matrix <3 00> <400> 17 aatatrarac agctacaata ttccagggcc artcacttgc catttctcat aacagcgtca gagagaaaga actgactgar acgrtttgag atg Met aca gcc atc ttg Thr Ala Ile Leu gaa cga gaa aaa Glu Arg Glu Lys wtt ttt gtg ttcc Xaa Phe Val Phe gca Ala aga Arg cc t Pro gtg gct gtw ggt Val Ala Val Gly agt atc agt gac Ser Ile Ser Asp is tac cca tat cca Tyr Pro Tyr Pro aag aaa Lys LYS -15 ttc cca Phe Pro 1 agc gat Ser ASP ttt cgc Phe Arg gtt ctc ctc ctg Val Leu Leu Leu gtc tct caa gac Val Ser Gin Asp gaa tta gct tca Glu Leu Ala Ser cca ctt cca cca Pro Leu Pro Pro atc Ile cag Gin ggr Gly att Ile 113 161 209 257 305 354 414 474 526 cca .ttt cca aga ttV cca t99 ttt aga cgt aan ttt cct Pro Phe Pro Arg Phe Pro Trp Phe Arg Arg Xaa Phe Pro 45 50 cct gaa tct gcc cct aca act ccc ctt cct agc gaa aag Pro Glu Ser Ala Pro Thr Thr Pro Leu Pro Ser Glu Lys att cca ata Ile Pro Ile taaacaaraa ggaaaagtca crataaa caaaattcct gttaata gccaatact ttagtga <210> 18 <211> 17 <212> PRT <213> Homo Sapiens <220> <221> SIGNAL <222> 1. .17 <223> Von Hiine m, c.ct ggtcacctga aattgaaact aaa raaaaacaaa tgtaattgaa tct Cctttaataa acatgaaagc gagccacttc cttgaaraat atagcacaca gcattctcta aaaaaaaaaa aa atriY score 8.2 WO 99/25825 WO 9925825PCT/IB98/01862 seq LLLITAILAVAVG/FP <300> <400> 18 Met Lys Lys Val Leu Leu Leu Ile Thr Ala Ilie Leu Ala Val Ala Val .1 5 10 Gly <210> 19 <211> 822 <212> DNA <213> Homo Sapiens <220> <221> misc feature <222> 260. .464 <223> blastn <221> misc-feature <222> 118. .184 <223> blastn <221> misc-feature <222> 56. .113 <223> biastn <221> misc-.feature <222> 454. .485 <223> biastn <221> misc-feature <222> 118. .545 <223> biastn <221> misc-feature <222> 65. .369 <223> biastn <221> misc..feature <222> 61. .399 <223> blastn <221> misc-feature <222> 408. .458 <223> biastn <221> misc-feature <222> 60. .399 <223> blastn <221> misc-feature <222> 393. .432 <223> blastn <221> sig...peptide <222> 346. .408 <223> Von Heijne matrix <300> <400> 19 actcctttta. gcataggggc ttcggcgcca gcggccagcg ctagtcggtc tggtaagtg ctgatgccga gttccgtzctc tcgcgccttt tcctggtccc aggcaaagcg gasgnagat( ctcaaacggc ctagtgcttc gcgcttccgg agaaaatcag cggtctaatt aattcctct gtttgttgaa gcagttacca agaatcttca accctttccc acaaaagcta attgagtac cgttcctgtt gagtacacgt tcctgttgat ttacaaaagg tgcaggtatg agcaggtct aagactaaca ttttgtaaa ttgtaaac

C

9 a 9 Y dd"C-CgE cag caa ggc ccc agt ttc ctt cct tca gcc ctt Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu -10 gct gct ttc ata ttt tca tac att act gca gta Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala Val 1 5 10 gac ccg gct tta cct tat arcc agt gac act ggt Asp Pro Ala Leu Pro Tyr Ile Ser Asp Thr Gly 25 tagaa atg tgg tgg ttt Met Trp Trp Phe gta att tgg aca tct Val Ile Trp-Thr Ser aca ctc cac cat ata Thr Leu His His Ile aca gta gct. cca raa Thr Val Ala Pro Xaa 120 180 240 300 357 405 453 501 WO 99/25825 PCTIIB98/01862 aaa tgc tta ttt'ggg gca atg cta aat att gcg gca gtt tta tgt caa 549 Lys Cys Leu Phe Gly Ala Met Leu Asn Ile Ala Ala Val Leu Cys Gin 35 40 aaa tagaaatcag gaarataatt caacttaaag aakttcattt catgaccaaa. 602 Lys ctcttcaraa acatgtcttt acaagcatat Ctcttgcatt g9cttcaca ctgttgaatt 662 gtctggcaat aetctgcag tggaaaattt gatttarmta gttcttgact gataaatatg 722 gtaaggtggg ctttcccccc tgtgtaattg gctactatgt cttactgagc caagttgtaw 782 tttgaaataa agatatga gagtgacaca aaaaaaaaaa 822 <210> 20 <211> 21 <212> PRT <213> Homo Sapiens <220> <221> SIGNAL <222> 1. .21 <223> Von Heijne matrix score seq SFLPSALVIWTSA/AF <300> <400> Met Trp Trp Phe Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu Val 11 5 10 Ile Trp Thr Ser Ala <210> 21 <211> 405 <212> DNA <213> Homo Sapiens <220> <221> misc-feature <222> complemene.(103. .398) <223> blastn <221> sig..peptide <222> 185. .295 <223> Von Heijne matrix <300> <400> 21 atcaccttct tccccatccc tstctgggcc agtccccarc ccagcccctc tcccgacctg cccagcccaa gtcagccttc agcacgcgct tttcgcaca, cagatattcc aggcctacct 120 ggcattccag gacctccgma atgatcc-ccc agtcccttac aagcgcttcc tggatgaggg 180 tggc atg gtg ctg acc acc ctc ccc ttg ccc tcc-gcc aac agc cct gtg 229 Met Val Leu Thr Thr Leu Pro Leu Pro Ser Ala Asri Ser Pro Val -30 aac atg ccc acc act ggc ccc aac agc ctg agt tat gct agc tc gcc 277 Asn Met Pro Thr Thr Gly Pro Asn Ser Leu Ser Tyr Ala Ser Ser Ala -15 ctg tcc ccc tgt ctg acc gct cca aak tcc ccc cgg ctt gcc atg atg 325 Leu Ser Pro Cys Leu Thr Ala Pro Xaa Ser Pro Arg Leu Ala Met Met 1 5 cct gac aac taaatatcct tatccaaatc aataaarwra raatcctccc 374 Pro Asp Asn tccaraaggg tttccaaaaa caaaaaaaaa a 405 <210> 22 <211> 37 <212> PRT <213> Homo Sapiens <220> <221> SIGNAL <222> 1. .37 <223> Von Heijne matrix score 5.9 seq LSYASSA.LSPCLT/AP WO 99/25825 WO 9925825PCTIIB98/01862 <300> <400> 22 Met Val Leu Thr Thr 1 5 Met Pro Thr Thr Gly Ser Pro Cys Leu Thr Leu Pro Leu Pro Ser 10 Pro Asn Ser Leu Ser 25 Ala Asn Ser Tyr Ala Ser Pro Val Asn Ser Ala Leu <210> 23 <211> 496 <212> DNA <213> Homo <220> <221> mi sc <222> 149.

<223> bias <221> misc* <222> 328.

<223> bias <221> misc <222> comp, <223> blasi <221> sig~j <222> 196.

<223> Von <300> <400> 23 aaaaaattgg attagccgtg cccggagata gcacacagac Sapiens feature .331 tn -feature .485 tn feature lement(182. .496) ~eptide .240 ieijne matrix tcccagtttt caccctgccg gcctaggccg tttaacgggg ggaccaaccg tcaggaatgc agacc atg ggg att ctg Met Gly Ile Leu cagggCtggc tggggagggc agcggtttag tgacacgagc ntgcagggcc gagtccaagg gaggaatgtt tttcttcgga ctctatcgag tct aca gtg aca gcc tta aca ttt Ser Thr Val Thr Ala Leu Thr Phe -10 gcc ara gcc ctg gac Ala Xaa Ala LeU ASP ggc tgc aga aat ggc att gcc cac cct gca agt Gly Cys Arg Asn Gly Ile Ala His Pro Ale Ser gag aag Glu Lys cac aga ctc gag aaa tgt agg gaa ctc gag asc asc cac tcg His Arg Leu Glu Lys Cys Arg Glu Leu Glu Xaa Xaa His Ser 120 180 231 279 327 375 424 484 496 gcc cca gga tca acc cas cac cga aga aaa aca acc aga aga aat tat Ala Pro Gly Ser Thr Xaa His Arg Arg Lys Thr Thr Arg Arg Asn Tyr 35 40 tct tca gcc tgaaatgaak ccgggatcaa atggttgctg atcaragccc Ser Ser Ala atatttaaat tggaaaagtc aaattgasca ttattaaata aagcttgttt aatatgtctc aaacaaaaaa aa <210> 24 <211> <212> PRT <213> Homo Sapiens <220> <221> SIGNAL <222> 1. <223> Von Heijne matrix score seq ILSTVTALTFAXA/LD <300> <400> 24 Met Gly Ile Leu Ser Thr Val. Thr Ala Leu Thr Phe Ala Xaa Ala 1 5 10 <210> <211> 623 <212> DNA WO 99/25825 WO 9925825PCTIIB98/OI 862 7 <213> Homo Sapiens <220> <221> sig..peptide <222> 49. .96 <223> Von Heijne matrix <300> <400> aaagatccct gcagcccggc aggagagaag gctgagcctt Ctggcgtc atg gag agg Met Glu Arg ctc gtc Leu Val cta acc ctg tgc Leu Thr Leu Cys acg acg eca gct Thr Thr Pro Ala ace Thr tgc gcc Cys Ala gtc agc Val Ser ctc ccg Leu Pro aac ctg Asn Leu ccg ccc Pro Pro ctg get gtg geg tct Lou Ala Val Ala Ser gct ggc Ala Gly agc tgc tac Ser Cys Tyr acc tgg tgc Thr Trp Cys cag tgc ttc aag Gin Cys Phe Lys age tgg acg Ser Trp Thr caa Gin gag Giu 25 aac Asn agc ccg ctg Ser Pro Leu gac Asp gtc tgc ate Val Cys Ile tcc Ser agc Ser gag gtg gtc Giu Val Val gte Val1 45 ccc Pro cc t Ser ttt aaa tgg Phe Lys Trp agt gta Ser Val cgc gte ctg Arg Val Leu atg aak ttc Met Xaa Phe agg cgc tgc Arg Arg Cys etc Leu gaa Glu aaa cgc tgt Lys Arg Cys gc c Al a 60 ccc Pro aga tgt ccc Arg Cys Pro tgg tcg ccg gcc Trp Ser Pro Ala atg gtg caa.

Met Val Gin Cgt tcc tgg Cys Ser Trp 75 etc Leu ggc Gly ctg Le u aac gac aac Asn Asp Asn gtg atc ac Val Ile Thzaec cca, cag Thr Pro Gin 105 153 201 249 297 345 393 441 489 tgc aac agg Cys Asn Arg gag ggg Glu Gly cgc tgg gcc Arg Trp Ala ggg ggg ctc Gly Gly Leu 100 agg Arg ctg Leu.

110 ctg Leu cag gac cct Gin Asp Pro teg Ser 115 tge Cys ggc ara aaa, Gly Xaa Lys acc Thr 120 aac Asn gtg cgg cca Val Arg Pro cag Gin 125 rgg Xaa ggg etc cca Gly Leu Pro Cct. ccc awt Leu Pro Xaa ccc ctc tgc Pro Leu Cys gaa ace cag Glu Thr Gin gaa Glu taacactgtg ggcgccecca Cctgtgcatt gggaecacra taaaetctca tgcccceaaa aaaaaaaaa <210> 26 <211> 16 <212> PRT <213> Homo Sapiens <220> <221> SIGNAL <222> 1. .16 <223> Von Heijne matrix score 10.1 seq LVLTLCTLPLAVA/

SA

etteaccce ttggaracaa <300> <400> 26 Met Glu Arg Leu Val Leu Thr Leu Cys Thr Lou Pro Lou Ala Val Ala 1 5 10 <ZU Iz <211> 84 <212> DN <213> Ho <220> <221> si <222> 32 8

A

mo Sapiens g-.peptide .73 WO 99/25825 PCT/IB98/01862 <223> Von Heijne matrix <300> <400> 27 aactttgcct tgtgttttcc accctgaaag a atg ttg tgg ctg ctc ttt ttt Met Leu Trp Leu Leu Phe Phe ctg gtg act Leu Val Thr got ttt aaa Ala Phe Lys gcc att cat gct gaa ctc tgt caa Ala Ilie His Ala Glu Leu Cys Gin cca ggt gca gaa aat Pro Gly Ala Clu Asn tat Tyr gtg aga ctt Val Arg Leu 15 gat acc aat Asp Thr Asn agt atc aga aca Ser Ile Arg Thr gct Ala 20 ttc Phe ctg gga gat aa Leu Gly Asp Lys gca Ala gcc tgg Ala Trp gaa gaa tac Giu Giu Tyr aaa gcg atg Lays Ala Met ttc tcc atg Phe Ser Met gtc cta ctt Val Leu Leu aca gac cot Thr Asp Pro aga Arg tgc Cys aaa L.ys gta got Val Ala gtt ccc aac Val Pro Asn aga Arg 50 agg Arg gca aca gaa.

Ala Thr Giu att tcc cat Ile Ser His ttt gtg gtt Phe Val Val 52 100 148 196 244 292 340 388 436 aat gta ac Asn Val Thr cag Gin ac c Thr gta tca ttc Val Ser Phe t gg Trp tca aaa aat Ser Lys Asn gcc ata Ala Ile caC His 80 aac As n ctt oct got Leu Pro Ala gtt Val1 gcc Ala gag gtg caa tca Giu Val Gin Ser aga atg aac Arg Met Asn cgg atc aac Arg Ile Asn gac Asp aat Asn 100 tcc Ser ttc ttt cta Phe Phe Leu aat Asn 105 caa act ctg Gin Thr Leu gaa Giu 110 gtg Val1 tta aaa atc Leu Lys Ile oct Pro 115 Ile aca ctt gca Thr Leu Ala oca ccc Pro Pro 120 atg gac oca Met Asp Pro tgc atc atc Cys Ile Ilie 140 caa cgt ada Gin Arg Xaa tc t Ser 125 ata Ile ccc atc tgg att Pro Ile Trp Ile ata ttt ggt Ile Phe Gly gtt gca act Val Ala Ile gca Ala 145 gaa Glu 130 ota Leu otg att tta Leu Ile Leu tca, Se r 150 gat~ Asp gtg ata ttt Val Ile Phe 135 ggg atc tgg Gly Ile Trp gac gct gaa Asp Ala Giu ara aag aac Xaa Lys Asn cca tot gaa Pro Ser Giu 155 rat aak Xaa Xaa 170 tgt gaa aac Cys Glu Asn atg Met 175 aca att gaa Thn Ile Glu aat Asn 180 ga t Asp atc ccc tct Ile Pro Ser ccc ctg gac atg Pro Leu Asp Met aag gga ggg Lys Gly Gly 190 aoc cct oto Thin Pro Leu cat act His Ile goc ttc atg Ala Phe Met aca Thin gat gag agg Asp Giu Aing ctc Leu 205 tgaagggctg ttgttctgct tootcaaraa attaaacatt tgtttctgtg tgaotgctga gcatcctgaa atacoaagag oagatcatat wttttgtttc accattottc ttttgtaata aattttgaat gtgcttgaaa aaaaaaaaaa <210> 28 <211> 14 <212> PR' <213> Hoi <2 <221> SI4

T

mo Sapiens

GNAL

<222> <223> 1.-14 Von Heijne matrix score 10.7 seq LWLLFFLVTAIHA/EL <300> <400> 28 WO 99/25825 WO 9925825PCT/IB98/01862 Met Leu Trp Leu Leu Phe Phe Leu Val 1 5 <210> 29 <211> <212> DNA <213> <220> <300> <400> 29 gggaagatgg agacagtatt gcctg <210> <211> 26 <212> DNA <213> <220> <300> <400> ctgccatgta catgatagag agattc <210> 31 <211> 546 <212> DNA <213> Homo Sapiens <220> <221> promoter <222> 1. .517 <221> transcription start site <222> 518 <221> protein bind <222> 17. <223> matinsetr praito Thr Ala Ile His Ala <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> name CMYB-.01 score 0.983 sequence tgtcagttg protein bind complement(18. .27) matinspector prediction name MYOD..Q6 score 0.961 sequence cccaactgac protein bind complement(75. ratinspector prediction name S8-.01 score 0.960 sequence aatagaattag protein-..bind 94. .104 matinspector prediction name S8.01 score 0.966 sequence aactaaattag prote in-.bind complement(129. .139) matinspector prediction name DELTAEF1O01 score 0.960 sequence gcacacctcag protein-bind complement(155. .165) rnatinspector prediction name GATA_C score 0.964 sequence agataaatcca WO 99/25825 WO 9925825PCT/IB98/01862 -<221> protein-bind 1 <222> 170. .178 <223> matinspector prediction name CMYB-01 score 0.958 sequence cttcagttg <221> protein bind <222> 176. .189 <223> matinspeccor prediction name GATA1.-02 score 0.959 sequence tgtagataggaca <221> protein bind <222> 180. .190 <223> matinspector prediction name GATA-.C score 0.953 sequence agataggacat <221> protein-bind <222> 284. .299 <223> matinspector prediction name TAL1ALPHAE47O01 score 0.973 sequence cataacagatggtaag <221> protein bind <222> 284. .299 <223> matinspector prediction name TALlBETAE47-01 score 0.983 sequence cataacagatggtaag <221> protein bind <222> 284. .299 <223> matinspector prediction name TALIBETAITF2_01 score 0.978 sequence cataacagatggtaag <221> protein-bind <222> complement(287. .296) <223> matinspector prediction name MYOD_.Q6 score 0.954 sequence accatctgtt <221> protein-bind <222> complementC302. .314) <223> -matinspector prediction name GATAl-04 score 0.953 sequence tcaagataaagta <221> protein-bind <222> 393. .405 <223> matinspector prediction name IKI-01 score 0.963 sequence agttgggaattcc <221> protein-bind <222> 393. .404 <223> matinspector prediction name IK2..03 score 0.985 sequence agttgggaattc <221> protein_bind <222> 396. .405 <223> matinspector prediction WO 99/25825 WO 9925825PCTIIB98/0I 862 name CREL_01 score 0.962 sequence tgggaattcc <221> protein bind <222- 423.-436 <223> matinspector prediction name GATAl-02 score 0.950 sequence tcagtgatatggca <221> protein-bind <222> complement(478. .489) <223> matinspector prediction name SRY-02 score 0.951 sequence taaaacaaaaca <221> protein bind <222> 466. .493 <223> matinspector prediction name E2F..02 score 0.957 sequence tttagcgc <221> protein-bind <222> complement(514. .521) <223> matinspector prediction name MZF1_01 score 0.975 sequence tgagggga <300> <400> 31 tgagtgcagt tcttgatttg gttattgact gataggacat a tcaggagaa atactttatc gaattgagga catcagtgat tttgttttag cttcat <210> 32 <211> 23 <212> DNA <213> <220> <300> <400> 32 gttacatgtc cctgctaat gaggtgtgct tgatagatac aaaaatgaca ttgagtagga gtcagc tcag atggcaaatg cgctgctggg agttgggtta ctattatttc aatctcccat ataagtacca tctggaaaac gagccttcct t tagaagcag tgggactaag gcatcgcctt agtttgttaa tggaactaaa tatgtggatt ggacaaaagc ctatagggaa gtggcaacgt ggagt tggga ggtagtgatc gggtcccctc tgtcattcaa ttagtttgat tatctatttc agggagatct aggca taaca ggagaaggga attccgttca agagggttaa aaacagattc atcttctatg ggttctatta t tcagt tgta tttttccaaa gatggtaagg agaggtcgta tgtgatttag aattgtgtgt ccatgaatct 120 180 240 300 360 420 480 540 546 gtaccaggga ctgtgac4 <210> 33 <211> 24 <212> DNA <213> <220> <300> <400> 33 ctgtgaccat tgctccc <210> 34 <211> 861 <212> DNA <213> Homo Sapiens <220> <221> promoter <222> 1. .806 <221> transcriptio c.at tgc aag agag n start site WO 99/25825 WO 9925825PCT/IB98/01862 12 <222> 807 <221> protein-bind <222> complement(60. <223> matinspector prediction name NFY..QE score 0.956 sequence ggaccaatcat <221> protein-..bind <222> 70.,.77 <223> maitinspector prediction name MZF1O01 score 0.962 sequence cctgggga <221> protein-bind <222> 124. .132 <223> matinspector prediction name CMYB-01 score 0.994 sequence tgaccgttg <221> protein_bind <222> complement(126. .134) <223> matinspector prediction name VMYB-02 score 0.985 sequence tccaacggt <221> protein bind <222> 135. .143 <223> matinspector prediction name STAT_01 score 0.968 sequence ttcctggaa <221> protein bind <222> complement(135. .143) <223> matinspector prediction name STAT_01 score 0.951 sequence ttccaggaa <221> protein bind <222> complement(252. .259) <223> matinspector prediction name MZF1_01 score 0.956 sequence ttggggga <221> protein-bind <222> 357. .368 <223> matinspector prediction name 1K2_0.

score 0.965 sequence gaatgggatttc <221> protein_bind <222> 384. .391 <223> matinspector prediction name MZF2.-01 score 0.986 sequence agagggga <221> protein-bind <222> complement(410. .421) <223> matinspector prediction name SRY-02 score 0.955 sequence gaaaacaaaaca, <221> protein-bind <222> 592. .599 WO 99/25825 WO 9925825PCT/IB98/OI 862 <223> matinspector prediction name MZF1_01 score 0.960 sequence gaagggga <221> protein bind <222> 618. .627 <223> matinspector prediction name MYODQ6 score 0.981 seque~nce agcatctgcc <c221> protein-..bind <222> 632. .642 <223> matinspector prediction name DELTAEFl-01 score 0.958 sequence tcccaccttcc c221> protein..bind c222> complement(813. .823) -223> matinspector prediction name S8.01 score 0.992 sequence gaggcaattat :221> protein-bind :222> cornplement(824. .831) :223> matinspector prediction name MZF1..01 score 0.986 sequence agagggga <300> <400> 34 tactataggg tgattggtcc cggtgaccgt c tcagagggc ggagcatgcc aaytcagggc gggatttcag ccaaa tcaag agtcggaact caagcagtgt tgggttctcg tgcc tgagct ttggaaccca tcctgatggt tctcttggga <210> <211> <212> DNA <213> <220> <300> <400> ctgggatgga <210> 36 <211> <212> DNA <213> <22 0> <300> <400> 36 gagaccacac <210> 37 <211>. 555 cacgcgcggt c tggggaagg tggattcctg taggcacgag t tcccccaac ccaascagaa gttagncagg gtaacttgct ccctaccact gagaacatgg cccaaagagc gtttggacaa atacctaggc cctttaggtt gcaatggtca cgacggccgg tctggctggc gaagcagtag ggaaggtcag cctggcttsc scacaggccc gtgagagggg cccttctgct ttcaggagag ctggtagagg atctgcccat aaatccaaac ttaCaggcca tgggcacaaa c gctgttctgg tccagcacag ctgttctgtt aggagaaggs yc ttggymam aktcntggct aggctctctg acgggccttg tggttttagg ctctagctgt ttcccacctt cccacttggc tcctgagcca atataattgc agcagagggc tgaggcattt tggatctggt aggsarggcc agggcgkt ty sma agca ca a gcttagtttt gtcttggctt cccgtggggc gtgcggggcc cccttctccc tactctggcc ggggcctctg ctc tcccctc atgtcagtaa aggtatctct agggacaggg cagtgagarg tgggmactcr tagcctgaat gttttgtttt gtcctcaccc tgttctgttc tgaaggggag accagaagct tggc ttcagc gaaattctct tcccattttc aggcacggta agc tagacaa WO 99/25825 PCT/IB98/01862 14 <212> DNA <213> Homo Sapiens <220> <221> promoter <222> 1..500 <221> transcription start site <222> 501 <221> protein_bind <222> 191..206 <223> matinspector prediction name ARNT_01 score 0.964 sequence ggactcacgtgcgc <221> proteinbind <222> 193..204 <223> matinspector prediction name NMYC_01 score 0.965 sequence acccacgtgctg <221> protein_bind <222> 193..204 <223> matinspector prediction name USF_01 score 0.985 sequence acccacgtgctg <221> protein_bind <222> complement(193..204) <223> matinspector prediction name USF_01 score 0.985 sequence cagcacgtgagt <221> protein_bind <222> complement(193..204) <223> matinspector prediction name NMYC_01 score 0.956 sequence cagcacgtgagt <221> protein_bind <222> complement(193..204) <223> matinspector prediction name MYCMAX_02 score 0.972 sequence cagcacgtgagt <221> protein_bind <222> 195..202 <223> matinspector prediction name USF_C score 0.997 sequence tcacgtgc <221> protein bind <222> complement(195..202) <223> matinspector prediction name USF_C score 0.991 sequence gcacgtga <221> proteinbind <222> complement(210..217) <223> matinspector prediction name MZF1_01 score 0.968 sequence cacgggga <221> proteinbind <222> 397..410 WO 99/25825 <223> matinspector prediction name ELK1_02 score 0.963 sequence ctctccggaagcct <221> protein-bind <222> 400.-409 <223> ratinspector prediction name CETSlP54..01 score 0.974 sequence tccggaagcc <221> protein_bind <222> complement(460. .470) <223> matinspector prediction name APIQ4 score 0.963 sequence agtgaccgaac <221> protein.bind <222> complement(460. .470) <223> matinspector prediction name APIFJ.Q2 score 0.961 sequence agtgactgaac <221> protein_bind <222> 547. .555 <223> ratinspector prediction name PADS-.C score 1.000 sequence tgtggtctc <300> PCTJIB98/01862 <400> 37 ctatagggca cgcktggt aggacagcat ttgtkaca kawaagctca gcaccggt aggaactgac ggactcac gagcagtcag acagtgcc cattcctgtc tgcattaq gttgctctgc ccatggtc cgtgtcttct gcctgcc ttttgcctcc tcaatttc tagctgtgtg gtctc <210> 38 <211> 19 <212> DNA <213> <22 0> <300> <400> 38 ggccatacac ttgagtgz <210> 39 <211> 19 <212> DNA <213> <220> <300> <400> 39 atatagacaa acgcacac <210> 40 <211> 1098 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 173. .2311 .cg tc .gc .gt tg ~ta .cc .cc .tc acggcccggg tggtc tactg ccatcacagg gctgctccgt ggatagagtg actcccaacc actgcagacc gctcacatcc ttgtcttagt ctggtctggt caccttccct gccggcagca ccccatgagc agagttcagc tagatgtgaa caggcactct cacacttgtg cccatcctct ctgtkgtgga ctgccgtgca cacacatccc tcagtggacc cagtaaatcc aacttagttc ccggaagcct ttcagtcact gttcccctgg gtcgggttga cttggccttt attactcaga tgtctatgta aagtgattgt tttctcatag ggaaatcacc gagttacaga ccagtttgtc 120 180 240 300 360 420 480 540 555 19 19 c :c WO 99/25825 WO 9925825PCTIIB98/01862 16 <223> Von Heijnd matrix score 4.19999980926514 seq MLAVSLTVPLLGA/MM <221> polyA..signal <222> 1063. .1068 <221> polyAsite <222> 1087.,.1098 <221> misc-.feature <222> 144. .467 <~223> homology id :AA057573 est <221> misc-feature <222> 510. .640 <223> homology id :AA057573 es t <221> misc-feature <222> 436. .523 <223> homology id :AA057573 es t <221> misc-feature <222> 708. .786 <223> homology id :AA057573 est <221> misc-feature <222> 635. .682 <223> homology id :AA057573 es t <221> misc-feature <222> 625. .1084 <223> homology id :N57409 est <221> misc-feature <222> 779. .1084 <223> homology id :R71351 est <221> misc-feature <222> 144. .506 <223> homology id :H12619 est <221> misc-feature <222> 90. .467 <223> homology id :T03538 est <221> misc-feature <222> 314. .523 <223> homology id MT4150 est <221> misc-feature <222> 567. .687 <223> homology id :T34150 est <221> misc..feature WO 99/25825 WO 9925825PCT/1B98/O1 862 <222> 686. .730 <223> homology id :T34150 est <221> misc..feature <222> 510. .553 <223> homology id :T34150 es t <221> misc-feature <222> 550. .579 <223> homology id :T34150 es t <221> misc..feacure <222> 144. .523 <223> homology id :N32314 es t <221> misc...feature <222> 510. .553 <223> homology id :N32314 es t <221> misc-feature <222> 352. .523 <223> homology id :T77966 es t <221> misc-.feature <222> 218. .351 <223> homology id :T77966 <221> <222> <223> <221> <222> <223> es t misc..jeature 510.-.553 homology id :T77966 es t misc-feature 550. .917 homology id :AA464128 es t <300> <400> agtgaggtgg tttctgcggg tgaggctggc gcecgtacca cgacagcgcc ggcccctgeg gccegcaagt egteacagac ggctaaggac ggcagctcct ttagcggcag agttttccga gct gtt Ala Val tct cet Ser Pro tct cte acc gtt ccc ctg ctt gga gcc Ser Leu Thr Val Pro Leu Leu Gly Ala tgagegaggc ggacgggctg gatgatggcc aggccccgga gtgaccttct tg atg ctg Met Leu atg atg ctg ctg gaa Met Met Leu Leu Glu 1 gaa ccc ccg ctc ttg Glu Pro Pro Leu Leu ata gat cca cag ect etc agc Ile Asp Pro Gin Pro Leu Ser 120 178 226 274 322 370 aaa Lys ctt ggt gtt ctg cat cca aat aeg Leu Gly Val Leu His Pro Asn Thr aag Lys ctg cga cag gca Leu Arg Gin Ala gaa Glu agg ctg Arg Leu ttt gaa aat caa ctt gtt gga ceg gag tee ata Phe Glu Asn Gin Leu Val Gly Pro Glu Ser Ile 45 gtg atg ttt act ggg aca gee get gge egg gte gca cat att ggg gat Ala His Ile GJly ASP gte aaa ctt gee aat 418 WO 99/25825 WO 9925825PCTIIB98/01862 Val Met Phe Thr*Gly Thr Ala Asp Gly 60 Arg Val Val. Lys Leu Giu Asn gg t Gly cga Arg gaa ata gag acc att gcc cgg ttt ggt Giu Ilie Glu Thr Ilie Ala Arg Phe Gly tcg Ser 80 ctg Leu ggc cct tgc aaa Gly Pro Cys Lys acc Thr ggt gat gag cct gtg tgt ggg aga Gly ASP Glu Pro Val Cys Gly Arg ccc Pro 95 tgc Cys ggt atc cgt Gly Ile Arg ggc agg Gly Arg 100 tat ttg Tryr Lou gcc caa tgg Ala Gin Trp gac Asp 105 tct ctt tgt ggc Ser Leu Cys Gly cga A.rg ata caa agg Ile Gin Arg gac Asp 110115 aag taaatccctg goaacgtgaa gtgaaactgc tgctgtcctc cgagacaccc attgagggga atttatctca gagggcacag ctattggacc ctggtggcag ggcggggc tg agctccgggg ctggacttct aaa agaaca tgtc ccgattctag a tgacgggcg agctgcggtt aaacaaccat atctgtttgt ggtac tgggt tatctgagag Ctttgtgaat cagcaaa tgg cctgctggag cccgaatgga ggccaggata ggagaacatg gggcatgtcg accctggatt gatcttacag caaagacgag tatgatactg gtCCagctgt cgaagagtct Cctggatttc accatccgcc aaaaggatga acactcagga &ctacctgct tgaccaggga ctcctgcaga acgtttctgg cagacaacat ctaaccctgg tttttaangg tgggaggaag tctggtgatg agtaaaagtt agactttgc cctgatgaag ccggcccagc gttttccatg taaaaaaaaa <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> 41 855

DNA

Homo sapiens sig..peptide 267. .371 Von Heijne matrix score 5.90000009536743 seq LCGLLHLWLKVFS/LK PolyA.s ignal 817. .822 P0iYA-.Site 842. .855 misc-feature 608.-811 homology id :M85769 est 466 514 562 615 675 735 795 855 915 975 1035 1095 1098 120 180 240 293 341 389 437 485 <300> <400> 41 acaatcagt t tcagtcagat catccc taca accgaacagg gtagcagtgg tat aga aa Tyr Arg As ctg tta ca Leu Leu Hi tgccaatacc tc actagtcaat at tgacagtgac aa aacagcacaa cc ttcagcacac tt t gtc agg tcc n Val Arg Ser :agaaacaa :caaatcat ~tgatgaac :tgggaccc :tggt atg Met atacctcgga caaatctttc tctaaagacc gtagatggcg gcattttagg cctcggacac tctcctgtag aaaattatat aggagtataa agacatgcag tacctctacg caaagtaaaa ttg act gtt aat gat gta cgt ttc Lou Thr Val Asn Asp Val Arg Phe aac cat ttc cca ttt gtt Asn His Phe Pro Phe Val -20 cga cta tgt ggt Arg Lou Cys Gly t tta tgg ctt aaa gtc ttt tct ctt aaa cag tta s Leu Trp Leu Lys Val Phe Ser Lou Lys Gin Leu aaa Lys tat Tyr aaa aaa Lys Lys 5 tct. tgg tct aag tat tta ttt gaa tcc tgt tgc tat agg agt ttg Ser Trp Ser Lys Tyr Leu Phe Glu Ser Cys Cys Tyr Arg Ser Leu 15 gtg tgt gtc ttc att taaacatacc tgcatacaaa gatggtttat Val Cys Val Phe Ile ttctatttaa tatgtgacat ttgtttcctg -gatatagtcc gtgaaccaca agatttatca WO 99/25825 PCT/IB98/01862 19 ccgtaaattg ttaaccattt tatgttcaga tatttttcaa taatatgaga agaaaatggg 605 tatttctcta gtttttacct agtttgcttt Cataacctta tatgttgaca caataattca cagagaagaa catttaaagg gttaatattt ttattgtggc ttctatttga aatgtgtcta aaaaaaaaaa <210> 42 <211> 1176 <212> DNA <213> Homo sapiens <220> <221> sig.peptide <222> 174..266 <223> Von Heiine marix aacatagaga Ccagcaagtg aatatatatg gaataaettg ttaaagataa actaattttt ttgaaacgtt ttcagataat atctatttga aaataaaatg Ctgtttattt aaaatgaaaa 665 725 785 845 855 <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> score seq WSPLSTRSGGTHA/CS polyAsignal 1144..1149 PolyAsite 1165..1176 misc-feature 886..1134 homology id :AA595193 est misc-feature 756..894 homology id :AA595193 est misc-feature 655..755 homology id :AA595193 est misc-feature 167..367 homology id :W81213 est misc-feature 66..172 homology id :W81213 est misc-feature 429..508 homology id :W81213 est misc-feature 756..894 homology id :AA150887 est misc-feature 536..643 homology id :AA1S0887 est <221> miscfeature <222> 655..755 WO 99/25825 PCT/IB98/01862 <223> homology* id :AA150887 est <221> misc-feature <222> 429. .643 <223> homology id :AA493644 es t <221> misc-feature <222> 655. .755 <223> homology id :AA493644 est <221> misc-.feature <222> 429. .643 <223> homology id :AA493494 est <221> misc-feature <222> 655. .755 <223> homology id :AA493494 est <221> misc-feature <222> 500. .643 <223> homology id :AA179182 est <221> misc-.feature <222> 655. .755 <223> homology id :AA179182 es t <221> misc-feature <222> 756. .847 <223> homology id :AA179182 est <221>- misc..feature <222> 3. .338 <223> homology id :HUM524FO5B es t <221> misc_feature <222> 334. .374 <223> homology id :HtM524FO5B es t <221> misc-feature <222> 886. .1134 <223> homology id :AA398156 est <221> misc-feature <222> 756. .894 <223> homology id :AA398156 es t <300> <400> 42 aaaaacaata ggacggaaac gccgaggaac ccggctgagg cggcagagca tcctggccag aacaagccaa ggagccaaga cgagagggac acacggacaa acaacagaca gaagacgtac 120 tggccgctgg actccgctgc ctcccccatc tccccgccat ctgcgcccgg agg atg 176 WO 99/25825 c cca gcc ttc r Pro Ala Phe 0 c tgg agc cct e Trp Ser Pro PCT/IB98/01862 agg gcc atg gat Arg Ala -25 ttg tcc Leu Ser Met Asp acc agg Thr Arg gct tca atg Ala Ser Met agt tct acg Ser Ser Thr aga caa Arg Glrn aga ccc Arg Pro Met gtg gag ccc cgc Val Glu Pro Arg -20 tcg ggg ggc act Ser Gly Gly Thr -5 agc ccc tgg tcc Ser Pro Trp Ser aga tgc gca aat Arg Cys Ala Asn gcc aaa ggc Ala Lys Gly cat gcg tgc His Ala Cys 1 caa ggg aac Gin Gly Asn tct ctc ccc Ser Lcu Pro tcc Se r t cc Ser a tc Ile agc Ser ccc tgg gca Pro Trp Ala 10 tcc ctg ctg Ser Leu Leu aca aag Thr Lys gac aaa gcc Asp Lys Ala att Ile aaa Lys 40 cc t Pro 25 ggc Gly ccc ttg tta Pro Leu Leu gc t Ala 45 cac His ggc Gly cat ccc tgc His Pro Cys ccc Pro s0 ttt tcc cct Phe Scr Pro ttc ccc tgt Phe Pro Cys ggc Gly 60 cca Pro agg gaa gtg Arg Glu Val tgg cct Trp Pro gaa tac ccc Glu Tyr Pro gaa gtg tca Glu Val Ser ctg agc aga Leu Ser Arg acc Thr tct Ser gct cct ctg Ala Pro Leu gag ctg ggg Glu Leu Gly ctc tct gag Leu 5cr Glu cac His 90 ttc ccc tgc Phe Pro Cys agc Ser gag Glu gcc acc tca Ala Thr agt cga gga Ser Arg Gly ang aaa ggt Xaa Lys Gly 224 272 320 368 416 464 512 560 608 656 712 772 832 892 952 1012 1072 1132 1176 ttg agt gat Leu Ser Asp 100 gtt cag Val Gin gct Ala 105 aag Lys cca gtc gtt Pro Val Val ggg gca gan adg cct Gly Ala Xaa Xaa Pro gcg ctc gkc Ala Leu Xaa 110 act Thr gct gaa acg Ala Glu Thr 115 ccc Pro 130 cca ccc tgacagcccc atcctcaaag actgtcttaa ttactcatgg caggttctag Pro Pro agacttaagg ggaaaag statctgcct wgtgttc catattttat aagaaca ttctgmtagg gtgtctt ttacccagtc ttcccyt ctgtattcag gstytgc ttaaaaattc ccgcaaa4 acataaaaat taataaai <210> 43 <211> 648 <212> DNA <213> Homo sapiens <220> <221> sig-.peptjde <222> 460. .555 <223> Von Hei-ine m ctg att ~aaa gtg amt ttt cta cat ct ttcaaggc t tgytat tt t ggcytgggtg c tg t rtggyt yttggatgs t aaagcaagcc taaagagcaa tttcaatgat caccacatgt gtgasgtgag cc tacccgkg tgttttgttt tyt taaccc t atgaggctgt tgttttcagt ggaaaaaaaa ctggtgctcc acagcaaaga tgggggcacw gccccyttat caggcaaacc tggagtttct yttttaggat aaaa ccznaccags t ccaataaaaa gtgggaagcc tttgctttgc tgtgttcccc gtttagggca tagaagaat t score seq F <221> polyA <222> 614..

<221> polyA <222> 635..

<300> <400> 43 aattctggcc tccttagagt taagactcat cccttttcta tggccttgga caaatgccag atrix

*SFMLLGMGGCLP/GF

-s ignal1 619 -si te 648 cagcttcttc tctccctcca gctacaagaa c tgagaggaa attaaaccac ttacggtgat cccagctcta ttagtagttg gttaaataag gtggaatgca caccaacaca gcgttcaaca tcctgcttcc tcttagggtc tttcccgaag c tccgacaag cttttggatt tcc ttat ttc ctccatctcc tgtttctggg tcacacagct gataaggttt atcagaaggt cagtctttat tataggattc gagccctgcc agcctctcat tattgtgagc ggaaggagtg gacgcctttc WO 99/25825 PCT/IB98/01862 22 ctgaatcaca ggtgcfattgg ggtgcttcct cctccccagg actcccaccc aactttgtga 420 acacaaccca cttagaggag ttatctcagc acattatga atg ttg ggg acc acg 474 Met Lou Gly Thr Thr ggc ctc ggg aca cag ggt cct tcc cag cag gct ctg ggc ttt ttc tcc 522 Gly Leu Gly Thr Gin Gly Pro Ser Gin Gin Ala Leu Gly Phe Phe Ser -20 ttt atg tta ctt gga atg ggc ggg tgc ctg ccc gga ttc ctg cta cag 570 Phe Met Lou Leu Gly Met Gly Gly Cys Leu Pro Gly Phe Leu Lou Gin -5 1 cct ccc aat cga tct cct act ttg cct ga tcc acc ttt gcc cat 615 Pro Pro Asn Arg Ser Pro Thr Leu Pro Ala Ser Thr Phe Ala His 15 taaagtcaat tccccaccca taaaaaaaaa aaa 648 <210> 44 <211> 1251 <212> DNA '213> Homo sapiens <220> <221> sigpeptide <222> 79. .369 <223> Von Heijne matrix score 4 seq RLPLVVSFIASSS/AN <221> polyA.signal <222> 1217. .1222 <221> polyA..site <222> 1240. .1251 <221> misc-feature <222> 2. .423 <223> homology id :AA056667 est <221> misc-feature <222> 463. .520 <223> homology id :AA056667 es t <221> misc-feature <222> 418. .467 <223> homology id :AA056667 est <221> misc-feature <222> 159. .636 <223> homology id :AA044187 est <221> miscfeature <222> 629. .684 <223> homology id :AA044187 est <221> misc-feature <222> 5. .453 <223> homology id :AA131958 est <221> misc-feature <222> 446. .494 <223> homology id :AA131958 WO 99/25825 WO 9925825PCT/IB98/OI 862 23 est <221> misc-feature <222> 14.-.343 <223> homology id :W95957 es t <221> misc-feature <222> 323. .467 <223> homology id :W95957 est :2,2 1> misc..feature <222> .463...494, <223>. hom6olb~y*K id :W95957 es t <221> misc..feature <222> 14. .475 <223> homology id :W95790 es t <221> misc-feature <222> 410. .876 <223> homology id :AA461134 es t <221> misc-feature <222> 974. .1195 <223> homology id :AA595195 es t <221> misc-feature <222> 769. .982 <223> homology id :AA595195 est <221> misc-feature <222> 1208. .1237 <223> homology id :AA595195 es t <221> misc-feature <222> 223. .522 <223> homology id :AA041216 es t <221> misc-feature <222> 518. .636 <223> homology id :AA041216 est <221> misc-feature <222> 774. .1127 <223> homology id :N494607 est <221> misc-feature <222> 690. .765 <223> homology id :N494607 est <221> misc-feature <222> 833. .1195 WO 99/25825 WO 9925825PCTIIB98/01862 <223> homology id :AA076410 est <300> <400> 44 aaagtgacag gagagaaggg cggagagaac caggsagccc agaaacccca ggcgtggaga ttgatcctgc t.frP~ Id v '-6 aag tta Lys Leu got gga Asp Gly cca agt ge.t Pro Ser Val gta cct gtt Val. Pro Val Met gaa Giu w-y gat Ala Asp -95 ggg etc Gly Leu -80 gac cta aag ega Asp Leu Lys Arg ttc Phe gtg ValI ttg tat aaa Leu Tyr Lys tca gat ago Scr Asp Arg cat gcc att His Ala Ile gtt Val1 gc t Ala ttg cga cct Leu Arg Pro att aaa Ile Lys -65 ttc tta Phe Leu ctt tcc Leu Ser *gtg gca aat Val Ala Asn tcc act ttt Ser Thr Phe -45 aaa aat aaa Lys Asn Lays gac Asp gcc Ala at gct cca gagi Asn Ala Pro Glu cat His ctt gca aca Leu Ala Thr -55ca Acp caa AspGi gga. agc aaa Gly Ser Lys aac acc tac Asn Thr Tyr ttc ata gcc Phe Ile Ala aag gag ctt LYS Glu Leu ctt Leu cag Gin agt atc atc Ser Ile Ile gtg gtt caa Val Val Gin ttt Phe -15 aat Asn -30 aat Asn cgt tta cct Arg Leu Pro t tg Leu gtc Val1 agc agc agt gcc Ser Ser Ser Ala 1 tgt tac tat Cys Tyr Tyr gtg gtg agt Val Val Ser agc cta gaa Ser Leu Glu gtg gaa att Val Glu Ile aca gga cta att Thr Gly Leu Ile gct cca Ala Pro 15 ttg ttt gaa gaa Leu Phe Giu Glu ctg aga caa gtt Leu Arg Gin Val 159 207 255 303 351 399 447 500 560 620 680 740 800 860 920 980 1040 1100 1160 1220 1251 20 tct taatctgaca gtggtttcag tgtgtacctt atcttcatta taacaacaca Ser atatcaatcc agcaatcttt cccccttttc caacttatac tgctatatt tctggtgta atggttcagt ctatcacagc ttctattcag tggattagaa ccaattgtac aatatgccca taaggacata tttcttca atggcttggt aaaagtaata ttgcagtata gatgaatatt atgaaaatga aaattatagc gtgttttaat gtttgtgtgt atgttagtat gtatgtaaac aaatagtatt tttaaaagta <210> 45 <211> 1524 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 160. .231 agactacaat taaagaacta aggtctttct tcccatggag tcaaactggt ggcttgcaga gattatgttt aaatcagtac actaatcagt taatgttttt tcttcataaa atgatagtac aaaaaaaaaa aatgctttta gcatatagat tatttagtga t tag tc tgg t acattgatcc a taaagccaa tatttctttg aatcactaac ttgattattc tcctcaaact atttaaatac agccattttt tccatgtgc t gtaatttata gatctaggga caccagatat acttgagccg ctttttattg cattgagtga tttcctttgt tcagagggtg ctgctttctg aattcgttat ttcatatgtg caagaaaggg gatagatcag taccacagaa gga tgagaga ttaagtgctg tgaataataa ggtacataaa acatattatt Ctgctcttta taaccaatca tctgtttcca agtaaaaata <223> Von Heiine matrix score 5.69999980926514 seq ILGLLGLLGTLVA/ML <221> polyA~signai <222> 1510.-1515 <221> polyA-site <222> 1506. .1519 <221> misc-feature <222> 1048. .1504 <223> homology id :AA552647 WO 99/25825 WO 9925825PCT/1B98/OI 862 <221: <222- <223> <221> <222> <.223> <221> <222> <223> <22 1> <222> <223> <221> <222> <223> <221> <222> <223> es t misc-feature 597. .846 homology id :AA345449 est misc-.feature 39. .93 homology id :AA345449 est misc-feature 113. .149 homology id :AA345449 est misc-feature 98. .400 homology id :T86266 est misc-feature 1210. .1489 homology id :T86158 es t mi sc..feature 954. .983 homology id :AA1167O9 es t <300> <400> agccgcttgt actctgaaat ggagcagtcc ggccacccac gassgattag ctgaagacgc agacacttgt aaggaggaga gaagtcagcc tggcagagag aggtgctcaa ggragcaaag agcttcagcc tgaagacaag ttctactgag aggtctgcc atg gcc tct ctt ggc Met Ala Ser Leu Gly ctc caa ctt gtg Leu Gin Leu Val Ctg gtt gcc atg Leu Val Ala Met ggc Gly ctg Leu aca Thr tac atc cta Tyr Ile Leu Ctc ccc agc Leu Pro Ser 5 gca gtt ggc Ala Val Gly ggc ctt Gly Leu -10 tgg aaa Trp Lys ttc tc Phe Ser ctg ggg ctt t-tg Leu Gly Leu Leu aca Thr gcc agc Ala Ser tgt gcc CYS Ala 1 att gtg Ile Val aca cac Thr His aca agt tct Thr Ser Ser aag ggc ctc Lys Gly Leu tat gtc ggt Tyr Val Gly tgg atg gaa Trp Met Glu 20 agc aca ggc Ser Thr Gly atc acc cag Ile Thr Gin gac Asp atc tat agc Ile Tyr Ser ctt Leu 120 174 222 270 318 366 414 462 510 558 606 Ctg ggc ctg Leu Gly Leu 35 gc t Ala gac atc cak Asp Ile Xaa gc t Ala 55 tgc Cys cag gcc atg Gin Ala Met atg gtg Met Val aca tcc agt Thr Ser Ser atg ara tgc Met Xaa Cys gcg gta gca Ala Val Ala tcc tcc ctg Ser Ser Leu gcc Ala 70 gaa Glu att atc tct Ile Ile Ser gtc ttc tgc Val Phe Cys cag Gin 85 t tc Phe tcc cga gcc Ser Arg Ala aaa Lys ctc Leu gtg gtg ggc Val Vai Gly gac aga gtg Asp Arg Val ctg gga ttc Leu Gly Phe att cct gtt ggt gga gtc Gly Gly Val gcc tgg aat ttt Phe 100 ctt atc ctt gga Ile Leu Gly ggc Gly 105 cgg cat ggg atc cta gac ttc tac tca WO 99/25825 PTI9/16 PCT/IB98/01862 Ile 110 cca Pro Pro Val Ala'Trp Asn Leu His Gly ctg geg cct Leu Val Pro gac Asp 130 tee Ser atg aaa ttt Met Lys Phe Ile Leu Arg Asp 120 att gga gag get Ile Gly Glu Ala Phe Tyr Ser gag Giu 135 c tg Leu 125ta ccc Tac 1e40 y tcg ggc at Leu Gly Ile tge ccc tcc Cys Phe Ser 160 cac caa gc Tyr Gin Ala at Ilie 145 ccc Ctg tcc Ser Leu Phe tcc Ser 150 aca get gga Ile Ala Giy tgC tea tcc cag Cys Ser Ser Gin aat cgc ccc aac Asn Arg Ser Asn tac Tyr 170 agg Arg ac ate cc Ile Ile Leu 155 tac gat gcc Tyr Asp Ala ccc 99gceaa Pro Gly Gin 702 750 798 846 caa cct ct Gin Pro Leu 175 Cctcc c Pro Pro gee Ala 180 gag Glu agg age cc Arg Ser Ser aaa gee aag Lys Val Lys ag c Ser 195 tcc aat cc Phe Asn Ser tac Tyr age ccg aca ggg Ser Leu-Thr Gly 190 gCg cgaagaacca ggggecagag etggggggcg gcgggeceg tgaaaaacag Val1 tggacagcac eecgagg etgcgaggg tagacg gcaaeageac geaggec caeccgcg etcccc gccaggamcc agaggac ccaatccaa eccacg gscyccagct cacegee.

aaecamcey eeaagce yttgttacga etccaca aeggcatcea gggaaca eatttaaaaa aacaaaa <210> 46 <211> 610 <212> DNA <213> Homo sapiens <220> <221> sig...pepcide <222> 106. .201 gee act gaa gee ccc ace ggg tee cg gaa aaa aeaggcgagg ceggeeaccg ttgecaagga ccaagtcccc tYtgcccck gacccctgc ga tgggaagg e teeaaagaa tceagamcaa ageagga ege aaaaa gacaccacca gaccgagcaa tgctcgccat aacccecaac ggcccamccg gaccaaagae agaagcagcg amtgaecggc tctgcgcacg aggatgggag ctggacgeg aggeagaaa c gecagececct e cgaaac ccc ggacteac ceteeetccg gccccys egg cccggaaec aaccgaaaea gacaggaagg tcagaaggeg gggggctagt etgtttcce atccctcaa cceaaaeca gcgaggccg geattcet ecateecact aaaeca te t cagcctggga 959 1019 1079 1139 1199 1259 1319 1379 1439 1499 1524 <223> Von Heijne matrix score 8.80000019073486 seq VPMLLLIVGGSFG/LR <221> polyA signal <222> 577. .582 <221> polyA_site <222> 598. .610 <221> misc-feature <222> 68. .167 <223> homology -id :AA531561 est <221> misc-.feature <222> 166. .262 <223> homology id :AA531561 est <221> misc-feature <222> 423. .520 <223> homology id :AA531561 <221> <222> <223> est misc-feacure 518. .564 homology id :AA531561 WO 99/25825 WO 9925825PCTIIB98/OI 862 27 est <221> misc-feature <222> 276.-313 <223> homology id :AA531561 est <221> misc-feature <222> 41. <223> homology id :tA531561 est <221> misc..feature <222> 41. .262 <223> homology id :AA535454 est: <221> misc-feature <222> 423. .520 <223> homology id :AA535454 es C <221> misc-feature <222> 518. .564 <223> homology id :AA535454 est <221> misc-feature <222> 276. .313 <223> homology id :AA535454 est <221> misc-feature <222> 46. .262 <223> homology id :H81225 es t <221> misc-feature <222> 2. .39 <223> homology id :H81225 es t <221> misc-feature <222> 455. .493 <223> homology id :H81225 est <221> misc-feature <222> 276.-313 <223> homology id :H81225 est <221> misc-feature <22-2> 423. .458 <223> homology id :H81225 est <221> misc-feature <222> 53. .262 <223> homology id :AA044291 est <221> misc feature <222> 423. .520 WO 99/25825 WO 9925825PCT/IB98/01862 <223> homology id :AA044291 <221> <222> <223> <221> <2 22 <223> <221> <222> <223> est misc-.feature 518. .564 homology id :AA044291 est misc..feature 276. .313 homology id :AA044291 est misc-.feacure 125. .262 homology id :W47031 est <300> <400> 46 aaagtgagtt aaggacgtac gcgctaggcc cgcttggagt tcgtcttggt gagagcgtga tctgagccga tggaagagct stgccgagat ttgggagtct cactc atg ttt gca ccc Met Phe Ala Pro gcg gtg atg Ala Val Met ccc atg ttg Pro Met Leu tct caa atc Ser Gin Ile cgt Arg Leu gct ttC cgc aag Ala Phe Arg Lys aac Asn -20 ggt Gly aag Lys tc t Ser act ctc ggc Thr Leu Gly ttt ggt ct Phe Gly Leu tat gga gtc Tyr Gly Val cgt gag ttt Arg Glu Phe 117 165 213 261 ctg att gtt Leu Ile Val cga tat gat Arg Tyr ASP 10 gct gtg aag agt Ala Val Lys Ser atg gat cct gag Met Asp Pro Glu gaa Glu aaa aaa ccg LYS LYS Pro aaa Lys gag aat aaa eta tct Glu Asn Lys Ile Ser gag tcg gaa Glu Ser Glu gga agt etc tgt tgaagggcta Ctatctttcc ttggcccrttc tccctrtgttg Gly Ser Ile Cys ggactc ttggga gggaag tgactc aaaaa <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> :aatc Laaat ratc tgc t Laaa 47 tccagactat ctccccagag aatcttgtca caaagactcc aagtttgatg actggaagaa tgacctcctc caaggaagaa atccaggaaa gattcttttt tccttttttt ttttaaataa aggcttggct ttaagctttg tattcgagga cccaggcctt gccttaagac taagacaact aaatactatt aactggaaaa 1370

DNA

Homo sapiens Sig-peptide 359. .466 Von Heijne matrix score 7.80000019073486 seq LTFLFLHLPPSTS/LF polyA..signal 1334. .1339 polyA_site 1357. .1370 misc..feature 113. .420 homology id :R79290 est WO 99/25825 WO 9925825PCTfIB98/01862 29 <221> misc featUre <222> 406. .482 <223> homology id :R79290 est <221> znisc-feature <222> 199. .420 <223>- homology id :R81173 esr- <221> misc-feature <222> 406. .514 <223> homology id :R81173 es t <221> misc-feature <222> 2. .269 <223> homology id :R81277 es t <221> misc..feature <222> 406. .646 <223> homology id :R74123 est <221> misc..feature <222> 647. .682 <223> homology id :R?4123 es t <221> misc-feature <222> 439. .646 <223> homology id :AA450228 est <221> misc-feature <222> 647. .739 <223> homology id :AA450228 est <221> misc-feature <222> 406. .646 <223> homology id :R02473 es t <221> misc-feature <222> 406. .604 <223> homology id :T71107 est <221> misc-feature <222> 71. .282 <223> homology id :C06030 est <221> misc-feature <222> 319. .365 <223> homology id :C06030 est <221> misc-feature <222> 2. .57 <223> homology WO 99/25825 WO 9925825PCTIIB98/O1 862 <221~ <222- <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <22 2> <223> <221> <222> <223> id :C06030 est misc-feature 1173. .1277 homology id :N54909 est misc..feature 1080. .1177 homology id :N54909 est misc-feature 1273. .1356 homo logy id :N54909 est misc-feature 1173. .1277 homology id :AA196824 est misc-feature 1080. .1177 homology id :AA196824 est misc-feature 1273. .1356 homology id :AA196824 est <300> <400> 47 acaaggcaga CCtttgctgg cc tgt tecac taacagacc t caggtcattt tagtggggtg atg aac ac Met Asn Th ccc att tg~ Pro Ile Tr gcttctgaat ti aggaaggtac a caagtgagag a~ ccctacagct gi ggagaacaag t~ gaattcagaa g~ httt gag cca r Phe Glu Pro tcaggcctt cattccagag cagggtgaa aggtactta taggaacta gctttagta aaatttgaa gctgawgs tg ctcttgtacc ctgtcccaga gtagtttaaa gaccagatca CCCtCtcgtg tacttggggg tcctgttcag gc tgaggcaa gtagtaactg tgggtggtct gccaggcctt atctccttgg ccaggtgcat ggggatttct ctactgtatt gac Asp -30 gcC Ala agc ctg gct gtc Ser Leu Ala Val att Ile ttt Phe gct ttc ttc ctc Ala Phe Phe Leu acc ttc cca Pro SThr Phe~ tcc acc agt cta Ser Thr Ser Leu tct Ser -15 ttt Phe ctt aca ttt Leu Thr Phe ctc cat cta Leu His Leu att aac tta gca Ile Asn Leu Ala cct ctt ggc Pro Leu Gly tgc gac ttt Cys Asp Phe

I

ttg att Leu Ile gcc cta Ala Leu ttg ctt ctt Leu Leu Leu 20 tcc tat ttg Ser Tyr Leu ttC tgt Phe Cys gaa atc cct Giu Ile Pro tct att Ser Ile atg gat cca Met Asp Pro aaa Lys 35 aga Arg gga caa ata aag ggc Gly Gin Ile Lys Gly gga gga tat act aag Gly Gly Tyr Thr Lys aac aga att gag ttt Asn Arg Ile Glu Phe taatgaagcc atcasgtcaa gaaatcCaac tagacaaata ctggagtgca gtggtatgat tCtcctgcct cagtctcctg atttttgtgt ttttggtaga 120 180 240 300 358 406 454 502 550 598 651 711 771 831 891 951 aaa aca aaa Lys Thr Lys gggtcacatg ccaataaaca ataaattttc cagaagaaat aagtagagct tatgaaatgg ttcagtaagg atgagcttgt tttgtttttt taaagacgga gtctcgctct gtcactcagg cttggctcac tgtaacctcc gcctcccggg ttcaagccat agtagctggg attgcaggtg cgtgccacca ticctggcta WO 99/25825 PCT/IB98/01862 gacagggttt caccacgttg gtcgggctgg 1011 cct.tggcctc ccaaagtgat gggattacag gatttttaaa gtatgttcca gttctgtgtc gaaaaggtca tggggaagca gaggtgattc ccttattgtc taggccactt gtgaagaata ctctctagc ttacaatgga ccttttgaa aaatgtcaaa actaattttt ataataaatg <210> 48 <211> 791 <.212> DNA <213> Homo sapiens <220> 31 tctcgggctC ctgacctctt gatccgcctg atgtgagcca atggttggaa atggctctgt tgagtcagtt ctgggaaaca ~tattttca Ccgtgcctag gacagagtag ggaat.ttgag attgccagcc ccr.tgtctgc ca tygaaaaa ccaaggatga gaaggatatg gtgaatggtt attcacttta aaaaaaaaa 1071 1131 1191 1251 1311 1370 <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig-paptide 191. .286 Von Heijne macrix score 8.80000019073486 seq VPMLLLIVGGSFG/LR polyA..signal 755. .760 POlYA&Site 780. .791 misc-feature 361. .531 homology id :W73841 es t misc-.feature 210. .347 homology id :W73841 es t misc feature 548. .637 homology id :W73841 est <221> misc-feature <222> 181. .210 <223> homology id :W73841 est <221> misc-feature <222> 361. .530 <223> homology id :HSU74317 est <221> misc-feature <222> 238. .347 <223> homology id :HStJ74317 est <221> misc-feature <222> 568. .637 <223> homology id :HSU74317 es t <221> misc-feature <222> 698. .733 <223> homology id :HSU74317 est <221> misc-feature WO 99/25825 PCT/TB98/01862 32 <222> 361. .531 <223> homology id :W47031 est <221> misc-feature <222> 210. .347 <223> homology id :W47031 est <221> misc-foeiture <222> 148.-.210 <223> homology id :W47031 est <221> misc-feature <222> 548. .600 <223> homology id :W47031 est <221> misc-.feature <222> 129. .347 <223> homology id :AA044118 est <221> misc-feature <222> 437. .531 <223> homology id :AA044118 es t <221> misc-feature <222> 361. .454 <223> homology id :AA044118 est <221> misc-feature <222> 176. .347 <223> homology id :AA293342 est <221> misc-feature <222> 361. .531 <223> homology id :AA293342 est <221> misc-feature <222> 548. .605 <223> homology id :AA293342 est <221> misc-feature <222> 361. .531 <223> homology id :AA531561 est <221> misc-feature <222> 153. .252 <223> homology id :AA531561 est <300> <400> 48 aacaagtatg ttacgatggc tcgattgctt ttgcctagcg gaaaccattc actaaggacc gagcaccaaa taaccaagga aaaggaagtg agttaaggac gtactcgtct tggtgagagc 120 WO 99/25825 PCT/1B98/OI 862 33 aggcccgctt ggagtccga gccgatggaa gtgagctgct gagattggg agtccgcgct 180 gagttcacc atg ttt gca ccc gcg gtg atg cgt gcc ttt Met Phe Ala Pro Ala Val Met Arg.Ala Phe -25 cgc aag aac Arg Lys Asn 229 aag act Lys Thr ctc ggc tat gga Leu Gly Tyr Gly gtC ccc atg Val Pro met t tg Leu ttg ccg etc gtc gga Leu Leu Ile Val Gly ggc tct tc Ser Phe ggc aa Gly Lys tc te Ser Leu ggt ccc Gly Leu 1 cg c Arg gag ttc tct .caa acc cga tat gat gcc Glu Phe Ser Gin Ile Arg Tyr Asp Ala gtg aag Val Lys ecg gat ccc gag ctt gee aaa ae ccg Met Asp Pro Glu Leu Glu Lys Lys Leu 20 gag tcg gaa tat gag aee etc eee gec Glu Ser Glu Tyr Glu Lys Ile Lys Asp ace Lys Ccc Ser gag eat aaa ate, GlU Asn Lys Ile aeg cct gat gac Lys Phe Asp Asp tgg Trp aeg eat ettc ga 9g ccc agg cct Lys Asn Ile Arg Gly Pro Arg Pro tg Trp gat CCt gee Asp Pro Asp etc cc 277 325 373 421 469 518 578 638 698 758 791 caa gga age aat cce gaa age etc Gin Gly Arg Asn Pro Glu Ser Leu aag Lys act aeg ace act Thr Lys Thr Thr tgactcgcc gactcccc tcccccc ectcatce agcggae acggacaaea kcaacct ccccacgt ggecgea aaaaacgcge aceaccg <210> 49 <211> 1433 <212> DNA <213> Homo sapiens <220> ccc ttttataa~~ eeactcec egg aaetcccgg cccacggaaa kcc ecceeaggc acgtccagg egc tgeeacgctg gCceccgcat ccc ceaaeeeeea aa aactggactc cetaacatac ctcggacacg ggcaecccgg tccctatact tcccagceaa kaccctacac cntcgaaa <221> Sig-pepcide <222> 346. .408 <223> Von Heijne matrix score seq SFLPSALVIWTSA/AF <221> PolyA-signal <222> 1400. .1405 <221> polyA site <222> 1420. .1433 <221> misc-feature <222> 268. .634 <223> homology id :W02860 esc <221> misc-feature <222> 118. .564 <223> homology id :N27248 est <221> misc-feature <222> 268. .697 <223> homology id :N44490 es c <221> misc-feature <222> 582. .687 <223> homology id :AA274731 est <221> mise...feature WO 99/25825 WO 9925825PCTIIB98/OI 862 34 <222> 65. .369 <223> homology id :H94779 est <221> misc..feature <222> 471. .519 <223> homology id :H94779 est <221> misc-feature <222> 61. .399 <223> homology id :H09880 est <221> misc-feature <222> 408. .452 <223> homology id :H09880 est <221> misc-feacure <222> 484. .699 <223> homology id :H04537 est <221> misc-feature <222> 685. .772 <223> homology id :H04537 est <22 1> misc-feature <222> 454. .486 <223> homology id :H04537 est <221> misc-feature <222> 410. .439 <223> homology id :H04537 est <221> misc-feature <222> 572. .687 <223> homology id :AA466632 est <221> misc-feature <222> 260. .444 <223> homology id :AA459511 est <221> misc-feature <222> 449. .567 <223> homology id :AA459Sll est <221> misc-feature <222> 117. .184 <223> homology id :AA459511 est <221> misc..feature <222> 260. .464 <223> homology id :H57434 WO 99/25825 WO 9925825PCTIIB98/OI 862 <221> <222> <223> <221> <222> <223> <221> <222> 42 23 est misc-feature 118. .184 homology id :H57434 est misc-feature 56. .113 homology id :H57434 est misc-.feature 454. .485 homology id :H57434 est <300> <400> 49 acccctttta gcat ctgatgccga gttc ctcaaacggc ctag gtttgttgaa gcag cgttcctgtt gagt aagactaaca ttc cag caa ggc ctc Gin Gin Gly Leu gct gct ttc ata Ala Ala Phe Ile 1 gac ccg gc-t tta Asp Pro Ala Leu aggggc cgtctc tgcttc ttacca acacgt gtgaag t tcggcgccj tcgcgtcttt gcgcttccg( agaatcttcz tcctgttgat ttgtaaaacz Igcggccagcg cta t tcctggtccc agg I agaaaatcag cgg accctttccc aca -ttacaaaagg tgc gaaaacctgt tag tca gcc ctt gta Ser Ala Leu Val act gca gta aca Thr Ala Val Thr gccggtc tggtaagtgc caaagcg gasgnagatc tctaatt aattcctctg aaagcta attgagtaca aggtatg agcaggtctg aa. atg tgg tgg ttt Met Trp Trp Phe agt ttc ctt Ser Phe Leu ttt tca tac Phe Ser Tyr cc t Pro -10 att Ile att Ile ctc Leu tgg aca tct Trp Thr Ser cac cat ata His His Ile gct cca gaa Ala Pro Glu 5 tat Tyr atc agt gac act Ile Ser ASP Thr ggt Gly aca gta Thr Val aaa tgc tta Lys Cys Leu gct acc att Ala Thr Ile gag aac gtt Giu Asn Val ttt Phe tat Tyr gca atg cta Ala Met Leu aat Asn gcg gca gtt tta Ala Ala Val Leu tgc att gtt cgt tat Val Arg Tyr caa gtt cat gct Gin Val His Ala c tg Leu gta Val1 atc atc aaa Ile Ile Lys Ctg agt Leu Ser t ta Leu 70 tc t Ser aag gct ggc Lys Ala Gly ctt Leu tgt tta gga Cys Leu Gly att gtg gca Ile Vai Ala cct Pro aac Asn 90 gc t Ala ttc cag Phe Gin tac ctt Tyr Leu agt cct gaa Ser Pro Glu ctt gga ata Leu Gly Ile gaa aac aac Giu Asn Asn tgg tat ggg Trp Tyr Gly 110 aaa tgc agc Lys Cys Ser 125 gtt gtt ggt Val Val Giy 120 180 240 300 357 405 453 501 549 597 645 693 741 789 837 891 951 1011 1071 1131 ttt tgc tgc Phe Cys Cys aag tgg agc Lys Trp Ser tgt Cys 105 cc t Pro ctc att ata Leu Ile Ile cca aaa tcc Pro Lys Ser 130 tat ctg gtg Tyr Leu Val 145 cagtggcaat ttatgcgctt ttttttcctg acatggatta tat Tyr 115 aa t As n tgt tca gac Cys Ser Asp cat His 120 ctt Leu ttc cta cca Phe Leu Pro ggc aaa aca Gly Lys Thr ctg gat cag act Leu Asp Gin Thr tgg agt aag Trp Ser Lys tgc Cys tagcatgctg acttgctcat cagttttgca tttgggactg atttagaaca gaaactccat cacatgatca ctactgcagc agaatggtct acttacattc gtgattttca gaaaatttcc accctctatg acactgcacc ttgCcctatt tggaaccccg aggacaaagg atgtcatttt ccttctttgg ttacgggtgg aagccaactt aacaatgaac gaacacggct WO 99/25825 PCTIIB98/OI 862 36 aaaatatttc tgtaantgan ttastgastt actttccags aagatattag atgaaaggat 1191 Ctcagggant tggggaaang gttcacagaa aanccactta antcaaggct gacagstaac gaaagaagcc atttgcatag attattytaa Cctatgccta tactttttta tytcagaaaa aa <210> S0 <211> 1158 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 214. .339 <223> Von Heiine matrgttgcttavt acgtga tgaa aggatatcat taaagtcaaa tcttcatcrt tgctgataat Caagaagamt agactatgaa gaanattttc caggaaacat at taaaaaca aaaaaaaaaa <221> <222> <221> <222> <221> <222> <223> <22 1> <222> <223> score 6.09999990463257 seq AILLLQSQCAYWA/LP polyA..signal 1133. .1138 POlYA.site 1146. .1158 misc..feature 840. .968 homology id :H64717 es t misc-feature 858. .968 homology id :H65208 est 1251 1311 1371 1431 1433 120 180 234 282 330 <300> <400> aarttgagct tggggactgc ctcatctt ggatttgaaa tgrsagtgta mtggattatt aacagtccat gtgggtgatt agctgtgggg gt tgagagca CCttgggcct cagctctgat agatttcagt gcattgcctc ccctgggtgc gcatgttttg cccactgaaa ctcatcctgs gaatgacttg aatgtttccc cgcctgagct ggg atg tgt ttt cca gag cac aga Met Cys Phe Pro Glu His Arg aga Arg cgc Arg caa atg tat att Gin Met Tyr Ile caa Gin -30 tgt Cys gat aga ctg Asp Arg Leu gct ata cta Ala Ile Leu caa gga cga Gin Giy Arg gac tct Asp Ser -25 tta ctc Leu Leu gtc acc agg aga Val Thr Arg Arg gca Ala caa tct cag Gin Ser Gin tgt gcc Cys Ala ctt atg tat tgg gcg ctt Tyr Trp Ala Leu gaa ccg cgt aca ctt gat ggg gga. cat Giu Pro A-rczf 1 5 X10 xhSL caa tgatggctct ctcctgctcc aagatgtgca agaggctgac cagggaacct Gin ui M'et atatctgtga atgtgcttcc aagagctttc ggc caagtac cagaataaaa actttaaaca gtccaaatat accthtgaca cacattgatg aaagaaataa Cgaggcagca actgcattyt aaaaaaa aatccgcctc agaggagccc atgtgcaaga ttaccacaga a takgagt ta cacttccccc gttttggaca acaaagtcya ctacatytgt gcagkycagg gaamtgcc tg agcc tgggwg aaaggggaga aaaggtacgc ggcaaggaaa gtgaatcttc ttttagttaa tcacaaaagc catatttatt tgttytrtttt attttatagg ctcagtggct agccccaggg acagagcaag gccaggtgtt aaatgcttac ctgattatct aaagaaatga kaataaaata cc tgtgaagg aaatggaata actatgccct taccctatgt catgcctgta t tcaagac tg actYtgttta caagaaggcg t taaagaggg tgagtaaatg ntcattaaat ttgataatta atgttttgtt aatagtamtt aataccttts taggtgtttt atcctagcat cag tgagc ta aaataaaaaa gtggtactgc gccaaggggc ccagcctttg tatttcagrt ttgtattatt cacatataat gaaccc tggc atcagttatc gggggataga tttgggaggc tgawggcacc agagaaaaaa 491 551 611 671 731 791 851 911 971 1031 1091 1151 1158 WO 99/25825 PCTIIB98/01862 37 <210> 51 <211> 850 <212> DNA <213> Homo sapiens <220> <221> sig..,peptide <222> 372. .437 <223> Von Heijne matrix score 6.09999990463257 seq LI'LTCLFWPLAAL/NV <221> polyA..Signal <222> 812. .817 <221> polyA-.Sie <222> 838. .850 <221> misc..feature <222> 128. .424 <223> homology id :N78012 es t <221> misc-feature <222> 61. .128- <223> homology id :N78012 es t <221> misc-feature <222> 483. .554 <223> homology id :N78012 est <221> misc-feature <222> 417. .464 <223> homology id :N78012 est <221> misc-feature <222> 460. .500 <223> homology id :N78012 est <221> misc-feature <222> 577. .612 <223> homology id :N78012 es t <221> misc-feature <222> 612. .649 <223> homology id :N78012 es t <221> misc..feature <222> 546. .577 <223> homology id :N78012 est <221> misc-feature <222> 29. .63 <223> homology id :N78012 est <221> misc-feature -<222> 128. .294 <223> homology id :W37233 WO 99/25825 WO 9925825PCT/IB98/01862 <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> est misc-feature 370. .509 homology id :W37233 est misc-.feature 505. .591 homology id :W37233 est misc..feature 293. .330 homology id :W37233 est misc..feature 22..57 homology id :W37233 est misc..feature 95. .128 homology id :W37233 est misc..feature 128. .326 homology id :AA186399 es t misc..feature 418. .605 homology id :AA186399 est misc-feature 326. .423 homology id :AA186399 es t misc-feature 39. .128 homology id :AA186399 eat mi sc.f ea ture 206. .640 homology id :W52489 es t <300> <400> 51 agacact.tcc tggtgggatc cgagtgaggc gacggggtag gggttggcgc tcaggcggcg accatggcgt atcacggcct cactgtgcct Ctcattgtga tgagcgtgtt ctggggcttc gtcggctttc ttggtgcctt ggttcatccc taagggtcct aaccggggag ttatcattac catgttggtg acctgttcag tttgctgcta tctcttttgg ctgattgcaa ttctggccca actcaaccct ctctttggac cgcaattgaa aaatgaaacc atctggtatc tgaagtatca ttggccttga ggaagaagac atgctctaca gtgctcagtc tttgaggtca cgagaagaga atgccttcta g atg caa aat cac ctc caa acc aga cca ctt ttc ttg act Met Gln Asri His Leu Gin Thr Arg Pro Leu Phe Leu Thr -15 tgc ctg ttt tgg cca tta gct gcc tta aac gtt aac agc aca ttt gaa 120 180 240 300 360 410 458 WO 99/25825 WO 9925825PCT/IB98/01 862 Cys Leu Phe Trp Pro Leu Ala Ala Leu 39 Asn Val Asn Ser Thr Phe Glu tgc ctt att cta caa tgc agc gtg ttt tcc ccc gcccc ccctt gca ccc Cys Leu Ilie Leu Gin Cys Ser Val Phe Ser Phe Ala Phe Phe Ala Leu 10 15 egg tgaatcacgt gcctccacaa cctgaaccgt gccgacccca caaaacgacc Trp atgtactcce ctgagacaga agatgccgtt cCctgagag atacgttacc ctcccccc aatcgtgga tttgaaaatg gctcccgcct tctcacgcgg gaaccagtga agtgtttag aactgctgca agacaaa caa gactccagtg gggtggtcag taggaaaaca cgtcagag gaagaaccac ctcaacagaa tcgcaccaaa ctatactcec aggatgaatt tctctc gccatcccc ggaataaata CCCtcctcct ttycatgcaa aaaaaaaaaa a <210> 52 <211> 1107 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 132. .215 <223> Von Heijne matrix 506 559 619 679 739 799 850 9 t <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <22 2> <223> <221> <222> <223> score 3.59999990463257 seq PLSDSWALLPASA/GV polyAsignal 1069. .1074 P0lYA-Site 1094. .1107 misc-feature 177. .392 homology id :W80978 est misc-feature 425. .542 homology id :W80978 est misc-feature 43. .114 homology id :W80978 es C misc-feacure 387. .441 homology id :W80978 est misc-feature 113. .165 homology id :W80978 esemisc-feacure 551. .590 homology id :W80978 est misc-.feacure 166. .314 homology id :AA043154 est <221> misc-feacure <222> 27. .181 WO 99/25825 WO 9925825PCT/IB98/O1 862 <223> homology id :AA043154 est <221> misc-feature <222> 425. .564 <223> homology id :AA043154 est <221> misc-jeature <222> 387. .441 <223> homology id :AA043154 est <221> misc..feature <222> 309. .352 <223> homology id :AA043154 est <221> misc..feature <222> 549. .580 <223> homology id :AA043154 est <221> misc..feature <222> 601. .1071 <223> homology id :AA126732 es t <221> misc-feature <222> 576. .605 <223> homology id :AA126732 est <221> misc-feature <222> 387. .477 <223> homology id :AA161280 est <221> misc-feature <222> 292. .362 <223> homology id :AA161280 est <221> misc-feature <222> 46. .113 <223> homology id :AA161280 est <221> misc-feature <222> 217. .277 <223> homology id :AA161280 est <221> misc-feature <222> 113. .160 <223> homology id :AA16128O est <221> misc-feature <222> 173. .217 <223> homology id :AA161280 est WO 99/25825 WO 9925825PCT/1B98/OI 862 <300> <400> 52 aacaactecc cgctgcttac egcccgtgac ggccccaceg agcggtgtcc tgagccgatt acagctaggt agtggagcgc ctgggtgcag gagacagccg gagtcgctgg gggagctccg cgccgccgga c atg tgg agg ctg ctg gct cgc gct agt gcg ccg cte ctg Met Trp Arg Leu Leu Ala Arg Ala Ser Ala Pro Lou Leu cgg Arg gta Val1 gtg ccc ttg tca Val Pro Leu Ser aag aca ctg ctc Lys Thr Leu Leu ga t Asp cca Pro tcc tgg gCa ctc ctc ccc gee agt gct ggc Ser Trp Ala Lou Leu Pro Ala Ser Ala Gly gta cca agt Val Pro Scr 10 ttt gaa gat gcC Phe Glu Asp Val gaa aaa ccc Glu Lys Pro gta aga aga Val Arg Arg aag Lys ett aga ttt Leu Arg ?he gaa agg gea eca Giu Arg Ala Pro ctt Leu gga Gly tec att ect Ser Ile Pro gtg cca aaa Val Pro Lys cet tec act Pro Ser Thr gaa cct 888 Glu Pro Lys gaa get Glu Ala 88 t As n gaa Glu agt gac ata Ser Asp Ile 120 170 218 266 314 362 410 458 506 554 602 650 aeg gag kikk Thr Glu Xaa gg t Gly aca Thr 55 tg Trp gge aat ttt Gly Asn Phe gca Ala 60 atg Met ttg gea ttg Leu Ala Leu ggt Gly gge tac ctg Gly Tyr Lou eat His gac Asp ggc cac ttt Gly His Phe atg cgc etg Met Arg Leu aca ate Thr Ile aae cge tet Asn Arg Ser gcc cct ttc Ala Pro Phe 100 gga gge aaa Gly Gly Lys ccc aag aac Pro Lys Asn atg Met 90 a8a Lys ttt gce ata tgg Phe Ala Ile Trp eec ate act Pro Ile Thr cge Arg 105 eac His agt gtt ggg Ser Val Gly ega gta eca Arg Val Pro cge atg ggg Arg Met Gly aag get ggc Lys Ala Gly ggt get att Gly Ala Ile tac gtg aca Tyr Val Thr 115 cgc rnww Arg Xaa cc t Pro 125 ttt Phe gww gta gag Xaa Val Glu 130 ggt Gly atg Met 135 gtt Val1 ggg cgt tgt gina Gly Arg Cys Xaa gaa gaa gtg Glu Glu Val ttc ctet gac Phe Leu Asp cag Gin 150 act Thr gee eac aag ttg Ala His Lys Leu tty gca gca Phe Ala Ala aag get Lys Ala 160 gtg age ege Val Ser Arg gaa mgt aae Glu Xaa Asn 180 mae atg etg Xaa Met Leu ggg Gly 165 aae Asn yta gag aag Leu Glu. Lys atg Met 170 aca Thr aaa gat caa Lys Asp Gin gag gaa aga Glu Glu Arg 175 gee act gee Ala Thr Ala cag aae c Gin Asn Pro ttt gag ega Phe Glu Arg ata Ile 190 gge ata egg Gly Ile Arg etg age eca Leu Ser Pro 195 aag ggg Lys Gly 210 tat gac ttg Tyr Asp Leu 205 main cgt gtg Xaa Arg Val ace cac Thr His aaa tamn tgg Lys Xaa Trp gge Gly 215 tty tac atg Phe Tyr Met tagtgagtgt aggagat ceecteagce tacecaci attaaataaa atttaaa <210> 53 <211> 500 <212> DN'A <213> Homno sapiens <220> <221> sig..peptide <222> 199. .288 aac tgtatatagg staetgaaag aag :ga agtytttggg tagetyttaa gcc aaa aegatgttat ttgttgattt aaa cat eactteagga aaaaaaaaaa aaa gattytg ataamta aagaaaa eatttytatt aggageagca cwg tatt tt t 944 1004 1064 1107 WO 99/25825 WO 9925825PCT/IB98101862 42 <223> Von H-eijne matrix score 5.59999990463257 seq IVSVLALIPETTT/LT <221> polyA..signal <222> 464. .469 <221> polyA..site <222> 489. .500 <221> misc-feature <222> 197. .412 <223> homology id :AA429945 es t <221> misc-feature <222> 61. .195 <223> homology id :AA429945 est: <221> misc-.feature <222> 425. .488 <223> homology id :AA429945 est <221> misc-feature <222> 197. .412 <223> homology id :AA455042 est <221> misc-feature <222> 61. .195 <223> homology id :AA455042 es t <221> misc-feature <222> 425. .488 <223> homology id :AA455042 Cs t <221> misc-feature <222> 207. .412 <223> homology id :W93646 Cs t <221> misc..feature <222> 58. .195 <223> homology id :W93646 es t <221> misc-feature <222> 425. .488 <223> homology id :W93646 Cs t <221> misc-feature <222> 197. .412 <223> homology id :AA516431 Cs t <221> misc-feature <222> 90. .195 <223> homology id :AA516431 est <221> misc-feature WO 99/25825 WO 9925825PCT/1B98/01862 <222> 425. .488 <223> homology id :AA516431 est <221> misc-feature <222> 52. .195 <223> homology id :W38899 est <221> misc-feature <222> 197. .324 <223> homology id :W38899 est <221> misc..feature <222> 443. .477 '223> homology id :W38899 est :221> misc-feature :222> 197. .338 :223> homology id :W52820 est :221> misc..feature :222> 71. .195 :223> homology id :WS2820 est :221> misc-feature 222> 339. .401 :223> homology id :W52820 es t 221> misc-feature 222> 425. .469 223> homology id :W52820 est 221> misc-feature 222> 40. .195 .223> homology id :W19506 est <300> <400> 53 agagctgtnn cnsaagtagg tcgcagaacc tactcaggca ctgcagacgc gatggataac ggagggcggt gctccgc gccagctgag aagagtt gtgcagccga aaataaa tgaaaggCca cgtgayag aca aca gta ttc atg Thr Thr Val Phe Met acc aca aca ttg aca Thr Thr Thr Leu Thr atg Met ctc Leu gt t Val1 ctg cgg ctg gat Leu Arg LeU ASP mgm ggtggcggdht gag ggaaagtgct q aca tcgccccttct att atc aac tca Ile Ile Asn Ser -25 ttg gca ctg ata Leu Ala Lieu Ile :gctatcgct ~ccgctgggt :gcttcagtg ctg gta Leu Val cca gaa Pro Glu aca gca Thr Ala atc gta tct Ile Val Ser gta tgc Val Cys aat ccc Asri Pro 1 tgt ctt gcc gac Cys Leu Ala Asp agc ggt cct tac Ser Gly Pro Tyr ggt gga Gly Gly 5 ggg gcc Gly Ala ggg Gly ttt gca ctt Phe Ala Leu gtg Val1 120 180 231 279 327 375 423 ctt att tac cgg aag ctt ctg ttc Leu Ile Tyr Arg Lys Leu Leu Phe 20 cag Gin aaa aag cct gtg Lys Lys Pro Val cat His gaa aaa aaa gaa Glu Lys Lys Giu WO 99/25825PC/B8O86 PCT/IB98/01862 44 35 40 gtt ttg taattttata ttacttttta gtttgatact aagtattaaa catatttetg Val Leu tattcttcca aaaaaaaaaa a <210> 54 <211> 765 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 293. .385 <223> Von Heiine matrix <221> <222> <221> <222> <221> <222> <223> score 4.40000009536743 seq TCCHLGLPHPVRA/PR polyA-signal 733. .738 polyAsite 752. .765 misc..feature 310. .576 homology id :HUM426A07B est <300> <400> 54 aaaccttgtt tgaggggcga aaaceg tagg tttcccccca tgaaagagag ge tagggac gggaaaagt kacgcggtc agcgaaccg gc tagaag t C gggeggtttg cg~ t tcctcaggt gt~ a gaaaggcgac gg~ rg gatgggaagt gac .t ccgcttgcca gec ctt gtc tca ctt Leu Val Ser Leu ggc etc cca eac Gly Leu Pro His gcaaccgt ;g tgggga ;etgtegg :ttcaatg Igectcet gggcactgct gagggaggcg agt tggaaag agattgaact tagtagagcg gaatttgaat ga tgccggng ggacgcc tgg tcagctggat ga atg agt Met Ser gee etc Ala Leu cgc cet 479 500 120 180 240 298 346 394 442 490 535 595 655 715 765 aat acc eac acg Asn Thr His Thr ace tgc tgt cac Thr Cys Cys His ctt Cct cgc gta Leu Pro Arg Val gtg Val1 etc Leu cat ccg cac ccg His Pro His Pro gte cgc get ccc Val Arg Ala Pro gaa ccg tgg Glu Pro Trp 10 gee atg aat Ala Met Asn -5 gat cet Asp Pro tee ttc Ser Phe agg tgg cag Arg Trp Gin eta aat gag LeU Asn Glu 1 gac tea gag eta Asp Ser Glu Leu agg Arg tge Cys tat eea eag Tyr Pro Gin agg ace tta Arg Thr Leu egg tea teg Arg Ser Ser eeg Pro agg Arg gaa gca teg Glu Ala Ser tgaaeetgat agattgel atttetgaaa agaeeat tagaatttag atttaggl tgggaeeaae tttatgg <210> 55 <211> 584 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 130. .189 <223> Von Hei-ine m :ga ttttatetta ttttatectt 1ca gataaeeaca aatatcaaga :tt ectteetgct tceacetec aat aaataagctg agctgeaaaa aga tgt gat Arg Cys Asp gacttggtac aagtegtctt t tegaataag waaaaaaaaa etc Leu aagttttggg eagtat taag gaaacgtett atrix score 3.5 seq KFCLICLLTFIFH/HC <221> polyk.signal <222> 546. .552.' WO 99/25825 WO 9925825PCT/IB98/01862 <221> polyA-Site <222> 572. .584 <300> <400> aagacgcgcc ggtttctgcg acgcagttag cgcagtctgc tttggtgaat acacgattt g gtgcagccgg ggtttggtac cgagcggaga ggjagatgcac acggcactcg agtgtgagga aaaatagaa atg aag gta cat atg cac aCa aaa ttt tgc ctc att tgC ttg Met Lys Val His Met His Thr Lys Phe Cys Leu Ile Cys Leu ctg aca ttt act Lou Thr Phe Ile ctt cat cat tgc aac cat tgc cat gaa gaa cat gac Phe His His CYS Asn His Cys His Giu Giu His Asp cat ggc His Gly gag cca Giu Pro cct gaa gcg Pro Glu Ala agc aaa ttt Ser Lys Phe ctt cac Leu His tca aag Ser Lys aga cag cat Arg Gin His 20 caa gct gct Gin Ala Ala cgt gga atg aca Arg Gly Met Thr gaa ttg Giu Leu gaa aat gaa Glu Asn Giu cat act gaa Tyr Ile Glu ttt tt~t ggt Phe Phe Gly aaa Lys ctt ttt gag Leu Phe Glu 35 tat Tyr ggt gaa aat Gly Giu Asn gga Gly ctt Leu aaa aaa tac Lys Lys Tyr aga tta tcc Arg Leu Ser gga gag aga Gly Glu Arg 120 171 219 267 315 363 411 459 507 567 584 ttg gag aaa Leu Glu Lys aaa gta Lys Val Ct: Leu 65 cat His aca aac ttg Thr Asn Leu ggc Gly gtt gag att Val Giu Ile cat His aa t Asn 80 ggc Gly gag gat ctc, Glu Asp Leu, cac gat cat gtt His ASP His Val tct Ser aca Thr tta agg tat Leu Arg Tyr agt tca aga Ser Ser Arg ggg Gly .100 gca Ala ttt tca ctc Phe Ser Leu taaccaccca gcattcc Wtccacaaaa aaaaaaa <210> 56 <211> 1387 <212> DNA <213> Homo sapiens <220> cat aatcatttaa attcagaaaa tcaaaactgt gaccagtgta <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> sig..peptide 191. .325 Von Heijne matrix score 4.59999990463257 seq VLVYLVTAERVWS /DD polyA.s ignal 1348. .1353 poiyA_site 1374. .1387 misc..feature 1258. .1372 homology id :AA417826 est <221> misc-feature <222> 791. .887 <223> homology id :AA417826 es t <221> misc-feature <222> 94. .524 <223> homology id :AA235826 es t <221> misc..feature <222> 44. .94 WO 99/25825 WO 9925825PCT/IB98/O1 862 46 <223> homology id :AA235826 est <221> misc-feature <222> 1258. .1372 <223> homology id :AA236941 esf: <221> misc-feature <222> 935. .1279 <223> homology id :AA480326 est <221> misc-feature <222> 1258. .1372 <223> homology id :AA480326 esf: <221> misc..feature <222> 724. .1148 <223> homology id :AA234245 est <221> misc..jeature <222> 944. .1279 <223> homology id :AA479344 est <221> misc-feature <222> 1258. .1372 <223> homology id :AA479344 es t <221> misc..feature <222> 1070. .1212 <223> homology id :AA133636 est <221> misc..feature <222> 1258. .1372 <223> homology id :AA133636 est <221> misc-feature <222> 938. .1054 <223> homology id :AA133636 est <221> misc..feature <222> 94. .436 <223> homology id :AA133635 est <221> misc-feature <222> 32. .94 <223> homology id :AA133635 est <221> misc-feature <222> 895. .1273 <223> homology id :AA479453 est WO 99/25825PCIB8O86 PCT/IB98/01862 <221> <222> <223> <221> <222> <223> misc-feature 1258. .1371 homology id :AA253214 est misc..feature 94..268 homology id :AA482378 est <300> <400> 56 acteCcaggc Ccteeecgaaa cgccggc tgc egggteeaec tgggccagca caceeggcag gctetgtcct ggaaacaggc aecttccccg cttctggata tgaavattca agctgcttge tgggagccag gagagcectg aggagcagtc acteagtage acg aac tgg agt atc tec gag gga etc ctg .agt Met Asn Trp Ser Ile Phe Giu Gly Leu Leu Ser ttcaacgggc tgagtceeat agctgaegcg ggg gtc aac aag tac Asn Lys Tyr ace ttc cgc Ile Phe Arg tcc aca, gcc ttt Ser Thr Ala Phe gtg ctg geg tac Val. Leu Val Tyr ggg Gly -25 ctg Leu cgc ate tgg ctg Arg Ile Trp Leu gtg aeg gcc gag Val Thr Ala Giu tct ctg gtc ttc Ser Leu Val Phe cgt gtg tgg agt Arg Val Trp Ser ccc ggc tgc tee Pro Gly Cys Ser gat gac Asp Asp cac aag gac His Lys Asp 5 -10 ttc gac Phe Asp 1 aac Asn tgc aat act Cys Asn Thr 10 ttc cct gtg Phe Pro Val cgc cag Arg Gin tcc cat Ser His gtc tgc ttt Val Cys Phe gat gag ttc Asp Giu Phe atc ctg gtg Ile Leu Val gcc ctg eag Ala Leu Gin cac gtg gcc His Vai Ala ett Leu 25 tgc Cys ccc tea ctg Pro Ser Leu gtg Val1 ctc Leu ega cgc ctc tgg Arg Leu Trp gtg gtc atg Val Val Met gaa gcc cat Glu Ala His tac egg gag Tyr Arg Glu ggg gag Gly Giu gt t Val1 55 cc Leu gag aag agg Giu Lys Arg cac His ggc Gly aac agt ggg Asn Ser Gly ggg Gly cgc Arg 70 tat Tyr tac ctg aac Tyr Leu Asn ccc Pro 75 gtg Val aag aar cgg Lys Lys Arg gg t Gly 120 180 229 277 325 373 421 469 517 565 613 661 709 757 805 853 901 949 etc tgg tgg Leu Trp Trp aca Thr C tc Leu gtc tgc agc Val-Cys Ser eta Leu tca Ser ttc aag geg Phe Lys Ala agc gtg Ser Val gac atc gcc Asp Ile Ala etc cct cct Leu Pro Pro 115 gac tgc ttc Asp Cys Phe ttt Phe 100 gtg Val1 tat gtg ttc Tyr Val Phe cac His 105 gca Ala ccc tac ccc Phe Tyr Pro gtc aag tgc Val Lys Cys cac His 120 tea Ser gat cca tgt Asp Pro Cys ccc Pro 125 t te Phe aaa tat atc Lys Tyr Ile 110 aat eta gtg Asn Ile Val acc etc ttc Thr Leu Phe atc ccc aag Ile Ser Lys 130 atg gtg Met Val ccc Pro 135 ate Ile gag aeg eac Glu Lys Asn att Ile 140 ac Asn gcc aca gct Ala Thr Ala 145 ate Ile gee Ala 150 aag Lys tgc etc etg Cys Ile Leu etc gtg gag Leu Val Glu tac ctg gtg Tyr Leu Val age Ser 165 kge xaa aga tge cac Arg Cys His gag Giu 170 ec Pro ctg gea gca Leu Ala Ala get caa gee Ala Gin Ala kge aaa caa Xaa Lys Gln 195 aca ggt cat Thr Gly His cac His 185 ggk Gly cay get ace Xaa Asp Thr acy Thr 190 c tg Leu egg aaa Arg Lys 175 ttt tee Phe Ser ggn tea Gly Ser gac ytc ytt Asp Xaa Xaa teg Ser 200 gac ytc ate Asp Xaa Ile WO 99/25825 WO 9925825PCT/IB98/01862 48 gac agt cat cyt cct ytc tta cca gac cgc CCC cga gac cat gtg aag Asp Ser His Xaa Pro Xaa Leu Pro Asp Arg Pro Arg Asp His Val Lys 210 215 220 aaa acc aty ttg tgaggggctg cctggamtgg tytggcaggt tgggcctgga Lys Thr Ile Leu 225 tggggaggct ytagcatyty tcataggtgc aacctgagag tgggggagct aagccatgag gtaggggcag gcaagagaga ggattcagac gytytgggag ccagttccta gtcctcaamt ccagccacct gccccagsth gacggcamtg ggccagrttcc ccctytgsty tgcagstcgg ttcctttty tagaatggaa atagtgaggg ccaatgccca gggttggagg gaggagggcg ttcatagaag aacacacatg cgggcacctt catygtgtgt ggcccactgt cagaacttaa taaaagtcaa mtcatttgct ggttaaaaaa aaaaaaaa <210> 57 <211> 1385 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 141. .251 <223> Von Heijne matrix <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> score 4 seq PLSLDCGHSLCRA/CI PolY&..signal 1354. .1359 polyk..site 1375. .1385 misc-.feature 1183. .1240 homology id :AA463623 est misc-.feature 176. .239 homology id :AA258927 est misc-feature 803. .854 homology id :AA286417 es t misc-feature 1183. .1213 homology id :AA608077 est 1049 1109 1169 1229 1289 1349 1387 120 173 221 269 317 <300> <400> 57 aacacccacc aagccatcca gaagccaggg gag gtg ac Glu Val Th.

cta gac tg Leu Asp Cy.

c tggct t ttc ggggtcttta aagcag tgca tgt ccc at Cys Pro 11 ttcacctctt caaccaggag ccgagatttc tgttgctctg accagaagag agaggagagc ctcaggagtt aggaccagaa atg gct tca aaa atc ttg ctt aac gta caa. gag Met Ala Ser Lys Ile Leu Leu Asn Val Gln Glu :c tgc ctg gag ctg ttg aca gaa ccc ttg agt .e Cys Leu Glu Leu Leu Thr Glu Pro Leu Ser t ggc cac s Gly His agc Se r -20 ctc tgc cga gcc tgc atc act gtg agc aac Leu Cys Arg Ala Cys Ile Thr Val Ser Asn -D 1 aag gag gca gtg acc agc atg gga gga aaa agc agc tgt cct gtg tgt Lys Glu Ala Val Thr Ser Met Gly Gly Lys Ser Ser Cys Pro Val Cys 15 ggt atc agt tac tca ttt gaa cat cta cag gct aat cag cat cgg gcc WO 99/25825 WO 9925825PCTIIB98/01862 Gly Ile Ser Tyr Ser Phe Glu His Leu 49 Gin Ala Asn Gin His Arg Ala aac ata gtg Asn Ile Val aag aag aga Lys Lys Arg gag aga ctc aag Glu Arg Leu Lys 45 gat ctc tgt gat Asp Leu Cys Asp gag gee aag teg Glu Val Lys Leu age Ser aaa Lys cca gac aat ggg Pro Asp Asn Gly cat cat gga His His Gly ege Cys gag Giu ccc Leu ctc cta cc Leu Leu Leu aag gag gat Lys 0Th ASP agg Arg cac His gte ace cgc Val Ile Cys egg Trp 80 cac His tge gag egg CyB Glu Arg tee cag Ser Gin gag cac cgt ggt Glu His Arg Gly cac aca ggc His Thr Gly cct Pro gga gga age Gly Gly Ser att caa gga Ile Gin Gly 100 gaa gga aga Giu Gly Arg aeg tea gga Met Ser Gly 105 gga gga age Gly Gly Ser gaa ace cca ggc agt ccc caa gag gce gaa Giu Thr Pro Gly Ser Pro Gin Glu Ala Giu egagaagceg gaagctgaca ecagagaaga gaaaacttcc 413 461 509 557 605 654 714 774 834 894 954 1014 1074 1134 1194 1254 1314 1374 1385 120 eggaagtacc aggeaca agcaccaa aeaaega aegeeggata agecege gageecace cagacge.

aegageggaa tcatgaa aagaaaeega agactget ggaaeegaea gctgtce tcegaatckt gtcctte ecagegecat aateatg tgggaagtgg aegegec eccegecaca cgaagta tacagaccec caeeegg aaaaaaaaaa a <210> 58 <211> 1497 <212> DNA <213> Homo sapiens <220> aac gga aga gga atg ace 9gt cag geg caa ege a ta tgagagacaa geagagagag ggc egagga e gegeeggage gagegagac ccaegeccca ceaccggge aagatcagag ekbttgggat gaaaac egee ege tagaaga ctgggttata agga tacaaa eegcaaagae gagcagtec cagtggecaa tggaggc cga .gacgagea ggaegecaca acaagcgaea cccaaeattt tggatccegg tgcgcaaaey gggttacaga cagaaeecga tggaagaaga agcagaagca caa eggagect aaaageeaaa ggaegccgcr cegaateag tcegtgecaa bccesSgg gggcataeeg gtcaaaatbt ataaatgtaa tcagcetaga agaaaagaag ge tggegaga geegeaggac aaeggetcc aaegteeaga ecaacctaaa ceeggccec gaaacattae tagaacata C e eaeaccaaa gtatggtgcc <221> sig-.pepeide <222> 212. .268 <223> Von Heijne matrix score 8.60000038146973 seq LLWLALACSPVHT/TL <221> polyA..signai <222> 1465. .1470 <221> polyA-site <222> 1489. .1497 <221> misc-feature <222> 958. .1110 <223> homology id :W72124 ese <221> misc-feature <222> 1362. .1488 <223> homology id :W72124 ese <221> misc-feacure <222> 1202. .1312 <223> homology id :W72124 est <221> misc..feature WO 99/25825 WO 9925825PCTIIB98/O1 862 6 <222> 1115.-1190 <223> homology id :W72124 est <221> misc...feature <222> 1312. .1370 <223> homology id :W72124 est <221> misc..feature <222> 653. .942 <223> homology id :AA009415 est <221> misc...feature <222> 454. .605 <223> homology id :AA009415 est <221> misc-feature <222> 598. .639 <223> homology id :AA009415 est <221> misc-feature <222> 805. .1032 <223> homology id :AA088502 eat <221> misc-feature <222> 633. .807 <223> homology id :AA088502 est <221> misc-feature <222> 598. .639 <223> homology id :AA088502 es t <221> misc-feature <222> 564. .605 <223> homology id :AA088502 est <221> misc-feature <222> 653. .807 <223> homology id :A.A183.148 es t <221> misc-feature <222> 907. .1046 <223> homology id :AA181148 est <221> misc-feature <222> 475. .605 <223> homology id :AA181148 est <221> misc..feature <222> 598. .639 <223> homology id :AA181148 WO 99/25825 WO 9925825PCT/1B98/01862 <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> est misc-feature 1069. .1190 homology id :AA181149 est misc..feature 1362. .1475 homology id :AA181149 misc..feature 1202. .1312 homology id :AA181149 est misc..feature 1312. .1370 homology id :AA181149 est <300> <400> 58 atccggcgcg ctggagcg.t gcagatttcc anssagaaga agactgggtg cctgggagct cgaggttgga ccctactgtg tr.ccggccgt cagagaagga gaggcagcca acacacc tac gcgtttgtgg Ccgccggcc gcnagtggtc atggaatggg ccgtt.tcagc ctggccagcc c atg cgg aca ctc ttc Met Arg Thr Leu Phe tCccgacat ctggggtcaa ctctggaccc aac ctc Asn Leu 120 180 232 280 328 ccc tgg Ctt gcc ctg gcc tgc agc cct gr.t cac Leu Trp Leu Ala Leu Ala Cys Ser Pro Val His -5 tca gat gcc asa aaa ccg cct caa aga cgc tgc Ser Asp Ala Xaa Lys Pro Pro Gin Arc Arg Cys act acc ccg tca aag Thr Thr Leu Ser Lys 2.

tgg aga aga gtc agt Trp Arg Arg Val Ser 10 is ttt cag ata agc cgg tgc aar acc ggg gtt tgg tgg tgacggacct Phe Gin Ile Ser Arg Cys Lys Thr Gly Val Trp Trp caaagctgag acactttgct caccaaggtc acgtggccgt agtgtggttc ggggatgtac ttgggagca gagatgtttg agctgtcagg aagcatgcca gacttacgat gatttccgga.

gaccgtggtc caggtggcaa ccagctgcta agccagaagc cctgcaccag gcccggctgc cgaccagctg ggcatgttca tttcagcctc atgacctacg gtcctgggtt cgagcctgcg tectcctggg gctcaacttc ctgttgtcgg ggccaggtac gggacagcca ggcctcagag tcgtcttcta cccaaccctg ggCgttgggg tctctatytg taggtgggca ttgcggcctc gcaggtgtga aatacaggcc <210> 59 <211> 1570 <212> DNA <213> Homo sapiens <220> <221> Sig-.peptjde ttgagcatcg tgggctatgt agt.tcacaca aggtcacggg agggcc tgca, acgtct taga agaaccagca gcgtgggcc t tggccctcct cgcacaagga actactctac tccaggtee t tatggtatgg atccagacac cacttcttcg aagtccctgc ggagctgggc cgcggtggac tccactccgt cagctactgc cactccatgg gatctcaeccc ctccacgac catagtgcct cagtgaggat tttcgatggc catccacatg ggtcatcccg grtttgagcag agcgcatcag ggacccggaa actacgcgac tgaadggaec agtacaagaa aggtgcgggc cagggcctgg gtgttyttt ttgcaaaaaa tcggcaaagg aacagccatg gtctggctgc gtggaccaag cggctcctgt gaga tagagg ttcgtggtgg ctcacccact cctgccatca ctggcccceg cctggcccta gtccaagtgg ctccaaggat acaggccccg gagccgcagt tggagctggc actacttyta ytaagccatg aaa cccgggacag gctaegatgt agt tgaagag ggtggatgcg ttgaggactg age tgagcaa aggtc tggaa tggccgaggc cccccgggac tgctggatgg atgcacccct cgaagcaaaa gcccgtgagc ggaatggtgt gggaggcacg ccgggagct~g cgacctgety gagtgagtga 434 494 554 614 674 734 794 854 914 974 1034 1094 1154 1214 1274 1334 1394 1454 1497 WO 99/25825 52 <222> 147. .248 <223> Von Heiine matrix score 4.30000019073486 seq QLFAFLNLLPVEA/DI <221> polyA..signal <222> 1538. .1543 <221> polyA..site <222> 1558. .1570 <221> misc-feature <222> 466. .968 <223> homology id :AA506103 esc <221> misc-feature <222> 142. .664 <223> homology id :AA237105 est <221> misc..feature <222> 114. .269 <223> homology id :AA317201 est <221> misc-feature <222> 2. .122 <223> homology id :AA317201 est <221> misc-feature <222> 401. .443 <223> homology id :AA317201 es t <221> misc..feature <222> 103. .385 <223> homology id :T80259 est <221> misc-feature <222> 21. .120 <223> homology id :T80259 es t <221> misc-feature <222> 109. .459 <223> homology id :N32697 est <221> misc-feature <222> 45. .87 <223> homology id :N32697 est <221> misc-feature <222> 92.-122 <223> homology id :N32697 est <221> misc-feature <222> 1220. .1409 <223> homology id :AA449621 est PC'rIIB98/OI 862 WO 99/25825 WO 9925825PCTIIB98/01862 53 <221> misc-feature <222> 928. .1092 <223> homology id :AA449621 est <221> misc-feature <222> 1178. .1222 <223> homology id :AA449621 est <221> misc-feature <222> 1220. .1545 <223> homology id :N34685 es t <221> misc..jeature <222> 1168. .1222 <223> homology id :N34685 est <221> misc-feature <222> 1220. .1545 <223> homology id :N22990 est <221> misc-feature <222> 1178. .1222 <223> homology id :N22990 est <221> misc-feature <222> 114. .325 <223> homology id :AA330462 est <221> misc-feature <222> 18.-122 <223> homology id :AA330462 eat <221> misc-feature <222> 135. .475 <223> homology id :HUMEST5H12 est <300> <400> 59 agtcgtccct gctagtactc cgggctgtgg gggtcggtgc ggatattcag tcatgaaatc agggtaggga cttctcccgc agcgacgcgg ctggcaagac tgtttgtgtt gcgggggccg 120 gaacttcaag gtgattttac aacgag atg ctg ctc tcc ata ggg atg ctc atg 173 Met Leu Leu Ser Ile Gly Met Leu Met ctg tca gcc aca caa gtc tac acc atc ttg act gtc cag ctc ttt gca 221 Leu Ser Ala Thr Gin Val Tyr Thr Ile Leu Thr Val Gin Leu Phe Ale.

-20 -15 ttc tta aac cta ctg cct gta gaa gca gac att tta gca tat aac ttt 269 Phe Lieu Asn Leu Leu Pro Val Glu Ala Asp Ile Leu Ala Tyr Asn Phe 1 gaa aat gca tct, cag aca ttt gat gac ctc ccc gca ara ttt ggt tat 317 Giu Asn Ala Ser Gin Thr Phe Asp Asp Leu Pro Ala Xaa Phe Gly Tyr 15 aga ctt, cca gct gaa ggt tta aag ggt ttt tta att, aac tca aaa cca 365 Arg Leu Pro Ala Giu Gly Leu Lys Gly Phe Leu Ile Asn Ser Lys Pro WO 99/25825 WO 9925825PCTIIB98/O1 862 gag Giu tc t Ser aat As n gcc tgt gaa CCC Ala Cys Giu Pro 30 ata Ile gtg cct cca Val Pro Pro ggc act ttc Gly Thr Phe 45 gtg Val1 cca gta Pro Val 50 ctt gat Leu Asp aaa gac aat Lys Asp Asn t ca Ser tta act ara Leu Ile Xaa tgt aat Cys Asn ttt gat Phe Asp ata aag gtt Ile Lys Val cac aat gtt His Asn Val gag gta eta Giu Val Leu tta Leu go t Asp gca cag aga Ala Gin Arg gca Ala 80 act Ile tac aag gca Tyr Lys Ala tee gat gac Ser Asp Asp etc Leu 95 att Ile age aeg gga Ser Met Gly tee Ser 100 att Ile gcc eta gt Ala Ile Val aec gac at Asn Asp Ile ggt gee tea Gly Glu Ser 509 557 605 653 aag aaa act Lys Lys Ile 105 tea get Ser Ala gac Asp 110 gat Asp cca tct gtc Pro Ser Val agt tet ctg Ser Ser Leu 120 ct Leu aaa Lys 125 gaa Giu gaa tte aea Giu Phe Thr tak Xaa 130 t eg Leu aaa ggg gge Lys Gly Gly ate eta gee Ile Leu Val ttt agt ott Phe Ser Leu gee tee tee Giu Tyr Tyr eta Leu 150 eec tte etc Pro Phe Leu aeg ate eca Met Ile Thr 170 eet cgt ae Leu Arg Lys etc Ile 155 aaa Lys geg gge ate Val Gly Ile egt Cys 160 age Arg ate teg ata Ile Leu Ile ttg tee agg Leu Ser Arg get Asp 175 aaa Lys cat age get His Arg Aia a ga Arg 180 aae Lys gte att tte Val Ile Phe 165 age aac age Arg Asn Arg tte eag aaa Phe Lys Lys gee eea ett Asp Gin Leu ett ec gte Leu Pro Val 185 gga get Gly Asp cat His 195 get Asp gag tat gee Glu Tyr Asp 200 gga Gly gee Val1 205 ate Ile gee at tge Ale Ile Cys gag tat gee Glu Tyr Glu get Asp 215 gee aaa etc Asp Lys Leu ett ccc tge Leu Pro Cys get tat cat Ala Tyr His tgc aag Cys Lys 230 tgt gte gao Cys Val Asp egg eaa ea Arg Gin Lys 250 age age eae Ser Ser Gin ec Pro 235 gtt Val1 gee Glu eta act aaa Leu Thr Lys acc Thr 240 ggc: Giy aaa acec tge Lys Thr Cys gte cct tet-cae Val Pro Ser Gin 255 gaa eat gaa geg Giu Asn Giu Val get tea gee Asp Ser Asp tcc Ser 260 ec Pro eea gtg ege Pro Val Cys 245 gac eca gac Asp Thr Asp tea etg aga Leu Leu Arg 749 797 845 893 941 989 1037 1085 1133 1181 1223 1283 1343 1403 1463 1523 1570 aca gae cat Thr Giu His 265 oct tta Pro Leu 270 cc Thr 27.5 ggg Gly gne tte tgt Xae Phe Cys 280 ant Xaa cag Gin 285 cag Gin ego cee rge Cys Pro Xaa eak eat gac Xaa His Asp eamn tt Xea Phe 290 aga ate Arg Ile get tta nte Ala Leu Xaa eec get c Pro Ala His ant Xaa 300 gaa Glu att eag act Ile Gin Thr ast Xaa 310 gae gac gac Glu Asp As9 tgaaateaee eatageaeat ggtaeataee ttttegtece tgceaegaae agcyttyt <210> 60 <211> 1022 get act gac Asp Thr Asp 305 ag t Ser get gea gee Asp Ala Glu gaatgaeg aeegtetgac gtaectgat ytecegteta atecttcatt ttggaatgaa tegtggteca ttteagaaga tttttgotcc eteaaettee eactaaceat agetetgcca gttgeegcet tgattggttt ettaaagat tgaeaeagge egeetggtc aaacaaaaaa aa-tggtgaae etttcccttt c tycgtegea cttttgatyt tgtaee teaa aaeaa gggattaeaa eeeatgatta etaecatt ggtatcec gcetcaettc WO 99/25825 WO 9925825PCTJIB98/OI 862 <212> DNA <213> Homo sapiens <220> <221> <222> <223> sig-peptide 112. .237 Von Heiine matrix score 7.19999980926514 seq ILFSLSFLLVIIT/FP <221> polyA..signal <222> 976. .981 <221> polyk..site <222> 1010.-1022 <300> <400> aatactttct cctctcccct ctcccaagca catctgagtt agctccaaac ccatgaaaaa ttgccaagta taaaagcttc gctgccgtt cttcacactt tcaagaatga g atg gat Cct Ser gtc Val1 agg gtg tct tca Arg Val Ser Ser cct Pro -35 ctt Leu gag aag caa gat Glu Lys Gin Asp 888 Lys -30 tgg Trp gag aat ttc Giu Asn Phe atc ctg ttt Ile Leu Phe aac aat aaa Asn Asn Lys cgg Arg gtg Val1 ggt gta .tgt Gly Val Cys ggc Gly -15 ccc Pro Met Asp gtg ggt Val Gly tcc ctc Ser Leu atg tgc Met Cys tct ttc ctg Ser Phe Leu ttg aag atc Leu Lys Ile cgc atc caa Arg Ile Gin ttg Leu att Ile gc t Ala atc att aCC ttc Ile Ile Thr Phe atc tcc ata tgg Ile Ser Ile Trp aag gag tat Lys Giu Tyr 15 gac aaa gcc Asp Lys Ala 1 gaa cgt Giu Arg aag ggg Lys Gly gct gtt gta Ala Val Vai cca ggt ttg Pro Glv Leu ttc cgt ctg gga Phe Arg Leu Gly atc ctg gtc Ile Leu Val cca Pro tgc ata gat Cys Ile Asp 30 ttt Phe gtc aag gtt Val Lys Val gac Asp 50 aga Arg ctc Leu ctg Leu cga aca gtt Arg Thr Val 40ttg ath tcy ThrCy aac att cct Asn Ile Pro gta gat gga Val Asp Gly gct aat gtc Ala Asn Val cca Pro gtt Val1 gag atc ctc Giu Ile Leu gac tcc gta Asp Ser Vai gtc tat tac Val Tyr Tyr tat agt gct Tyr Ser Ala gtc Val1 ctg Leu act act cag Thr Thr Gin tca gca gtg Ser Ala Val gct caa acc Ala Gin Thr 117 165 213 261 309 357 405 453 501 549 597 645 693 741 789 837 885 aac gat gtc Asn Asp Val gca aca ttt Ala Thr Phe act ctg Thr Leu ctg Leu 100 tcc Ser aga aat gtc Arg Asn Val 105 gga Gly t ta Leu 110 gcc Al a aca cag acc Thr Gin Thr cag atc tta Gin Ile Leu cga gaa gag Arg Glu Glu cat agc atc His Ser Ile cag Gin 130 cga Arg tta ctt gat Leu Leu Asp gat gcc Asp Ala 135 acc gaa ctg Thr Giu Leu cgg att ccc Arg Ile Pro 155 acc C9g gaa Thr Arg Glu tgg Trp 140 gtg Val1 atc cgg gtg Ile Arg Vai gtg gaa atc Val Glu Ile cag ttg cag Gin Leu Gin atg gca gcc Met Ala Ala gag Giu 165 gga Gly aaa gat gtt Lays Asp Val 150 gct gag gcc Ala Giu Ala gaa atg agt Giu Met Ser gcg aga gcc Ala Arg Ala ctt gca gct Leu Ala Ala 170 gcc tcc Ala Ser gaa Giu 180 ctg Leu aaa tcc ctg Lys Ser Leu gcc tcc atg Ala Ser Met 185 ata gtg Val1 195 ttg gct gag tct Ala Giu Ser ccc Pro 200 acc gct ctc cag ctg tac ctg cag acc agc acg gta gcc WO 99/25825 WO 9925825PCTIIB98/01862 Ile Ala Leu Gin Leu Arg Tyr Leu Gin 56 Thr Leu Ser Thr Val Ala Thr 210 215 ctg ccc atg aat ata cta gag Leu Pro Met Asn Ile Leu Giu gag aag aat Giu Lys Asn tct Ser 220 acg att gtg ttt Thr Ile Val Phe cc t Pro ggc att Gly Ile 9gt ggc gtc agc tat gat aac cac aag aag ctt Giy Gly Val Ser Tyr Asp Asn His Lys Lys Leu 235 240 245 gtcctc ttgcggtagt cagctaaaaa aaaaaaaa 230 cca aat aaa Pro Asn Lys gc Ala <210> <211>~ <212> <213> <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> :gag 61 1022 *615

*DNA

*Homo sapiens sig..peptide 239. .316 Von Heijne matrix score 3.90000009536743 seq ITWVSLFIDCVMT/RK polyAsignal 586. .591 POlYA..site 603 .615 misc-feature 341. .574 homology id :AA453275 es t misc..feature 174. .332 homology id :AA453275 est misc-.feature 85. .171 homology id :AA453275 est misc-feature 341,..574 homology id :AA149631 est misc-feature 170. .339 homology id :AA149631 est misc-feature 43 123 homology id :AA149631 est misc-feature 88. .339 homology id :AA588414 est misc-feature 341. .574 homology id :AA588414 WO 99/25825 WO 9925825PCT/1B98/01 862 57 est <221> risceature <222> 1. .345 <223> homology id :AA156847 est <221> misc...feature <222> 342. .414 <223> homology id :AA156847 est <221> misc-.feature <222> 341. .574 <223> homology id :AA501739 es t <221> misc..feature <222> 110. .339 <223> homology id :AA501739 est <221> misc-feature <222> 341. .574 <223> homology id :AA131792 est <221> misc-feature <222> 153. .259 <223> homology id :AA131792 est <221> misc..feature <222> 259. .339 <223> homology id :AA131792 est <221> misc-feature <222> 59. .338 <223> homology id :AA131842 est <221> misc-feature <222> 344. .415 <223> homology id :AA131842 est <221> misc-feature <222> 400. .434 <223> homology id :AA131842 est <221> misc-feature <222> 341. .574 <223> homology id :AA152042 es t <221> misc-feature <222> 183. .339 <223> homology id :AA152042 est <300> <400> 61 WO 99/25825 WO 9925825PCTlIB98/01862 atctttgaag aagaagaagt tgaatttatc 58 agtc;tgcctg tcccagagtt tgcagatagt cagcctattt agatcttaac 120 ttatgccacc cagaaaccta 180 gatcctg ctggata ctggagtc atg agc Met Ser gtt tot Val Ser :ca acattgttca tgactttaac aagaaactta igt gctatgtgat ccctctgaac acttccattg ac ttattaacat caaggctgga acctatttgc aca tgg tta tta, ctg atc gca ttg aaa, Thr Trp, Lou Lou Lou Ile Ala Lou Lys 20 ctcagtccta tctgattc aca ttg atc acc tgg Thr Leu Ile Thr Trp 00 c As n tta ttt atc gao tgt gec atgj aca agg aaa ctt aca aac tgc Loeu Phe Ile Asp Cys Val Met Thr Arg Lys Loeu Thr Asn Cys -5 1 ago gao act att a80 ggt att cog 800 cgt gao gcc agc aat Arg Glu Thr Ile Ly's Gly Ile Gin Lys Arg Glu Ala Ser Asri got Ala tgt ttc gca Cys Phe Ala Ott Ile cgg cat ttt Arg His Phe 15 gad aac Glu Asn a88 ttt gco Lys Phe Ala gtg Val1 gad aot tta Glu Thr Leu 286 334 382 430 479 539 599 615 ott tgt tct Ile Cys Sex acagcataac gtaagtagca cocaaaaaaa <210> 62 <211> 804 <212> DNA <213> Homo <220> tgaacagtca agaaaaacat tottgaggaa, aattaatotc cccacccttt acattttgtg cagtgattat tttttaaogt Cttctttcat.

aacagggctt taotatcttt tcatotcatt aattcaatta aaaocattao aaaaaa sapiens <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig...peptide 157. .345 Von Heijne matrix score seq GLVCAGLADMARP/AE polyk..signal 771. .776 polyA..site 791. .804 misc-feature 244. .789 homology id :AA576425 est misc-feature 286. .790 homology id :AA236527 est misc-feature 287. .790 homology id :AA435919 est misc-.feature 520. .790 homology id :AA165350 est <221> misc-feature <222> 389. .522 <223> homology id :AA165350 est <221> misc-feature WO 99/25825 WO 9925825PCT/IB98/01862 59 <222> 336. .386 <223> homology id :AA165350 est <221> misc-feature <222> 326. .790 <223> homology id :AA490322 est <221> misc..feature <222> 326. .790 <223> homology id :AA490310 est <221> misc..feature <222> 515. .780 <223> homology id :AA164559 es t <221> misc..feature <222> 325. .522 <223> homology id :AA164559 est <221> misc..feature <222> 350. .790 <223> homology id :AA427895 est <221> misc-feature <222> 378. .790 <223> homology id :AA532390 es t <221> misc-feature <222> 186. .382 <223> homology id :AA082259 est <221> misc-feature <222> 61. .141 <223> homology id :AA082259 est <221> misc-feature <222> 426. .478 <223> homology id :AA082259 est <221> misc-feature <222> 29. .61 <223> homology id :AA082259 est <221> misc-feature <222> 389. .790 <223> homology id :AA157009 est <221> misc-feature <222> 425. .790 <223> homology id :AA034912 WO 99/25825 WO 9925825PCT/IB98/01862 <221> <222> <223> <221> <222> <223> est misc-feature 186. .430 homology id :AA428006 est misc..feature 59. .132 homology id :AA428006 est <300> <400> 62 aacagcgggc cgtggcagga gctcccaccc cga ggc ctG Arg Gly Leu atg ctg ccc Met Leu Pro agggaaagcc aaagegacta ggctgcchaa gcgggaagg gctcccctt( ggatccctcc cgg gcc acc tac Arg Ala Thr Tyr cac His -50 Ccg Pro 9 taceccaggc gag gttgtcagcc egg I gcggcg atg tcg met Ser cgg ctc ctc get Arg Leu Leu Asp ttg tac eac cat Leu Tyr Asn His aggcgga cgcgagtcgc gacgaga ecacegccac gcc gcc ggt gcc Ala Ala Gly Ala aaa gtg gag ctg Lys Val Glu Leu gag aaa ttg Giu Lys Leu aga aca Arg Thr agg Arg -35 gc t Ala cca Pro gca ggt ccc Ale Gly Pro gtt ttc ttc Val Phe Phe gc t Ala tg Trp -20 atg Met cca att atg Pro Ile met aaa Lys -15 gee Glu tgg ggg ttg gtg Trp Gly Leu Val aea ctt agc ace Lys Leu Ser Thr tgt Cys gc c gge ttg Gly Leu cee tct gc 't Gin Ser Ala ctt gte ett Leu Val. Ile gct gat Ala Asp gtt ttg Val Leu ett ccg Ile Pro atg gct ace Met Ala Thr 15 aae eat tgg Lys Asn Trp gcc ega cct gce Ala Arg Pro Ala ggg ttt att tgg Giy Phe Ile Trp tca Ser gtc Val age tac tca Arg Tyr Ser eat ttc ttt Asn Phe Phe 120 174 222 270 318 366 414 462 510 557 617 677 737 797 804 agt ctg ttt Ser Leu Phe gtg ggg Vel Gly gca gca gge Ala Ala Gly caa Gln gcc Ala 45 a Lys 30 tct Ser cag ctt ttt Gin Leu Phe gc t Ala att Ile tgg ega tat Trp Arg Tyr gaa cte aaa Glu Leu Lys gca cac aaa Ala His Lys taaeegagtt cctgatcacc tgaeceatct egatgtggac aaaaccettg ggecctagtt egceeagcte ectgtgtgtt tegeeggcac tgtaactggt gaaaaatgce gcaeectttt aeteacagtc tctctacatg gatattagta acecttttct eccatttgtc cgtaateaa aeaaa <210> 63 <211> 792 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 194. .253 <223> Von Heiine matrix tattatttgg ttattgetae egctagttct tgattceate acttaeggea cttetctatg catecttgct cgtaaaaaaa <221> <222> <221> <222> <221> <222> score 12.3999996185303 seq ALLLGALLGTAWA/ RR P0lYA...Signel 768. .773 PolYA-site 780. .792 misc-feature 154. .428 WO 99/25825 WO 9925825PCT/IB98/01862 61 <223> homology id :R22491 est <221> misc..feature <222> 104. .160 <223> homology id :R22491 est <221> misc..feature <222> 47. .218 <223> homology id =A136163 est <220. misc..feature <222> 265. .403 <223> homology id :AA136163 es e <221> misc-.feature <222> 3. <223> homology id AA136163 est <221> misc-feature <222> 123. .265 <223> homology id :N57089 est <221> misc..feature <222> 47. .127 <223> homology id :N57089 est <221> misc...feature <222> 282. .323 <223> homology id :N57089 est <221> misc-feature <222> 128. .403 <2230 homology id :AA314970 est <221> misc-feature <222> 138. .403 <223> homology id :AA314807 est <221> misc-feature <222> 164. .403 <223> homology id :A271811 est <221> misc-feature <222> 163. .385 <223> homology id :AA103053 est <221> misc-.feature <222> 154. .403 <223> homology id :AA042016 es t WO 99/25825 PCT/IB98/01862 62 <221> misc-feature <222> 2. .250 <223> homology id :AA315322 est <221> misc-.feacure <222> 154. .403 <223> homology id :AA470189 est <221> misc-feature <222> 217. .403 <223> homology id :AA462839 es t <221> misc-.feature <222> 154. .403 <223> homology id :AA120322 est <221> misc..feature <222> 163. .403 <223> homology id :W71694 es t <221> misc-feature <222> 164. .385 <223> homology id :AA250603 est <221> misc..feature <222> 266. .403 <223> homology id :AA036242 es t <300> <400> 63 aaggcggtcg ccgggacacc ccgtgtgtgg caggcggcga asgctctgga gaatcccgga cagccctgct ccctgcagcc aggtgtagtt tcgggagcca ctggggccaa agtgagagtc 120 cagcggtctt ccagcgcttg ggccacggcg gcggccctgg gagcagaggt ggagcgaccc 180 cattacgcta aag atg aaa ggc tgg ggt tgg ctg gcc ctg ctt ctg ggg 229 Met Lys Gly Trp Gly Trp Leu Ala Leu Leu Leu Gly -15 gcc ctg ctg gga acc gcc tgg gct cgg agg agc cgg gat ctc cac tgt 277 Ala Leu Leu Gly Thr Ala Trp Ala Arg Arg Ser Arg Asp Leu His Cys 1 gga gca tgc agg gct ctg gtg gat gaa cta gaa tgg gaa att gcc cag 325 Gly Ala Cys Arg Ala Leu Val Asp Glu Leu Glu Trp Glu Ile Ala Gln 15 gtg gac ccc aag aag acc artt cag atg gga tcc ttc cgg atc aat cca 373 Val Asp Pro Lys Lys Thr Ile Gin met Gly Ser Phe Arg Ile Asn Pro 30 35 gat ggc agc cag tca gtg gtg gag gta act gtt act gkt tcc ccc aaa 421 Asp Gly Ser Gin Ser Val Val Glu Val Thr Val Thr Xaa Ser Pro Lys 50 aca aaa gta gct cac tct ggc ttt tgg atg aaa att cga ctg ctt aaa 469 Thr Lys Val Ala His Ser Gly Phe Trp, Met Lys Ile Arg Leu Leu Lys 65 aaa gga cct tgg tct taatagaaaa tgaagraaaa cagactcaga aaaaaagatt 524 Lys Gly Pro Trp, Ser tbggctctgt ctcawtttgg aaagct~cgc a tccccaatgc aactttgctt 584 cctggctgca aaccyttaat acytttgttt Ctgctgtaga aatttgttag ccaaaacawg 644 WO 99/25825 PCTIIB98/01862 63 ggagtcctga twcagcaacc ccttcttcca caatccacca tgactggttt ttaatgtamc 704 acttggggta tacatgcaaa accatccgtt cmaaaatctg aatycggagc ttaaaaattt 764 aaaaatgaaa aacchaaaaa aaaaaaaa 792 <210> 64 <211> 832 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 148. .207 <223> Von Heijne matrix score 12.3999996185303 seq ALLLGALLGTAWA/RR <221> polyk..Signal <222> 789. .794 <221> polyA..site <222> 820. .832 <221> misc-feature <222> 258. .553 <223> homology id :AA435303 est <221> misc-feature <222> 117. .219 <223> homology id :AA435303 es t <221> misc..feature <222> 552. .645 <223> homology id :AA435303 est <221> misc-feature <222> 217. .258 <223> homology id :AA435303 est <221> misc-feature <222> 258. .553 <223> homology id :AA314807 es t <221> misc-feature <222> 92. .258 <223> homology id :AA314807 est <221> misc-feature <222> 258. .554 <223> homology id :AA314970 es t <221> misc-feature <222> 82. .258 <223> homology id :AA314970 est <221> misc-feature <222> 258. .553 <223> homology id :AA547310 est WO 99/25825 WO 9925825PCTIIB98OI 862 <221> <222> <223> <221> <222> <223> <221> <222> <223> <22 1> <222> <223> <22 1> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> misc-feature 119. .258 homology id :AA547310 es t misc-feature 359. .553 homology id :AA565602 est misc-.feature 552. .683 homology id :AA565602 est misc-feature 684. .751 homology id :AA565602 est misc-feature 742. .783 homology id :AA565602 est misc..feature 364. .553 homology id :AA136094 es t misc-feature 552. .683 homology id :AA136094 est mi sc-eature 684. .751 homology id :AA136094 es t misc-feature 258. .461 homology id :AA136163 est misc-feature 2..172 homology id :AA136163 est misc-feature 216. .258 homology id :AA136163 est <300> <400> 64 aggagaatcc ccaaagtgag aggtggagcg cggacagccc tgctccctgc agccaggtgt agtttcggga gccactgggg agtccagcgg tcttCcagcg cttgggccac ggcggcggcc ctgggagcag accccattac gctaaag atg aaa ggc tgg ggt tgg ctg gcc ctg Met Lys Gly Trp Gly Trp Leu Ala Leu 120 174 222 ctt ctg ggg gcc ctg ccg gga acc gcc tgg gct cgg agg agc cag gat WO 99/25825 WO 9925825PCT/IB98/01862 Leu Leu ctc cac Lieu His Gly Ala Leu Leu Gly Thr Ala Trp, Ala Arg Arg tgt gga gca tgc agg get Ctg gtg gat gaa act aga Cys Gly Ala Cys Arg Ala Leu Val Asp Glu Thr Arg Ser Gin Asp atg gga aat tgC cca Asn Cys Pro gg t Gly ccc caa gaa gac Pro 0Th Glu Asp tca gat ggg Ser Asp Gly gat caa tee aga Asp Gin Ser Arg etc aga ggc cca Lau Arg Gly Pro tgg cag eca gtc agt ggt gga ggt Trp Gin Pro Val Ser Gly Giy Gly gc Ala cet cac aga get get gga gga gat atg Pro His Arg Ala Ala Gly Gly Asp Met ate ttt ccg Ile Phe Pro tta tgc ceg Loeu Cys Pro tgacegga tg gt acgtgcagtg ga eteagatat gg atgaaeecat ta agcgaaeaga ct ggagcagcec Ce ttttatatat aa aaaaaaa 270 318 366 415 475 5-35 595 655 715 775 832 daggagtatg gggaaca ggceggaatg gagaac ageggeacce teaagbt tgaattcttt tcegag tetttgtgae eatgccc aeactggett gatggat tatgttttae tgaaatt <210> 65 <211> 721 <212> DNA <213> Homo sapiens <220> gat cag tg agg tge cac tgatcctc e tgaaetggae cgtgtgggaa etgacaatgt actategge eccaggnaa tgaaaaact acecategec etacaaggca cattgtggag taaagacaaa atgatgagct gggaaaatgg gaaaecaaaa agaac tac tcegaac gaatacga ctttgcag atgaacca tggeaatg gtscaaaa <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig...peptide 156. .230 Von Heijne matrix score seq MFAASLLAMCAGA/EV poiyk..signal 706. .711 P0lYA-site 709. .721 misc-feature 351. .688 homology id :H98648 est mise feature 289. .353 homology id :H98648 est mi sc-eature 274. .641 homology id :AA181022 est mi sc.feature 255. .286 homology id :AA181022 est misc-feature 242. .641 homology id :AA143192 est misc-feature 261. .646 homology WO 99/25825 WO 9925825PCTIIB98/O1 862 66 id :AA594850 est <221> misc-feature <222> 165. .474 <223> homology id :AA563681 est <221> misc...feature <222> 1. .74 <223> homology id :AA563681 <221> misc-feature <222> 261. .643 <223> homology id :AA287457 est <221> misc..feature <222> 352. .646 <223> homology id :N22567 est <221> misc-feature <222> 299. .354 <223> homology id :N22567 es t <221> misc..feature <222> 265. .303 <223> homology id :N22567 est <221> misc-feature <222> 30. .165 <223> homology id :AA186657 est <221> misc-feature <222> 270. .349 <223> homology id :AA186657 est <221> misc-feature <222> 213. .261 <223> homology id :AA186657 est <221> misc-feature <222> 165. .214 <223> homology id :AA186657 est <22> misc-feature <222> 346. .387 <223> homology id :AA186657 est <221> misc-feature <222> 52. .400 <223> homology id :HSC1EDO8l est <221> misc...feature WO 99/25825 WO 9925825PCT/1B98/OI 862 <222> 398. .436 <223> homology id :HSClEDO81 est <221> misc-.feature <222> 171. .316 <223> homology id :AA143136 est <300>.

<400> atctgggtc ccc tgcagt t gcccacagtt cggcctgctc gcmgetccgct Cctccgccc ttagacctgt cgcggwacag tctctattag agcgcgtgca tagaggcagai ccccccctcc tagagcctgc cgacc atg ccc gcg ggc Met Pro Ala Gly tgcccagcat kaggagtgaa gtg ccc Val Pro atg tgc Met Cys atg tcc acc tac Met Ser Thr Tyr gca ggg gca gaa Ala Gly Ala Glu ctg Leu gtg Val1 aaa atg ttc gca Lys Met Phe Ala gcc agt Ala Ser -10 etc ctg gcc Leu Leu Ala gtg cac agg tac tac cga ccg gac Val His Arg Tyr Tyr Arg Pro Asp ctg aca ata Leu Thr Ile cct gaa Pro Glu ctg aaa Leu Lys 1 att cca Ile Pro cca aag cgt Pro Lys Arg gga gaa ctc aaa acg gag ctt *ttg gga Gly Glu Leu Lys Thr Glu Leu Leu Gly 120 173 221 269 317 365 418 478 538 598 658 718 721 gaa aga aaa cac aaa cct caa gtt tct caa cag gag gaa Glu Arg Lys His Lys Pro Gin Val Ser Gin Gin Glu Glu ccc Leu aaa taaccatgcc aagaatcg tgaataatat aagtctaaa tatgtattc Lys ttaatttatt gcatcaaact acttgcccc aagcacttag tctaatgcta actgcaagag gaggtgctca gtggatgttt agccgatacg ttgaaattta attacggttt gattgatatt tcttgaaaac tgccaaagca catatcatca aaccatttca tgaatcacggt ttggaagatg tttagtcttg aatataacgc gaaatagaat atctgtaagt ctactatatg ggttgccc atttcatata aattaagaaa ttatttaaaa. actatgaact aggtttcatt aaaaaaaaaa gaa <210> 66 <211> 531 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 272. .397 <223> Von Heiine matrixe <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> score 4.59999990463257 seq RIPSLPGSPVCWA/WP polyA..signal 503. .508 polyA..site 518. .531 misc-feature 235. .517 homology id :AA524403 est misc-feature 52. .208 homology id :AA524403 est mi sc-eature 259. .517 homology WO 99/25825 WO 9925825PCTIIB98/01862 68 id :N93660 es t <221> misc-feature <222> 85. .207 <223> homology id :N93600 est <221> misc-feature <222> 353. .517 <223> homology id :AA594610 es t <221> misc-feature <222> 258. .363 <223> homology id :AA594610 est <221> misc-feature <222> 105. .207 <223> homology id :AA594610 est <221> misc-feature <222> 202. .517 <223> homology id :AA074748 est <221> misc..feature <222> 116. .153 <223> homology id :AA074748 es t <221> misc-feature <222> 167. .202 <223> homology id :AA074748 es t <221> misc-feature <222> 258. .517 <223> homology id :N93603 est <221> misc-feature <222> 208. .251 <223> homology id :N93603 est <22 l>-misc-feature <222> 163. .202 <223> homology id :N93603 est <221> misc-feature <222> 90. .125 <223> homology id :N93603 es t <221> misc-feature <222> 125. .363 <223> homology id :HSPD04938 est <221> misc-feature WO 99/25825 WO 9925825PCTIIB98/O1 862 <222> 353. .517 <223> homology id :HSPD04938 <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> est misc-feature 28. .227 homology id :AA074804 est misc-feature 265. .310 homology id :A.A074804 eat misc-feature 227. .263 homology id :AA074804 est misc-.feature 352. .385 homology id :AA074804 est <300> <400> 66 aaaaggaaag aggaagt tga ctgctacgag tcggacggag ctcaacaaga aggtysggag aaggcccaga aagagaa tgg agcgcgagga ccatgcgcat Cgctcgcgag ggaggcctcc ctgttgcagt ctcggcggct tcgcatgaca atctcggacc acccaacctg gggcaaatgg ccggagctgg cggcgtcaga gcagctccag gagcgcgccc gacagcagct g atg gac gga cac tgg Met Asp Gly His Trp aaaggtgct t accgaccatg tgccggggat agaggcgc tg tcg gct Ser Ala *cgg cgc *Arg Arg ccc gtg Pro Val gc: Ala agg Arg ttc tct gca ctg Phe Ser Ala Leu acc Thr -30 cgt Arg gtg act gca atg tca Val Thr Ala met Ser -25 tcc tgg gct Ser Trp Ala agt tcc tca Ser Ser Ser agc Ser cca Pro cgg att cct tct ctg ccg ggg agc Arg Ile Pro Ser Leu Pro Gly Ser tgc tgg gcc tgg Cys Trp Ala Trp tgg tac ccg gac acc aca tcg ttt cca ttg agg Trp Tyr Pro Asp Thr T1hr Ser Phe Pro Leu Arg 5 1 tgc aga ggg aga gtc tgaccgggcc tccgtatctc t Cys Arg Gly Arg Val tttcagactt cattaaactt atgaccaaaa aaaaaaaaaa <210> 67 <211> 783 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 381. .629 <223> Von Hei-ine mat~rix gaccacgat ggcgcttacc <221> <222> <221> <222> <221> <222> <223> score 8.60000038146973 seq LELLTSCSPPASA/SQ polyA-.signal 736. .741 p0 lyA.s it e 770. .783 misc..feature 207. .263 homology WO 99/25825 WO 9925825PCTIIB98/OI 862 id :AA351230 est <300> <400> 67 agggacttcc agaactgtgc ccgggg tagg ggatggcgga cctttacccg tgatcgggtt ag tgLagaga cag gct gga Gin Ala Gly ccc gag ttc Pro Giu Phe ggcctcgctg tgggaagga t gttttgagcc gacgaaggac ggaggag tgg cccaaaccag ggcctctcac gcgtggacgt ttgtggtggg gcgtgttggt ccgcgctctc ggtagggcga ctggggctca cctccgcacc gttgtaggac cgtgggagct gccccacgcg gcctcgtcct gccaacggtC gcagcgcaga tgttggtgac cttcaaggat gtggcegtga agacagctgg acctggccca gaggaccctg taccgagagg agttggtcca cctgctagag catgggcagg agctgtggat atg cta cct gtg cag agt ttc act ctt gtt gcc Met Leu Pro Val Gin Ser Phe Thr Leu Val Ala gtg cag tgg cgc Val Gin Trp Arg cat His -65 tgc Cys ctc agc tCa Leu Ser Ser ctc agc Ctc Leu Ser Leu Ctg caa Leu Gin ctt ctg cct Leu Leu Pro aag gga ttc Lys Giy Phe agt agc tgg gat Ser Ser Trp Asp tac agg Tyr Arg 120 180 240 300 360 413 461 509 557 605 653 701 754 cgc cca cca Arg Pro Pro gag Glu cca Pro -35 cat His cc9 gct ggt Pro Ala Gly ttt gta ttt tta Phe Val Phe Leu gta Val acg ggg ctt Thr Gly Leu gtt ggc cag Val Gly Gin ggt ctt gaa ctc Gly Leu Glu Leu agt gct geg att Ser Ala Ala Ile tca tgt agt Ser Cys Ser gtg agc cac Val Ser His aat tta aga Asn Leu Arg ttg acc Leu Thr aca ggc Thr Gly cca Pro gtg Val1 gcc tct gcc tcc Ala Ser Ala Ser ccc ggc aaa aaa Pro Gly Lys Lys 1 aaa Lys ctg ctt aag Leu Leu LYS gtt gaa aag aaa Val Giu Lys Lys i5 aaw ttg ctg aeg Xaa Leu Leu Thr gra ata aaa acy taataaaact accacccgaa Xaa Ile Lys Thr ggaatgaaaa aaccaaaaaa aaaaaaaaa <210> 68 <211> 996 <212> DNA <213> Homo sapiens <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig..peptide 140. .205 Von Heijne matrix score 5.90000009536743 seq IILGCLJALFLLLQ/RK polyA.signal 965. .970 polyA-.si te 984. .996 misc-feature 676. .959 homology id :AA399103 est misc-feature 609. .679 homology id :AA399103 est.

misc-feature 225. .433 homology id :AA398040 WO 99/25825 WO 9925825PCT/IB98/01862 est <221> misc-feature <222> 43]. .563 <223> homology id :AA398040 <300> <400> 68 aacagttacg ccgtagcccai gaaggtgctg aaggagagct gcaaaagttg cagcagaaag gttgggagtc Ccgacaggtt Cagaaaagaa gcaagggacg gcaggactgt ttcacacttt tctgcttctg gacaaaaac aug gaa cta att tCC cca aca gcg act ata atc Met Glu Leu Ile Ser Pro Thr Val Ile Ile Ile ctg ggt tgc ctt Leu Gly Cys Leu aga ccc ccg tgc Arg Pro Pro Cys gct ctg ttc tta ctc ctt cag cgg aag aat ttg cgc Ala Leu Phe Leu Leu Leu Gin Az-g Lys Asn Leu Arg aag ggc tgg att Ly's Gly Trp Ile cct Pro 15 ata, Ile t99 att gga gtc Trp Ile Gly Val 9ga tt Gly Phe gak ttt ggg Xaa Phe Giy gta tgt ggt Val Cys Gly aaa Lys cgt Arg gec cct Cta gaa Ala Pro Leu Giu ttt Phe gag aaa gca GJlu Lys Ala aga atc aag Arg Ile Lys tgc ttt ctt Cys Phe Leu ggc ava cgg Gly Xaa Arg ggt Gly ctc cag agg aga Leu Gin Arg Arg caa Gin 120 172 220 268 316 364 417 477 537 597 657 717 777 837 897 957 996 45D ttt taaactttct ttcattgact cttaagtgca gggctagaac acggggaaca Phe tacctgcttg cctcaaa caacaatatc ctgtgca, catttttgga agcagag tgtgtatgtg ttaagta agtttgtgaa aatgagt attgcttgca cagttgg tatttgatga tgatgta tagtagatgt ttttgtc ccttattaat gtytttt, attgtytaat aaatttg <210> 69 <211> 657 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 183. .338 <223> Von Hiine m.

cta aaa act aat aa t tcc ctc t tg aag tat aaggatc tag ttttgcgaaa aacyyttcgt gttttcagta tccgtatcca gtacacaata cattttcamt aaagtatctt ttttattcaa gatattaaaa tcmtytctga gaaa tgaaa t atttttactt aytgggaaag aaytggagtt gacaggctyt acggcccgaa ttaaatgtyt tttccagtca aaaaaaaaa aktcctctac acaactgcmg cmtcgaagt t ataaagtgta aacaccaaag gtatttttag gagamtagta gagcacttta caaatatttt tsacrrttra cgtgcatcga aagt tccaaa atccaattta tattgtacaa ctgacgttgt atcctccttg aggaacagac atggtatttg <221> <222> <221> <222> <221> <222> <223> ariv score 3.79999995231628 seq VMLETCGLLVSLG/QS polyAsignal 620. .625 polyA site 644. .657 misc-feature 207. .263 homology id :AA357230 <300> <400> 69 agggacttcc ggcctcgctg gcgtggacgt ttgtggtggg gcgtgttggt ccgcgctctc agaactgtgc tgggaaggat ggtagggcga Ctggggctca CCtccgcacc gttgtaggac ccggggtagg gttttgagcc cgtgggagct gccccacgcg gcctcgtcct gccaacggtc gg atg gcg gag acg aag gac gca gcg cag atg ttg gtg acc ttc aag Met Ala Glu Thr Lys Asp Ala Ala Gin Met Leu Val. Thr Phe Lys 120 180 227 WO 99/25825 gat gtg get Asp Val Ala gcc Cag agg Ala Gin Arg PCTIIB98/OI 862 gtg acc ttt acc Vai Thr Phe Thr cgg Arg -30 gag Glu 72 -45 gag gag tgg Giu Giu Trp gtg atg ctg Val Met Leu aga cag Arg Gin gag acc Glu Thr ctg gac ctg Leu Asp Leu tgt ggg ett Cys Gly Leu acc ctg tac Thr Leu Tyr ctg gtt Leu Val tca eta ggg eaa Ser Leu Gly Gin atc Ile att tgg ctg Ile Trp Leu 5 Cat ata His Ile aaa ctg gee tca Lys Leu Ala Ser 1 cct Pro g9a agg aaa Gly Arg Lys ttc act aac Phe Thr Asn aca gaa aac eag Thr Clu Asn Gin tc:g cct gat gag Ser Pro Asp Giu aag Cc gag geg egg ttg get eca Lys Pro Glu Val Trp Leu Ala Pro 35 egacgccatc aaggaegc tggttctctg gtcctcaggc eggecceca tagggatgct cceccatgge tcaatccatc ctcecacctt aaaaaaaaaa <210> 70 <211> 416 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 140. .205 <223> VJon I4ejiine man-Jv 9gc ctg ttc GlY Leu Phe tccttCtte gggegccgca ggaataaatg gge gCC gca gcc cag Gly Ala Ala Ala Gin ttggetccagg Cttcggatt gceegaceg gggcagcagg CCtttc acaatgagaa <221> <222> <221> <222> <221> <222> <223> score 5.90000009536743 seq IILGCLALFLLLQ/RK polyA.s ignal 383. .388 P0lYA..Site 405. .416 misc...feaeure 225. .316 homology id :AA398040 ese 275 323 371 419 467 5 27 587 647 657 120 172 220 268 316 364 416 <300> <400> aaeagttacg aaggagagec geaaaagttg ccgtagccca cagaaaagaa gcaagggacg gaaggtgctg gaeaaaaac atg gaa eta Met Giu Leu cagcagaaag gttgggagtc cegacagget gcaggactgt ttcacaecttt tctgcttctg att ecc cca aca gtg ace ata atc Ile Ser Pro Thr Val Ile Ile Ile ctg ggt Leu Gly aga ccc Arg Pro gag tt Giu Phe tgc cec get Cys Leu Ala ccg tgc ate Pro Cys Ile ggg aaa gee Gly Lys Ala etg tee Leu Phe -5 aag ggc Lys Gly tta etc: Leu Leu ctt eag cgg Leu Gin Arg aag aat ttg cge Lys Asn LeU Arg tgg act cet egg att gga gtt Trp Ile Pro Trp Ile Gly Val gga ttt Gly Phe cctceta gaa Pro Leu Glu tat gga eca Tyr Gly Pro gtt act gaa Val Thr Glu <210> 71 <211> 543 ata Ile tee Phe 30 get Ala gag aaa gca Giu Lys Ala aga aec aag Arg Ile Lys atg acc tee Met Thr Phe ttt aca gtc Phe Thr Val aeg gga aac Met Gly Asn cga Arg gaa gga agg Glu Gly Arg aae taacgtgttt etaaaatcca aaaaaaaaaa a Asn WO 99/25825 <212> DNA <213> Homo sapiens <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig..peptide 129. .176 Von Heijne matrix score 4.80000019073486 seq SLFIYIFLTCSNT/SP polyk..signal 513. .518 PolYA-site 530. .543 misc..feature 264. .500 homology id :AA534039 est misc-.feature 205. .315 homology id :T82645 est mi se..fea cure 295. .382 homology id :T82645 est misc-feature 375. .405 homology id :T82645 est PCTIIB98/01862 73 caccttteag geagggasgn gatgagcttc aacatasgta tctattgaaa aggaagcagt 120 ate tat ata ttt ttg aca, tgt age 170 Ile Tyr Ile Phe Leu Thr Cys Ser <300> <400> 71 actgteceat tcctccccct acaacacaca cagcecaag agtggaggct gceacatcct gtgtatct acg att ata tct ctg ttc Met Ile Ile Ser Leu Phe aae ace tet Asn Thr Ser J. j-10 eca tet tat caa gga act caa Pro Ser Tyr Gin Gly Thr Gin agt Se r eta Leu gee eag tgg tgg Ala Gin Trp Trp cet Pro 20 t ta Leu ttg aca ggt agg Leu Thr Gly Arg ccc ggt etg ggt etc ec Leu Gly Leu Gly Leu Pro agg atg cag tge tge agg Arg Met Gin Cys Cys Arg 25 ttc ect ttt eec etc cac Phe Pro Phe Pro Leu His etc aca ac tee tgg gac Leu Thr Ile Ser Trp Asp ttt tgt ccc Phe Cys Phe ttg Leu ga t Asp caa aae tgt Gin Asn Cys ctt Leu 40 etg att eag Leu Ile Gin tgg get gag Trp Ala Giu ccc tgt gag Pro Cys Giu ctg gtt Leu Val 55 tat tet Tyr Ser ggg get teg Gly Ala Ser etc Leu ccc Pro taaecatact gtctcctcc eteectecct cecttgcct aaaccatcaa gtgaaaaaaa cccectgec acccagcagt taccecca gcatgectc ggcatatatt gtgcctcatt tatgctgcaa atataacatc aaaaaaaa <210> 72 <211> 605 <212> DNA <213> Homo sapiens <220> <221> sig..peptide WO 99/25825 WO 9925825PCTIIB98/01862 74 <222> 285. .341 <223> Von Heijne matrix score 5.59999990463257 seq PTLCVSSSPALWA/AS <221> polyA~signal <222> 575. .580 <221> polyA-Site <222> 592. .605 <221> misc..feature <222> 53. .296 <223> homology id :W07033 ese <221> misc..feature <222> 348. .432 <223> homology id :W07033 est <221> misc-feature <222> 435. .497 <223> homology id :W07033 est <221> misc-feature <222> 293. .337 <223> homology id :W07033 es t <221> misc-..feature <222> 521. .560 <223> homology id :W07033 est <221> misc-feature <222> 489. .520 <223> homology id :W07033 est <221> misc-feature <222> 15. .337 <223> homology id :AAl51004 est <221> misc-feature <222> 348. .412 <223> homology id :AA151004 es t <221> misc-feature <222> 434. .485 <223> homology id :AAI100O4 est <221> misc-feature <222> 83. .324 <223> homology id :AA476506 est <221> misc-feature <222> 347. .560 <223> homology id :AA476506 est WO 99/25825 PCT/IB98/01862 <221> misc-feature <222> 16. .347 <223> homology id :W56567 est <221> misc-feature <222> 350. .405 <223> homology id :W56567 est <221> misc-feature <222> 433. .470 <223> homology id :W56567 est <221> misc-feature <222> 15. .296 <223> homology id :AA147584 est <221> misc-feature <222> 348. .421 <223> homology id :AA147584 es t <221> misc-feature <222> 293. .337 <223> homology id :AA147584 eat <221> misc-feature <222> 419. .453 <223> homology id :AA147584 est <221> misc-feature <222> 2.-338 <223> homology id :AA281959 est <221> misc-feature <222> 350. .432 <223> homology id :AA281959 eat <300> <400> 72 aacgcctwta agacagcgga actaagaaaa gaagaggcct gtggacagaa caatcatgtc tgactccctg gtggtgtgcg aggtagaccc agagctaaca gaaaagctga kgaaattccg 120 cttccgaaaa. gagacagaca atgcagccat cataatgaag gtggacaaag accggcagat 180 ggtggtgctg gaggaagaat ttcagaacat ttccccagag gagctcaaaa tggagttgcc 240 ggagagacag cccaggttcg tggtttacag ctacaagtac gtgc atg acg atg gcc 296 Met Thr Met Ala gag tgt cct acc ctt tgt gtt tca tct tct cca gcc ctg tgg gct gca 344 Glu Cys Pro Thr Leu Cys Val Ser Ser Ser Pro Ala Leu Trp Ala Ala -10 agc gaa aca aca gat gat gta tgc agg gag taaaaacagg ctggtgcaga 394 Ser Glu Thr Thr Asp Asp Val Cys Arg Glu cagcagagct cacaaaggtg ttcgaaatcc gcaccactga tgacctcact gaggcctggc 454 tccaagaaaa gttgtctttc tttcgttgat ctctgggctg gggactgaat tcctgatgtc 514 tgagtcctca aggtgactgg ggacttggaa cccctaggac ctgaacaacc aaggacttta 574 aataaatttt aaaatgcaaa aaaaaaaaaa. a 605 WO 99/25825 WO 9925825PCT/IB98/01862 76 <210> 73 <211> 864 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 136. .444 <223> Von Heijne matrix score 4.90000009536743 seq VYAFLGLTAPSGS /KE <221> polyA~signal <222> 835. .840 <221> polyAsite <222> 851. .864 <221> misc..feature <222> 222. .456 <223> homology id :AA136758 est <221> misc..feature <222> 557. .648 <223> homology id ;AA136758 est <221> misc-feature <222> 501. .571 <223> homology id :AA136758 est <221> misc-feature <222> 130. .456 <223> homology id :AA393612 est <221> misc-feature <222> 88. .130 <223> homology id :AA393612 es t <221> misc-feature <222> 501. .538 <223> homology id :AA393612 est <221> misc-feature <222> 130. .458 <223> homology id :R59039 est <221> misc-feature <222> 71. .130 <223> homology id :R59039 est <221> misc-feature <222> 557. .716 <223> homology id :W48624 est <221> misc..feature <222> 365. .456 <223> homology id :W48624 WO 99/25825 PCT/IB98/01862 77 est <221> misc-feature <222> 501. .571 <223> homology id :W48624 est <221> misc-feature <222> 716. .751 <223> homology id :W48624 est <221> misc...feature <222> 222. .458 <223> homology id :AA136810 eat <221> misc-feature <222> 501.-581 <223> homology id :AA136810 es t <221> misc-feature <222> 587. .668 <223> homology id :AA136810 eat <221> misc...feature <222> 130. .419 <223> homology id :T35647 es t <221> misc feature <222> 59. .130 <223> homology id :T35647 eat <221> misc-feature <222> 557. .852 <223> homology id :HtMO93FO6A eat <221> misc-feature <222> 501. .571 <223> homology id :HLM093FO6A es t <221> misc-feature <222> 130. .384 <223> homology id :T35666 est <300> <400> 73 aaagttctcc ttccaccttc ccccaccctt ctctgccaac cgctgtttca gcccctagct ggattccagc cattgctgca gCtgctccac agcccttttc aggacccaaa caaccgcagc 120 cgctgttccc caggr atg gtg atc cgt gta tat att gca tct tcc tct ggc 171 Met Val Ile Arg Val Tyr Ile Ala Ser Ser Ser Gly -100 tct aca gcg att aag. aag aaa caa caa gat gtg ctt ggt ttc cta gaa 219 Ser Thr Ala Ile Lys Lys Lys Gln Gln Asp Val Leu Gly Phe Leu Glu -85 gcc aac aaa ata gga ttt gaa gaa aaa gat att gca gcc aat gaa gag 267 Ala Asn Lys Ile Gly Phe Glu Glu Lys Asp Ile Ala Ala Asn Glu Glu wo 99/25825 WO 9925825PCT/IB98/01862 cgg aag tgg Arg Lys Trp atg Met c tg Leu aga Arg gaa aat gta Glu Asn Val cc t Pro -50 ttc Phe -65 gaa aat agt Glu Asn Ser aat gaa agc Asn Glu Ser aca ggt aac Thr Gly ASn ggg gac tat Gly ASP Tyr gcc ttc tta Ala Phe Leu ccc Pro ga t Asp cca cct cag Pro Pro Gin att Ile cga cca gcc Arg Pro Ala cag tat cgc Gin Tyr Arg gca gtg tat Ala Val Tyr 315 363 411 459 gcc ttc tt Ala Phe Phe gaa gcc Glu Ala -20 cca tct Pro Ser aga gaa aat Arg GiU Asn aa t As n ggC tCg aca Gly Lou Thr gcc Ala ggt tca aag gas gca vgda agg GlY Ser Lys Glu Ala Gly Arg tgc sag cas sgc agc aag cca tgaaccttgs gcsctgtgct tttaagcatc Cys Lys Gin Ser Ser Lys Pro ctgaaaastg agtctcc aataggstta atgttga aatacaaaat taaastt taaaattata ttaatsa gcatacacca atgattt acctaagtgt acytgca <210> 74 <211> 1033 <212> DNA <213> Homo sapiens <220> att as t tga gta tac gcttttatsa satagat tag acattatggt gatatygtag aaagaaaaca ttacattaaa.

as tagcagaa ttgggttttc gattatggtg aaatagtgtt CCCttcctc aaggagaaag ttagctttgc acatgcaaac aggagaatgg gttacctgcc Cttytgccat agaaaaaaaa Sttcaaaaga amtcaaaa tg gatattaaca aagccatcct tamtatggca aa 570 630 690 750 810 864 <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig..peptide 200. .427 Von Heijne matrix score 4.69999980926514 seq LIVYLWVVSFIAS/SS PolyA..signal 1001. .1006 polyk..sjte 1022. .1033 misc-feature 55. .406 homology id :AA056667 est misc-feature 397. .487 homology id :AA056667 est misc-feature 527. .584 homology id :AA056667 est misc-feature 482. .531 homology id :AA056667 es t misc..feature 581. .634 homology id :AA056667 est.

<221> misc-feature <222> 397. .700 WO 99/25825 WO 9925825PCTIIB98/01862 79 <223> homology id :AA044187 eat <221> misc-feature <222> 222. .406 <223> homology id :AA044187 est <221> misc...feature <222> 693. .748 <223> homology id :AA044187 est <221> misc..feature <222> 68. .406 <223> homology id :AA131958 es t <221> misc...feature <222> 397. .517 <223> homology id :AA131958 est <221> misc-feature <222> 510. .558 <223> homology id :AA131958 est <221> misc-feature <222> 77. .531 <223> homology id :W95957 es t <221> misc-feature <222> 527. .558 <223> homology id :W95957 est <221> misc-feature <222> 397. .586 <223> homology id :AA041216 est <221> misc-feature <222> 286. .406 <223> homology id :AA041216 eat <221> misc-feature <222> 582. .700 <223> homology id :AA041216 est <221> misc-feature <222> 77. .406 <223> homology id :W95790 est <221> misc-feature <222> 397. .539 <223> homology id :W95790 est WO 99/25825 WO 9925825PCT/IB98/01862 <221> <222> <223> misc-feat'ure 474. .760 homology id :AA461134 est <221> rnisc...feature <222> 788. .940 <223> homology id :AA461134 <300> <400> 74 aagacgaggt catgaatcat gtgacggtgg cctgaagtga cagcggagag aaccaggcag tgcgagagaa gggggttcat catggcggat ccaagtgr.g aagggctcc atg cca tt~g Met Pro Leu Cttgaggagg aacctgtct taaagctgtc cccagaaacc ccaggcgtgg agattcgatcc gacctaaagc garttcttgta taaaaagtta ttg tgt cag ata gag atg gag tac Leu Cys Gin Ile Glu Met Giu Tyr ctg rtta tta aag tgg caa atg aca atg Ct~c cag jeu C tg Leu L.eu iLeu Lys Trp Gin -60 ct~t Leu Met Thr Met Leu Val Ser Tyr Gin -55 cag Gin agc at~g ct~t tgc Ser Met Leu Cys acc aag gaa gca Thr Lys Glu Ala cca Pro ttg ccc ttg Leu Pro Leu caa Gin gac Asp aac Asn 120 180 232 280 328 376 424 472 -35 ttg gac ttt Leu ASP Phe cca Pro aaa Lys ata aaa gt~a Ile Lys Val atc gtt tac Ile Val Tyr tca Ser -25 crt: Leu agg tgg ttc aat tta Arg Trp Phe Asn Leu agc agc agt gcc aat Ser Ser Ser Ala Asn tct gtt act ata Ser Val Thr Ile tgg gtg gtg agt Trp Val Val Ser aca oct acc Thr Pro Thr tt~c ata gcc Phe Ile Ala aca Thr -10 gga cta Gly Leu att gt~c agc ota gaa aag gaa Ile Val Ser Leu Glu Lys Giu Ott Leu gct cca tt~g ttt Ala Pro Leu Phe gaa Glu taatctgaca gtggttt agcaatcttt agactac caacttatac taaagag ttCtggtgt~a gggtctt Ctatcaacag Ctcccat tggatcagaa tcaaacti aatatgccca ggottgc tttttyttca gattatg aaaagtaata aaatcag <210> 75 <211> 499 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 68.-.133 <223> Von Heiine m~ ca a8 ct t~c tt ta, gaa ct~g aga caa Glu Leu Arg Gin .g tgtgtacctt at a gcatatagat gti t tatttagtga gat a gttagtctgg tc t acattgatcc act a ataaagccaa ctt t tatttyttt~g cat c aatcactaaa aa~ gt~t gt~g Val Val ct tca tta catgtgct %atttata :ctaggga icagatat :tgagccg ttttattg :tgagtga xaaaaaa gaa gt~t tct Glu Val Sei taacaacaca caagaaaggg gatagatcag tacoacagaa ggatgagaga ttaagtgctg tgaataataa ggaacataaa atatcaatcc ttgctatatt atggttcagt ttytattcag ccaattgtac taaggacata atggcttggt 574 634 694 754 814 874 934 994 1033 score 9.80000019073486 seq LVVFCLALQLVPG/SP <221> PolyA..signal <222> 472. .477 <221> polyA_site <222> 490. .499 <300> <400> aaaoagcagt gcctggtcaa acccagcaac ccrttggccag aacttaotca cccatcccac tgacaoc atg aag cot gtg ct~g cct ctc cag ttc ctg gtg gtg ttc tgc WO 99/25825 PCTIIB98/01862 81 Gin Phe Leu Val Val Phe Cys Met Lys*Pro Val Leu Pro Leu cta gca ctg cag Leu Ala Leu Gin 9t9 cct ggg agt Val Pro Gly Ser ccc aag Pro Lys cag cgt gtt Gin Arg Val ctg aag Leu Lys tat atc Tyr Ile cac ctg His Leu ttg gaa cct cca ccc tgc ata tca gca cct gaa aac tgt act Leu Glu Pro Pro Pro Cys Ile Ser Ala Pro Giu Asn Cys Thr 157 205 253 301 tgt aca atg cag Cys Thr Met Gin gat tgc gag Asp Cys Glu gga ttt cag tgc Gly Phe Gin Cys tcc Ser tcc ttc tgt ggg Ser Phe Cys Gly 30 ata Ile tgt C ys cgc Arg gtc tgt tca Val Cys Ser aac aga atc Asn Arg Ile aaa Lys cac His tca Ser 50 gtc Val1 ac ttt caa Thr Phe Gin Cag Lys aac As n aag ggc cc& Lys Gly Ser gaa Giu 65 atc atg cct Ile Met Pro gcc Ala tgaggcatat ttcctagatc Cttttgcctc aaggtattga gaagcaagaa actggaggcc ttggcaataa aggctaatct accaaaaaaa <210> 76 <211> 978 <212> DNA <213> Homo sapiens <220> tacgatgttt caatatctaa aaa tttCttggtc cacctttagg cctgcaaatc gtttttgagt 406 466 499 <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig...peptide 274. .399 Von Heijne matrix score 5.19999980926514 seq LLFDLVCHEFCQS /DD polyk..Signal 943. .948 polYA..Site 966. .978 misc-feature 335. .518 homology id :AA206225 est misc-feature 225. .274 homology id :AA206225 es t misc-feature 812. .861 homology id :AA206225 est misc-feature 186. .224 homology id :AA206225 es t misc-feature 708. .748 homology id :AA206225 est misc-feature 276. .314 homology id :AA206225 WO 99/25825 WO 9925825PCTIIB98/01862 82 est <221> misc-feature <222> 146. .176 <223> homology id :AA206225 est <221> misc-feature <222> 879. .909 <223> homology id :AA206225 est <221> misc..feature <222> 182. .518 <223> homology id :C15003 es t <221> misc..feature <222> 708. .748 <223> homology id :C15003 est <221> misc-feature <222> 182. .517 <223> homology id :HUM4O7El1B es t <221> misc-feature <222> 170. .202 <223> homology id :AA544037 est <221> misc...feature <222> 517. .595 <223> homology id :HTMOOTW17O es t <221> misc..feature <222> 596. .665 <223> homology id :HUMOOTW170 es t <221> misc-feature <222> 697. .748 <223> homology id :HUMOOTW170 est <221> misc-feature <222> '805. .861 <223> homology id :HtMOOTW17O est <221> misc-feature <222> 212. .369 <223> homology id :HM1l9EO8B es t <221> misc-feature <222> 406. .493 <223> homology id :HUM169EOB es t <221> misc...feature <222> 542. .595 WO 99/25825 WO 9925825PCTIB98/01862 <223> homology id :HUMOOTWi12 est <221> misc-feature <222> 697. .748 <223> homology id :HtJMO0TWii2 est <300> <400> 76 accaggaaca ttgacttgct taatgttaag tctgaagagc cgttctgaaa tccagctatt ggtgaaggtg azatgggtcag agccagtgt t atccagaatg tatgatagco ggggaggt tg aaatggggc t tcggcttgtg gcttgatgt: ttgcttcat tetgtcaagt tcaacaaatg tggacaagct ctttgatttg gatgagaaac gctcagcctc tggaccaacc ccaggaagag ccctgtatac ttgaagctgc caaacaagta tac atg cac att tta caa ctg ctt Met His Ile Leu Gin Leu Leu 120 180 240 294 342 390 act Thr gga Gly aca gtg gat gat Thr Val Asp Asp gga Gly aat Asn att caa gca att Ile Gin Ala Ile cat tgt cc: gac His Cys Pro Asp aaa gac at: Lys Asp Ile tg Trp ga t Asp tta ct: ttt Leu Leu Phe gac Asp ctt Leu gtc tgc cat Val Cys His tgc cag tct Cys Gin Ser cta gcc tct Leu Ala Ser gag caa gag Glu Gin Glu gat Asp 1 gtt Val1 tat Tyr cca gcc atc att Pro Ala Ile Ile caa gaa cag aaa aca gtg Gin Giu Gin Lys Thr Val ttt tca gtg Phe Ser Val 20 cta aag ata Leu Lys Ile 35 tct gcc atc Ser Ala Ile tat Tyr gcc Ala tca cag act Ser Gin Thr gaa aaa gta Glu Lys Val ga t Asp 40 Ctt cct cta att Leu Pro Leu Ile agc Ser gac Asp cca Pro ctc att cgg Leu Ile Arg gtc tta Val Leu gga gtc Giy Val caa aat atg Gin Asn Met cag tgt cag aaa Gin Cys Gin Lys aaa Lys gag aac tcg Glu Asn Sex aacccaagat ctaataattt tgagcaaaca gggtggaaga gaaaaaaamc sstgtbcaag <210> 77 gca Ala taacacagag gaaactaaaa ggactgattt gatttccact ttcaggcatt gaagtgttcc ttttattaaa ttcccaagtt aaaataaact tgaaaatctt aacaaaggag tctgcaatt atcctacgtg tgaaggt tca ttatttttct aaaaggatat acggtggc tc caaaarcctt aagttgataa gaas taaaaa cactgaaaaa tgttatggtg agggagtaaa cttctttcta ggcgcttgct rscktgaatg aaaaaaaa aagt ttc tg t ggaaggccgt tagcccctgt kgatgacttg ggaattact 690 750 810 870 930 978 <211> <212> <213> <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> 587

DNA

Homo sapiens sig-peptide 421. .465 Von Heijne matrix score 3.90000009536743 seq LVPLGQSFPLSEP/RC polyk-signal 553. .558 polyA..site 575. .587 misc..feature 182. .322 homology id :T35951 est WO 99/25825 WO 9925825PCTJIB98/OI 862 84 <221> misc..featire <222> 32. .132 <223> homology Ad ;T35951 est <221> misc-feature <222> 136. .193 <223> homology id :T35951 est <221> misc..feature <222> 182. .322 <223> homology id :T35949 est <221> misc-feature <222> 32. .132 <223> homology id :T35949 es t <220> misc..feature <222> 136. .193 <223> homology id :T35949 es t <221> misc-feature <222> 136,..299 <223> homology id :AA381111 est <221> misc-feature <222> 32. .132 <223> homology Ad :AA381111 est <221> misc-feature <222> 136. .322 <223> homology id :AA381001 est <221> misc-feature <222> 85. .132 <223> homology id AA381001 est <221> misc-feature <222> 182. .322 <223> homology id :H-SCZQEO41 est <221> misc-feature <222> 136. .193 <223> homology id :HSCZQEO41 est <221> misc..feature <222> 82. .132 <223> homology id :HSCZQEO41 est <221> misc-feature <222> 316. .428 <223> homology WO 99/25825 WO 9925825PCT/IB98/OI 862 id :AA477628 est <:221> misc..feature <222> 475. .554 <:223> homology id :AA477628 est <221> misc-feature <222> 182. .322 <:223> homology id :HSC34G011 est <:221> misc..feature <222> 136. .192 <223> homology id :HSC34G011 es t <221> misc-.feature <222> 41. .119 <223> homology id :AA090647 est <221> misc-feature <222> 136. .184 <223> homology id :AA090647 est <221> misc-feature <222> 316. .426 <223> homology id :AA505962 es t ':300> <400> 77 aatcatttt gaaaccagaa tkwgtgtgtk aggggaaccc gctcgtagcg agaagatgcc agagctattt atg cgt ctt Met Arg Le.

tgt ctt caa Cys Leu Glr acg gtt gtt Thr Val Val tcactcctcc gaaaaatatg gaagtycckg gctgttcaga ccgggccttc ccactccagc ccttccacag ctcctaggtc agacggggaa ctcaggtgtt gctgtgactg tctcctcgtc ctctggactg ggggccttgc acacrttttca tcatcgtgtg aggtacagtg cggctgcact a tcatccaga ggggctctct agggaagggt gtc ccc ttg ggc Val Pro Leu Gly -10 cct gtg aaa tgg 1Pro Val Lys Trp gtg agg act gag Val Arg Thr Glu taaaaaaaaa aaaa cag tca ttt ccc Gln Ser Phe Pro -5 gat cat aat cac Asp His Asn His 10 tgt gtg gaa gtt Cys Val Glu Val 25 gaaaaagaat ctgcatcctg atgtgtgtgc tgcctttggc tgtttgatcg tggtggcttg cagagaagct gcccttggct gcagccagtg tccgggaggc tcagtggctg aatgtccagc ccaggacttg acatcttaag ctc tCt gag cct cgg Leu Ser Glu Pro Arg 1 tgc ctt acc tcc ctc Cys Leu Thr Ser Leu ttt cat aaa ctt tgg Phe His Lys Leu Trp, atg ct Met Lc :2 10> ':211> ':212> ':213> <:220> <:221> <:222> <223> a u gt Val 78 400

DNA

Homo sapiens sig..peptide 198. .278 Von Heijne matrix score 4.90000009536743 seq CLLSYIALGAIHA/KI WO 99/25825 WO 9925825PCTIIB98/01862 <221> polyAsigftal <222> 364. .369 <221> polyA..site <222> 387.-400 <300> <400> 78 aactttgcct tctgactcca gctcctctcc Ccttcctgaa tgt cta atc Cys Leu Ile 888 atc tg Lys Ile Cys gggtgtcr.tg cgttctgcac attccggagg accagcttcc CCatcaga tggaaaccag atggggcaac ggggtggttc tagtgcagac tgtagctg acctctagcc tgctcatttc cagCtCagaa attctactaa tggcgttt aaaggaa atg aac agg gtc cct gct gat tct cca aat atg Met Asn Arg Val Pro Ala Asp Ser Pro Asri Met tgt tta ctg agt tac ata gca ctt gga gcc atc cat gca Cys Leu Leu Ser Tyr Ile Ala Leu Gly Ala Ile His Ala -10 aga aga gca ttc cag gaa gag gga aga gca aat gca aag Arg Arg Ala Phe Gin Glu Glu Gly Arg Ala Asn Ala Lys ag ca t t 120 180 230 278 326 375 1 acg ggc gtg aga gct tgg tgc ata Thr Gly Val Arg Ala Trp Cys Ile ttggaatagc caaaaaaaaa aaaaa <210> 79 <211> 1166 <212> DNA <213> Homo sapiens <22 0> cag cca tgg gcc Gin Pro Trp Ala aaa taaagtttcc Lys <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig..peptide 167. .229 Von Heijne matrix score 5.59999990463257 seq LVLSLQFLLLSYD/LF poiyk..signai 1133. .1138 P0lYk..Site 1154. .1166 misc..feature 22. .377 homology id :AA306911 est misc..feature 424. .540 homology id :AA306911 es t misc-feature 376. .424 homology id :AA306911 es t <221> misc-feature <222> 4. .458 <223> homology id :AA417777 es t <221> misc-feature <222> 10. .447 <223> homology id :AA236327 est.

<221> misc..feature <222> 279. .714 WO 99/25825 WO 9925825PCT/1B98/01 862 87 <223> homology id :AA410332 est <221> misc-feature <222> 680.. 893 <223> homology id :N32991 est <221> misc..feature <222> 881. .1023 <223> homology es t <221> misc-feature <222> 1056. .1109 <223> homology id :N32991 es t <221> misc-feature <222> 1122.-1153 <223> homology id :N32991 est <221> misc-feature <222> 1024. .1054 <223> homology id :N32991 est <221> misc..feature <222> 703. .893 <223> homology id :N24951 es t <221> misc-feature <222> 881. .1023 <223> homology id :N24951 es t <221> misc-feature <222> 1056. .1109 <223> homology id :N24951 est <221> misc-feature <222> 13.22.-1153 <223> homology id :N24951 est <221> misc-feature <222> 1024. .1054 <223> homology id :N'24951 es t <221> misc-feature <222> 225. .563 <223> homology id :AA455215 est <221> misc_feature <222> 544. .631 <223> homology id :AA455215 est WO 99/25825 WO 9925825PCT/IB98/01862 <221> <222> <223> misc-featuire 629. .660 homology id :AA455215 -est <221> misc-.feature <222> 680. .793 <223> homology id :N66437 est <300> <400> 79 aatgacaacc gacgttggag ttcaaaggaa ctagaagccc ggaaetgtgg gatgtgccct tttggaggtg Ctecctcagt tgggggcceg ctt aga tc Ser rca.

er :gecttag agcaagggaa ~agggaga cagccaggag aaacaga aggaag atg Met ctg cag ttc ctg ctg Leu Gin Phe Leu .Leu fteagctctca :ggttcctg :tc cag L.eu Gin etg tc Leu Ser aCC agt aac Thr Ser Asn tat gac ec Tyr Asp Leu 1 gte atc eag Val Ile Gin tac Tyr ttt Phe age ctg gtg etc Ser Leu Val Leu gtc aat tce ttc Val Asn Ser Phe gaa ctg Giu Leu etc Leu att Ile caa aag act cet Gin Lys Thr Pro ctt. gtg etc Leu Val Leu ate att ttc Ile Ile Phe ttc ate ate cag Phe Ile Ile Gin ga t Asp aac Asn gca gte ctc Ala Val Leu ttc Phe ate atc Ile Ile etc atg tte Leu Met Phe ttc Phe aag Lys ace tce gte Thr Ser Val 30cca Phe Gin 120 175 223 271 319 367 415 463 511 559 607 get ggc ctg Ala Gly Leu etg aca get Leu Thr Ala atg aac Itta Met Asn Leu gte Val1 gtg Val1 etc eta ttc Leu Leu Phe tte aaa ggg Phe Lys Giy ace ate ate Thr Ile Ile gte tgg gte Val Trp Val tac ttt gee Tyr Phe Ala ege tgg aaa Arg Trp Lys etc Leu tc Ser cag Gin age ate tee ctt Ser Ile Ser Leu aac age ttc ata Asn Ser Phe Ile aga eta gca gea Arg Leu Ala Ala eat His tgg aca gat gga Trp Thr Asp Gly ctt caa Leu Gin atg ctg ttt Met Leu Phe gtg ttg tac Val Leu Tyr tac Tyr t ge Cys 110 tac Tyr tte tat aaa Phe Tyr Lys gee gta aga Ala Vai Arg eta Leu 120 t te Phe gat cet cac Asp Pro His t te Phe 125 agg Arg cag gac tet Gin Asp Ser t tg Leu 130 tgaectettg teaeactgat tgeagggaga gttggeecta etagetgtgt teagcattca ateggtetee agtgcggcat.

cteteetetc tgtaceattc tcttaeectt gtgaagettt, gateteecag wtgaccaaag acegcagaea tttagatttt aataaattga attyttgtte <210> 80 <211> 754 <212> DNA <213> Homo sapiens <220> <221> sig-peptide <222> 180. .383 :g ege aag5 au Arg Lys ggatactttt tgcatgggea agaaggaaga cettcettg attctcectg tcetttagcc gatgegaaga tataceaag caaaaaaaaa atg caa gtt Met Gin Val Cetteetgat aaeagetgga tecccctet ccttetacct accggecttt tgggaeagaa gtgatagtta geactttaaa aaaa agaageeaca ettteeaagg tgcaeaatta etgttccaee et tgcegagg ggaec teceg egntgctcct aaaatgtttt tttgctgctt aaggt teaga gagtgtece ceetteette gttetgtgge gececcaaag gaetgatcae ataaatagag 712 772 832 892 952 1012 1072 1132 1166 WO 99/25825 WO 9925825PCT/IB98/01862 89 <223> Von Heijn6 matrix score 4.59999990463257 seq LPFSLVSMLV'rQG/LV <221> polyAsignal <222> 722. .727 <221> polyA..site <222> 743. .754 <221> misc-feature <222> 116. .450 <223> homology id :W68799 est <221> misc-.feature <222> 593. .710 <223> homology id :W68799 est <221> misc..feature <222> 18. .117 <223> homology id :W68799 est- <221> misc-feature <222> 561. .598 <223> homology id :W68799 es t <221> misc-..feature <222> 48. .511 <223> homology id :AA149518 est <221> misc-feature <222> 593. .673 <223> homology id :AA149518 est <221> misc-feature <222> 535. .710 <223> homology id :W80356 est <221> misc..feature <222> 256.-.405 <223> homology id :W80356 est <221> misc-feature <222> 432. .511 <223> homology id :W80356 est <221> misc..feature <222> 392. .437 <223> homology id :W80356 est <221> misc-feature <222> 535. .710 <223> homology id :W80631 est <221> misc-feature WO 99/25825 WO 9925825PCTIIB98/01862 <222> 289. .437 <223> homology id :W80631 es t <221> misc..feature <222> 432. .511 <c223> homology id :W80631 est <221> misc..feature <222> 343. .511 <223> homology id :AA142865 est <221> misc..feature <222> 535. .710 <223> homology id :AA142865 est <221> misc..feature <222> 256. .341 <223> homology id :AA142865 es t <221> misc..feature <222> 248.-511 <223> homology id :AA405876 est <221> misc-feature <222> 21. .271 <223> homology id :AA405876 est <22 1> misc-feature <222> 121. .450 <223> homology id :W68728 est <221> misc-feature <222> 592. .710 <223> homology id :W68728 est <300> <400> aagacaggtg gggtactcgg gaagetggag cgggceggcg ggcetgetgg gcttggcaac gagggacteg gcctcggagg ctgggtcaag gagtaagcag aggataaaca actggaagga atg get tea gcg tct gct cgt gga aac caa gat Met Ala Ser Ala Ser Ala Arg Gly Asn Gin Asp -60 gtgeagteac gggggagcga cgacccagac cacacagaca gagcaagcac aaagtcatc aaa gat gee cat ttt Lys Asp Ala His Phe cca aaa tca aaa etg Pro Lys Ser Lys Leu cea-cca eca Pro Pro Pro cac atc cac His Ile His age aag eag agc ctg ttg ttt tgt Ser Lys Gin Ser Leu Leu Phe Cys 120 179 227 275 323 371 419 aga gea gag Arg Ala Giu ate Ile -30 tca aag att &tg Ser Lys Ile Met gaa Clu tgt cag gaa Cys Gln Glu gaa agt ttc tgg aag aga gct ctg ect ttt tct Glu Ser Phe Trp Lys Arg Ala Leu Pro Phe Ser -15 -10 gtc ace eag gga eta gte tac caa ggt tat ttg Val Thr Gin Gly Leu Val Tyr Gin Gty Tyr Leu ett gta age atg ctt Leu Val Ser Met Leu gca get aat, tet aga Ala Ala Asn Ser Arg WO 99/25825 ttt gga tca Phe Gly Ser ctt gga aag Leu Gly Lys PCT/IB98/01862 1 ttg ccc Leu Pro 5 aaa gtt gca ctt Lys Val Ala Leu 20 ttt gaa Phe Glu gta tca tac ata gga gta Val Ser Tyr Ile Gly Val 35 cag ctc cgt ggg gct ggt Gln Leu Arg Gly Ala Gly so 91 get ggt Ala Gly tgc cag Cys Gin ttt ggt Phe Gly ga t Asp etc ttg gga ttt gge Leu Leu Gly Phe Gly agt aaa ttc eat ttt Ser Lys Phe His Phe cew aca gca Pro Thr Ala aagcatggat taagtgagaa tgactttcga agtttttaa ttaaaatriaa aatacttcta taacaggcac tgcetcc gggagactct cagcctt acctctgaat ttgtaca atgtvaaaaa aaaaaaa <210> 81 <211> 709 <212> DNA <213> Homo sapiens <220> tta cctgtgagga atgcaaaata cag Cttcctaaat tCtgtgtctg cat ttaaaatttc aaggtgtaet <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> sig...peptide 179. .298 Von Heijne matrix score 4.30000019073486 seq ITLVSAAPGKVIC/EM polyA.s ignal 680. .685 polyA..site 697. .708 mise...feature 137. .291 homology id :AA121372 es t misc-.feature 6. .91 homology id :AA121372 es t misc-feature 318. .397 homology id :AA121372 es t misc-feature 95. .132 homology id :AA121372 es t misejfeature 460. .501 homology id :AA121372 es t misc-feature 432. .465 homology id :AA121372 est misc-feature 284. .313 homology id :AA121372 est WO 99/25825 WO 9925825PCT/1B98/01862 92 <221> misc-featuire <222> 254. .670 <223> homology id :AA614605 est <221> misc-.feature <222> 392. .658 <223> homology id :T55234 est <221> misc..feature <222> 271. .327 <223> homology id :T55234 est <221> misc-.feature <222> 358. .670 <223> homology id :AA121362 eat <221> misc-feature <222> 312. .344 <223> homology id :AA121362 est <221> misc-feature <222> 2. .102 <223> homology id :T53974 eat <221> misc..feature <222> 150. .258 <223> homology id :T53974 est <221> misc-feature <222> 95. .171 <223> homology id :T53974 est <221> misc-feature <222> 322. .628 <223> homology id :HSP002295 est <221> misc..feature <222> 445. .670 <223> homology id :AA454502 es t <221> misc-feature <222> 2.-102 <223> homology id :R09314 est <221> misc-feature <222> 95. .171 <223> homology id :R09314 es t <221> misc..feature <222> 150. .222 <223> homology WO 99/25825 WO 995825PT/IB98/01862 id :R09314 est <300> <400> 81 aaaa tcgcgg ggcggagaag tccagctctt accaccgggg ctgccakctc ggtgcgggct cttcgccctt ccgaagttcg ttcttgcgca gcctgactcc tgtgtccttc aagcccaaag cggcctCttg cgctcctagg tttcactaac tctggactt gctggaaaac cgtccacg atg Met aec agc atg act Thr Ser Met Thr cag Gin -35 gag Glu tet Ctg C99 gag Ser Leu Arg Giu gtg Val1 -30 aag Lys ata aag gcc atg Ile Lys Ala Met acc Thr aag gct cgc aat Lys Ala Arg Asn gct gct cct ggg Ala Ala Pro Giv ttt Phe aaa Lys aga gtt ttg Arg Val Leu gga Gly -15 a tg Met att act Ile Thr ctt gtc tct Leu Vai Ser gaa gag cat Glu Giu His gtg att tgt gaa Val Ile Cys Giu acc aat Thr Asn gat aac Asp Asn gca ata Ala Ile ata tca Ile Ser ggc act ctc Gly Thr Leu 15 aca atg gct Thr Met Ala aaa gta gaa Lys Val Giu

I

cac ggc His Gly ggt ttg aca Gly Leu Thr gcc acg tta gta Ala Thr Leu Val ctg cta tgc Leu Leu Cys acg Thr 35 gaa agg gga gca Glu Arg Gly Ala gga Gly gtc agt gtc Val Ser Val 30 a tg Met ccc Pro 120 178 226 274 322 3-70 418 466 514 562 608 668 709 aac ata acg Asn Ile Thr tac Tyr s0 gtt Val1 atg tca cct gca Met Ser Pro Ala aaa tta Lys Leu gga gag gat Gly Giu Asp ctt gca ttt Leu Ala Phe ata gca caa Ile Ala Gin ata Ile ac c Thr att aca gca Ile Thr Ala cat His 65 acc Thr ctg aag caa Leu Lys Gin gga aaa aca Gly Lys Thr gga aaa tta Gly Lays Leu tct gtg ggt Ser Val Gly ctg Leu 80 aac aag gcc Asn Lys Ala aca Thr gga aga cac Gly Arg His aaa cac ctg gga Lys His Leu Gly a ac tgagagaaca Bn 00 agatttgac tcaaacaatt gcagaatgac ctaaagaaac ccaacaatga atatcaagta t~ gtaatttttg aaataaacta gcaaaaccaa aaaaaaaaaa g <210> 82 <211> 243 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 100. .171 <223> Von Heiine matrix <221> <222> <221> <222> <221> <222> <223> <221> <222> <223> <221> <222> <223> score 3.70000004768372 seq ILFNLLIFLCGFT/NY polyAsignai 211. .216 POIYA-.site 230. .243 misc-feature 2. .164 homology id :H64488 es t misc..feature 2..164 homology id :AA131065 es t misc-feature 5. .164 homology WO 99/25825 WO 9925825PCT/1B98/OI 862 94 id :AA224847 est <221> misc-feature <222> 10. .164 <223> homology id :AA161042 est <221> misc..feature <222> 2. .84 <223> homology id :AA088770 es t <221> misc..feature <222> 104. .164 <223> homology id :AA088770 es t <221> misc-.feature <222> 10. .164 <223> homology id :AAl00852 est <221> misc-feature <222> 79. .164 <223> homology id :AA146774 es t <221> misc..feature <222> 79. .164 <223> homology id :AA1d46605 est <221> misc-.feature <222> 109. .164 <223> homology id :AA299239 es t <221> misc-feature <222> 158. .207 <223> homology id :AA037885 est <221> misc-feature <222> 160. .207 <223> homology id :AA480512 est <221> misc-feature <222> 160. .207 <223> homology id :AA468030 es t <221> misc..feature <222> 160. .207 <223> homology id :AA420727 est <221> misc-feature <222> 160. .207 <223> homology id :AA574382 est <221> misc-feature WO 99/25825 WO 9925825PCT/IB98/01862 <222> 160. .207 <223> homology id :AA133048 <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> est misc..feature 200. .229 homology id :AA469266 es t misc..feature 200. .229 homology id :AA550735 est misc..feature 200. .229 homol1ogy id :AA601071 es t misc..feature 200. .229 homology id :AA225190 es t <300> <400> 82 aactcagtgg caacacccgg ttcagaactc actgccaaga gagctgtttt gtcctttgtg gagcctcagc agttccctct gccctgaaca ggagccacc atg cag tgc ttc agc Met Gin C ttc att aag 8CC Phe Ile Lys Thr ggC ttc acc aac Gly Phe Thr Asn atg Met~ tat Tyr atg atc ctc ttc aat ttg ctc atc Met Ile Leu Phe Asn Leu Leu Ile -10 acg gat ttt gag gac tca ccc tac Thr Asp Phe Glu Asp Ser Pro Tyr 5 Ys Phe Ser ttt ctg tgt Phe Leu Cys ttc aaa atg Phe Lys Met 114 162 210 243 cat aaa cct His Lys Pro

I

gtt aca Val Thr atg Met taaaaaaaaa aaaaa <210> 83 <211> 829 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 346. .408 <223> Von Heiine matrix score seq SFLPSALVIWTSA/AF <221> polyAsignal <222> 792. .797 <221> polYA-.site <222> 817. .829 <221> misc..feature <222> 260. .464 <223> homology id :H57434 est <221> misc-feature <222> 118. .184 <223> homology id :H-57434 est WO 99/25825 PCT/IB98/01862 96 <221> misc-featuire <222> 56.. 113 <223> homology id :H57434 est <221> rnisc...feature <222> 454. .485 <223> homology id :H57434 est <221> misc..jeature <222> 118. .545 <223> homology id :N27248 es t <221> misc..feature <222> 65. .369 <223> homology id :H94779 es t <221> misc-feature <222> 471. .519 <223> homology id :H94779 est <221> misc..feature <222> 61. .399 <223> homology id :H09880 es t <221> misc...feature <222> 408. .452 <223> homology id :H09880 est <221> misc..feature <222> 60. .399 <223> homology id :H29351 est <221> misc-feature <222> 393. .432 <223> homology id :H29351 eat <221> misc-.feature <222> 260. .444 <223> homology id :AA459511 eat <221> misc-feature <222> 449. .545 <223> homology id :AA45951.

est <221> misc-feature <222> 117. .184 <223> homology id :AA459511 est <221> misc-feature <222> 122. .399 <223> homology WO 99/25825 PTI9/16 PCT/IB98/01862 id :T74091 est <221> misc-feature <222> 393. .434 <223> homology id :T74091 est <221> misc..jeature <222> 61. .378 <223> homology id :HSC3CBO81 est <221> misc-.feature <222> 118. .399 <223> homology id :T82010 eat <221> misc-feature <222> 268. .545 <223> homology id :W02860 es t <221> misc-feature <222> 268. .545 <223> homology id :N44490 est <300> <400> 83 actcctttta ctgatgccga c tcaaacggc gtt tgttgaa cgttcctgtt aagactaaca gcataggggc gttccgtctc ctagtgcttc gcagt tacca gagtacacgt ttttgtgaag ttcggcgcca tcgcgtcttt gcgcttccgg agaatcttca tcctgttgat ttgtaaaaca gcggccagcg tcc tggtccc agaaaatcag accctttccc t tacaaaagg gaaaacctgt Ctagtcggtc tggtaagtgc aggcaaagcg gasgnagatc cggtctaatt aattcctctg acaaaagcta attgagtaca tgcaggtatg agcaggtctg tagaa atg tgg tgg ttt Met Trp Trp Phe gta att tgg aca tct Val Ile Trp Thr Ser cag caa ggc ctc agt ttc ctt cct tca gcc ctt Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu -10 gct gct ttc ata ttt tca tac att act gca gta aca ctc cac cat ata Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala Val Thr Leu His His Ile gac ccg gct tta cct tat atc agt Asp Pro Ala Leu Pro Tyr Ile Ser gac act Asp Thr 25 aat att Asn Ile 10 ggt Gly aca gta gct cca Thr Val Ala Pro gaa aaa tgc tta Lys Cys Leu ggg gca atg cta Gly Ala Met Leu gcg gca gtc tta tgt caa Ala Ala Val Leu Cys Gln aaa tagaaatcag gaagataatt caacttaaag aagttcattt catgaccaaa Lys ctcttcagaa acatgtcttt acaagcatat ctcttgtatt gctttctaca ctg gtctggcaat atttctgcag tggaaaattt gatttagcta gttcttgact tgg tggtaaggtg ggcttttccc cctgtgtaat tggctacsac gtcttacttg agc gtaagttgaa ataaaatgat watgagagtg acacavaaaa aaaaaaa <210> 84 <211> 674 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 177. .233 <223> Von Heijne matrix ttgaatt ataaata caagttg WO 99/25825PCIB8O86 PCT/IB98/01862 98 score 6.09999990463257 seq LALLWSLPASDLG/Rs <221> PolyA signal <222> 644. .649 <221> polyAsite <222> 663. .674 <221> misc-feature <222> 194. .592 <223> homology id :AA496246 est <221> mnisc...feature <222> 1. .100 <223> homology id :AA496246 est <221> misc-feature <222> 99. .202 <223> homology id :AkA496246 es t <221> misc-feature <222> 187. .592 <223> homology id :AA476481 eat <221> misc-feature <222> 594. .661 <223> homology id :AA476481 est <221> misc-feature <222> 188. .592 <223> homology id :AA496245 eat <221> misc-feature <222> 594. .661 <223> homology id :AA496245 eat <221> misc-feature <222> 194. .444 <223> homology id :AA476480 eat <221> misc feature <222> 1.-102 <223> homology id :AA476480 es t <221> misc-feature <222> 99. .187 <223> homology id :AA476480 eat <221> misc-feature <222> 437. .592 <223> homology id :AA505488 eat <221> misc-feature <222> 594. .661 WO 99/25825 WO 9925825PCT/IB98/01862 <223> homology id :AA505488 est <221> misc-feature <222> 441. .592 <223> homology id :AA554685 <221> <222> <223> <221> <222> <223> <221> <222> <223> es t misc..feature 594. .661 homology id :AA554685 est misc..feature 414. .503 homology id :AA215595 es t misc-feature 510.-.539 homology id :AA215595 est <300> <400> 84 ataagtgaac cagaccaccc gggtgggtgg actagaagca agctgctgca cagagcctgg tgatggcatc tttgggagta tgtccacaag cacagtgatg gtggccaggg cttccaggtt gccctggacg ggggt tggag ggctggccag c tagccacgg Cctggg atg Met agc ccc ggc Ser Pro Gly ct~g ggc Cgg Leu Gly Arg 1 gcc ttg gcc ct~t Ala Leu Ala Leu gtc att gct gga Val Ile Ala Gly c tg Leu ct~c Leu tgg tcc ctg cca gcc tct gac TrP Ser Leu Pro Ala Ser Asp tgg cca cac act ggc gtt ct~c Trp Pro His Thr Gly Val Leu at~c cac ttg gaa aca agc cag tct ttt ct~g caa Ile His Leu Glu Thr Ser Gin Ser Phe Leu Gin 20 25 agc ata ttt ccc ct~c tgt tgt aca tcg ttg ttt Ser Ile Phe Pro Leu Cys Cys Thr Ser Leu Phe 40 aca gt~g ggt gga ggg agg gt~g ggg tct aca ttt Thr Val Gly Gly Gly Arg Val Gly Ser Thr Phe ggt cag ttg acc aag Gly Gin Leu Thr Lys tgt gtt tgt gtt gt~a Cys Val Cys Val Val gtt gca tgagtcgatg Val. Ala gggcattttg tcgaaaataa ttagttctgt attgattaag gctaagaatt agggaacata ataaataaac atgcaaacct 120 179 227 275 323 371 420 480 540 600 660 674 ggtcagaact ttagtat gcaccttggt aactaaa ttactgtaaa agcttgg4 gcaagggggc tcctctg ttaaaaaaaa aaaa <210> 85 <211> 478 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 179. .319 <223> Von Hi-ine m acg catgcgtcct ctgagtg cc ctctaatagc tataaaq gtt tatttttgt~a ggacttae ttg gagtaatgta aattgta raca rgc t Lat~g La t t

V

<221> <222> <221> <222> score seq SALLFFARPCVFC/FK poly...signal 461. .466 poiyA_site 465. .478 WO 99/25825 WO 9925825PCTJIB98/01862 t00 <221> misc-.featture <222> 2. .464 <223> homology id :AA310996 est <221> misc..feature <222> 8. .464 <223> homology id :AA312901 est <221> misc-feature <222> 2. .416 <223> homology id :AA401411 est <221> misc..feature <222> 2. .349 <223> homology id :R64030 es t <221> misc-feature <222> 56. .464 <223> homology id :AA400108 es t <221> misc-feature <222> 126. .273 <223> homology id :AA010825 es t <221> misc-feature <222> 2. .147 <223> homology id :AA010825 est <221> misc-feature <222> 358. .435 <223> homology id :AA010825 est <221> misc-feature <222> 78. .464 <223> homology id :AA504732 est <221> misc-feature <222> 90. .441 <223> homology id :H60506 est <221> misc-feature <222> 59. .349 <223> homology id :AA346780 est <221> misc-feature <222> 2. .331 <223> homology id :AA2812.67 est <221> misc-feature <222> 6. .236 <223> homology WO 99/25825 WO 9925825PCTIIB98/O1 862 <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> <221> <222> <223> id :R35805 es t misc-feature 232. .284 homology id :R35805 est misc-.feature 41. .307 homology id :H13784 est misc..feature 2. homology id :H13784 est misc-.feature 64. .280 homology id :AA128122 est misc-feature 293. .349 homology id :AA128122 est misc-feature 332. .385 homology id :AA128122 est misc-fea ture 163. .420 homology id :AA555127 es t <300> <400> aagrtccttcg ttacactggg C taaaaaac t atg aga ctc Met Arg LeL ctt gag ggc Leu Glu Gl cgccctccc caacgtggtt tgaagaaatt gccctcccca ggaatgtatc aaaaaggact ccgacatcat tggc tcagaa gctccagt ctatgata gaagaaac cct cca gca ctg Pro Pro Ala Leu cct Pro tca gga tat act Ser Gly Tyr Thr gate Asp tt~g Leu ~tc ctgcteggat ita ccaaaccegg :cc cctagtgc tct act gct Ser Thr Ala ttt tcg tct Phe Ser Ser ctc gtt tac Leu Val Tyr cca gcc Pro Ala tat Tyr -25 t tc Phe ctg aac caa aag Leu Asri Gln Lys ttt gct aga ccc Phe Ala Arg Pro ctt Leu tgt Cys tca gca ctt Ser Ala Leu get ttt tgc tt Val1 Phe Cys Phe 120 178 226 274 322 370 418 478 aaa Lys gca agc aaa atg Ala Ser Lys Met aca tac tca Thr Tyr Ser ccc caa ttt Pro Gin Phe 10 ata acc cct Ile Ile Pro gag aac Glu Asn ttc caa Phe Gin ctt ccc Leu Pro tac cca aca ttt cca TIyr Pro Thr Phe Pro ctg cat ggg agg ttc Leu His Gly Arg Phe ttaatgaaaa aaaaacaaaa taagactgga attaeggtgc tagattagta aacatgactt <210> 86 <211> 952 <212> DNA <213> Homo sapiens <220> WO 99/25825 WO 9925825PCT/IB98/OI 862 <221> <222> <223> sig-peptide 112. .237 Von Heijne matrix score 7.19999980926514 seq ILFSLSFLLVIIT/FP <,221> PolyA signal <222> 910. .915 <221> polyA..site <222> 940.-.952 <300> <400> 86 aatactrttct cctcteeeee etcecaagea cactgag.t agctccaaac eeatgaaaaa ttgceaagta taaazagcttc gctgcctgc teaagaa tga gag aat ttc Giu Asn Phe tet Ser gtc Val1 agg gC9 CcC tca, Arg Val Ser Ser cet Pro -35 gag aag caa gat Glu Lys Gin Asp Ct tcacactt g atg gat Met Asp gtg 99t Val Gly aac aat Asn Asn tet ttc Ctg Ser Phe Leu aaa egg ctt 9gt gta tgt gge tgg Lys Arg Leu Gly Val Cys Gly Trp -15 ttg gtg atc att 8CC ttc Ccc ate Leu Val Ile Ile Thr Phe Pro Ile 1 ate ctg ttt tee ctc Ile Leu Phe Ser Leu tcc ata tgg atg tgc Ser Ile Trp Met Cys ttg aag att tgatcctggt eetgccatgc atrnt-mn- Leu Lys Ile tgaeeteega aeagttaett taactaetea ggtagatgga etaakgteaa egatgteeat tektagggae aeaggaeett ageatecaga etktaettga ggaaateaaa gatgttegga ggeeaeeegg gaagsgagag ateeetgaag teageeteca cetgeagacc ttgageacgg eatgaatata etagagggea ataaagcetg aggtcybctt <210> 87 <211> 131 <212> PRT <213> Homo sapiens <220> aaagi, 117 165 213 261 310 370 430 490 550 610 670 730 790 850 910 952 geaaea ttee gttgtetatt eaageaacat gteeeeagat tgatgecaee ttcecgtgea eeaaggteet tggtgstggs tagecacega t tggtggegt gcggtagtca teeacaagag acagaatcta ttctgetgge et taggetgg gaaetggtgg gt tgeagaga tgeagetgaa tgagtytec gaagaatttt eagstatgat aaaaaaaaaa ateeteacca tagtgetgte tcaaaceaet acgagaagag gggatccggg teeatggeag ggagaaatga atagctytec aegattgtgt aaeeacaaga aa rgagaetceg teageagtgg e tgagaaa tg ategeceata tggeeegagt cegaggs tga atgsttccaa agstgsgsta t teetbtgce agsttbscaa <221> SIGNAL <222> -13. i <300> <400> 87 Met Leu Ala V.

Leu Giu Leu Leu Ser Pro Leu Gly Ser Leu Thr Ile Asp Pro 10 Val Leu His Val Pro -5 Gin Pro Pro Asn Leu Leu Gly Ala Leu Ser Phe Lys Thr Lys Leu Arar Met Met Leu 1 Glu Pro Pro Arg 25 Gin Gin Ala Leu Phe Giu Glu Giu Asn Phe Leu Val Gly Asp Val Giu Asn Gly Lys Thr Arg Gly Arg Ala 100 Tyr Leu Lys Met Glu Thr Gly Thr Gly Pro 45 Ala Asp 60 Ala Arg Cys Gly Ser Ile Ala His0 i Ile Giu.Thr Ile 75 Val Gly Arg Val Phe Gly Ser Arg Pro Leu Val Lys Leu Gly Pro Cys Gly Ile Arg Gly Asp Giu Gin Trp Asp 105 Leu Cys Gly Cys Ile Gin Arg Asp 115 WO 99/25825 WO 9925825PCT/1B98/01862 <210> 88 <211> 63 <212>

PRT

<213> Homo sapiens <220> <221> SIGNAL <222> -1 <300> <400> 88 Mot LOU TIhr Val Asn Asp Val Arg Phe Tyr Arg Asn Val Arg Ser Asn -30 -25 His Phe Pro Phe Val Arg Lou Cys Gly Leu Leu His Lou Trp Leu Lys -10 -s Val Phe Ser Leo Lys Gin Leo Lys Lys Lys Ser Trp Ser Lys Tyr Leo 1 5 Phe Giu Ser Cys Cys Tyr Arg Ser Leo Tyr Val Cys Val -Phe Ile 20 <210> 89 <211> 163 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 89 Met Ser Pro Ala Ser Phe Trp Ser Ser Ala Ser Met Ile Ser Ser Thr Ser Thr Lys Asp Pro Ile Phe Ser Pro Glu Tyr Pro Ser Gbu Val Ser Gly Leo Ser Arg 100 Gly Val Glrf Pro .115 Pro Pro Pro 130 <210> <211> 52 <212> PRT Phe Arg Ala Met Asp Vol Pro Arg Arg Lys Pro Thr Ser Leu Val1 Ser Thr Pro Trp Ser Leo 25 Lys Gly 40 Pro Phe Ala Pro Ser Glu Asp Ala 105 Cys Lys 120 Arg Ala 10 Leo Pro Pro Leo His 90 Gly Ala Ser Ser Arg Leo Cys His 75 Xaa Ala Leo Glu Gly -5 Pro Cys Leo Gly 60 Pro Phe Xaa Xaa Pro Gly Trp, Ala Ala His Glu Pro Xaa Gly 125 Arg Thr Ser As n Gly Arg Leo Cys Pro 110 Thr Ala His Gln Ser His Giu Gly Ser Glu Ala Lys Ala Gly Leo Pro Val1 Ala Ser Xaa Glu Gly Cys 1 Asn Pro Cys Trp Thr Arg Lys Thr <213> Homo sapiens <220> <221> SIGNAL <222> -32..-1 <300> <400> Met Leo Gly Thr Thr Gly Leo Gly Thr Gin Gly Pro Ser Gin Gin Ala -25 Leu Gly Phe Phe Ser Phe Met Leu Leu Gly Met Gly Gly Cys Leu Pro -10 Gly Phe Leu Leu Gin Pro Pro Asn Arg Ser Pro Thr Leo Pro Ala Ser 1 5 10 WO 99/25825 PCT/B98/01862 Thr Phe Ala His <210> 91 <211> 124 <212> PR? <213> Homo sapiens <220> <221> SIGNAL <222> -97..-1 <300> <400> 91 Met Ala Asp Asp Leu Glu Gly Leu His Ala Ile Lys Val Ala Asn Phe Leu Ser Thr Phe Leu Ser Lys Asn Lys Val Gin Phe Asn Arg Ser Ala Asn Thr Gly 1 Leu Phe Giu Giu Leu <210> 92 <211> 230 <212> PRT <213> Homo sapiens <220> Lys Ile Asp -60 Ala Ser Leu Leu 5 Arg Arg Phe -90 Val Val -75 Asn Ala Leu Ala Ile Ile Pro Leu -10 Ile Val Gin Val Leu Ser Pro Thr Cys -25 Va1 Ser Val Tyr Asp Glu Asp -40 Tyr Va1 Leu Glu Lys Arg His -55 Gin Tyr Ser Glu 10 Ile Lys Asp Ala Gly Asn Phe Lys Ser Leu Gly Leu Ser Thr Ile Glu Pro Val Arg Lys Tyr Ala Leu <221> SIGNAL <222> -24..-i <300> <400> 92 Met Ala Ser Leu Gly Gly Leu Leu Gly Thr Ser Ser Tyr Val Gly Gly Leu Trp Met Glu Asp Ile Tyr Ser Thr Gin Ala Met Met Val Ile Ser Val Val Gly Ala Lys Asp Arg Val Gly Leu Leu Gly Phe 105 Arg Asp Phe Tyr Ser 125 Gly Glu Ala Leu Tyr 140 Ala Gly Ile Ile Leu 155 Asn Tyr Tyr Asp Ala 170 Pro Arg Pro Gly Gin Leu Gin Leu Val Gly Tyr Ile Leu Gly Vai Ser 15 Ala Leu Ser Xaa Va1 95 Pro Leu Gly Phe Gin 175 Pro Ala Ile Thr Gly Ser Cys 80 Ala Va1 Val Ile Ser 160 Ala Lys Met 1 Val His Leu Ala Thr Gly Ala Pro Ile 145 Cys Gin Val -15 Leu Thr Ser Pro 50 Ile Val Gly Trp Asp 130 Ser Ser Pro Lys Leu Ala Thr 35 Ala Ser Phe Vai Asn 115 Ser Ser Ser Leu Ser Pro Vai Gly Asp Ser Cys Phe 100 Leu Met Leu Gin Ala 180 Glu Scr Pro Pro Leu Gln Ser Ala Leu I Lys i Ser I Gin C 4 Ala I Cys Ser I Leu C Ile I 1 Glu 2 135 Leu 2 Arg S Ser S Ser 'I Val Va1 Gly Gly Va1 Ser Pro ,eu rhr .ys :ys I0 la.

le ~rg ;ly ~eu le :le ;er ;er ,yr Ser Gly Ile Ile Leu Gin Phe His Lys Phe Arg 165 Thr Phe WO 99/25825 PCT/1B98/01862 185 .190 Ser Leu Thr Gly Tyr Val 205 <210> 93 <211> 72 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -32..-1 <300> <400> 93 Met Phe Ala Pro Ala Val Gly Tyr Gly Val Pro Met Leu Arg Glu Phe Ser Gin 1 5 AsP Pro Glu Leu Glu Lys Ser Glu Tyr Glu Gly Ser <210> 94 <211> 91 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -36..-l <300> <400> 94 Met Asn Thr Phe Glu Pro 1 Pro Ile Trp Thr Phe Ser A -15 Pro Ser Thr Ser Leu Phe I 1 Pro Leu Gly Leu Ile Leu L Cys Asp Phe Ala Leu Ser I 3 Ser Ile Met Asp Pro Lys A 50 <210> 95 <211> 106 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -32..-i <300> <400> Met Phe Ala Pro Ala Val M Gly Tyr Gly Val Pro Met L Leu Arg Glu Phe Ser Gin I 1 5 Asp Pro Glu Leu Giu Lys L Ser Glu Tyr Glu Lys Ile L Ile Arg Gly Pro Arg Pro T Met Leu -10 Ile Lys lie Arg Ala Phe Arg LyS Asn Lys Thr Leu -25 Leu Leu Ile Val Gly Gly Ser Phe Gly -5 Arg Tyr Asp Ala Val Lys Ser Lys Met 10 Pro Lys Glu Asn Lys Ile Ser Leu Glu 25 Cys ~sp '30 la le *eu -yr 5 .rg Ser Leu Asn Leu 20 Leu Lys Arg -25 Leu Arg Leu Asp 40 Glu Leu Thr Leu 5 Ser Clu Thr Ala Leu Tyr Lys 25 Ser Asp Ala Phe Ala Phe Ile Lys Phe Ile Asp 10 Glu Lys Pro Va1 Leu -10 Arg Cys Pro Cys Arg Va1 Ala Asn Phe Asp Ile Ala Phe Phe Leu Phe Leu His Leu Pro Gly Gin Ile Lys Gly Gly Gly Tyr Thr Lys Asn Arg Ile Glu Phe et eu 10 le ,ys rys rp Lys Gly Val Lys Asp Leu Asn Gly Lys Ile Asp Leu Thr Phe Lys Leu Lys Gly Leu Gly Met Glu Asn Arg WO 99/25825 WO 9925825PCT/IB98/O1 862 Asn Pro Glu Ser Leu Lys Thr Lys Thr Thr <210> 96 <211> 172 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -21,.-1 <300> <400> 96 Met Trp Trp Phe Ile Trp Thr Ser Leu His His Ile Val Ala Pro Glu Val Leu Cys Ile Leu Ser Pro Glu Val Leu Gly Ile Gln Giu Asn Asn Leu Trp Tyr Gly 110 Pro Lys Cys Ser 125 Thr Val Val Gly 140 <210> 97 <211> 56 <212> PRT Gin Gin Gly -15 Ala Ala Phe 1 Asp Pro Ala Lys Cys Leu Ala Thr Ile 50 Glu Asn Val 65 Leu Ser Cys Pro Phe Cys Leu Ile Ile Pro Lys Ser 130 Tyr Leu Val 145 Leu Ser Phe Leu Pro Ser Ala Leu Val Ile Leu Phe 35 Tyr Ile Leu Cys Tyr 115 Asn Trp Phe Pro 20 Gly Val1 Ile Gly Thr 100 Val1 Giy Ser Ser 5 Tyr Al a Arg Lys Leu 85 Cys Cys Lys Lys Tyr Ile Met Tyr Leu 70 Ser Lys Ser Thr Cys 150 Ile Ser Leu Lys Asn Ile Trp Asp Ser 135 Thr Th r Asp Asn Gin Lys Val1 Ser His 120 Leu Ala Thr Ile Val1 Ala Ala Cys 105 Pro Leu Val Thr Gly Thr Ala Ala His Ala Gly Leu Asn Phe Ala Tyr Phe Leu Asp Gin <213> Homno sapiens <220> <221> SIGNAL <222> -l <300> <400> 97 Met Cys Phe Pro Glu His Arg -Arg Gin Met Tyr Ile Gin Asp Arg Leu -35 Asp Ser Val Thr Arg Arg Ala Arg Gin Gly Arg Ile Cys Ala Ile Leu -20 Leu Leu Gin Ser Gin Cys Ala Tyr Trp Ala Leu Pro Glu Pro Arg Thr -5 1 Leu Asp Gly Gly His Leu Met Gin <210> 98 <211> 46 <212> PRT <213> Homo sapiens <220> <221>

SIGNAL

<222> -22. i <300> <400> 98 Met Gin Asn His Leu Gin Thr Arg Pro Leu Phe Leu Thr Cys Leu Phe -15 Trp Pro Leu Ala Ala Leu Asn Val Asn Ser Thr Phe Giu Cys Leu Ile WO 99/25825 PCT/IB98/01862 107 Leu Gin Cys Ser Val Phe Ser Phe Ala Phe Phe Ala Leu Trp <210> 99152 <211> 251 <212> PRT <213> Homo sapiens <220> <221>

SIGNAL

<222> -l <300> <400> 99 Met Trp Arg Leu Leu Ala AraT Ala q- A'

Q

-ZD

-20 rro Leu Leu Ser Asp Ser Trp Ala Leu Leu Prc Leu Arg Val Pro Gly Val Lys Thr Leu Lys Giu Glu Leu Met Lys Gly Val Asp Gly 165 Asn Gly LeL Leu Pro Xaa His Asp Pro Ala Clu Gln 1.50 r'hr 'in Ile IPro Arg Lys Thr Trp Pro Ile Ile Met 135 Val Leu Asn Arg Val Phe Asn Giu Gly Lys Thr Asp 120 Gly Ala Giu Pro *Pro Ile *Leu Gly His Asn Arg 105 His Gly His Lys Trp 185 Ser 10 Giu Se r Asn Phe Met 90 Lys Tyr Arg Lys Met 170 rhr Phe Arg Asp Phe Giu 75 Phe Ser Leu 1.55 Arg ?he -5 Glu Ala Ile Ala 60 Met Ala Val1 Thr Xaa 140 Pro Lys Giu Pro Pro 220 Asg Pro Arg 45 Ile Met Ile Gly Pro 125 Phe Phe A~sp Arg T'yr 205 XCaa Ala Ser Ala Vai Ser Ile 15 Leu Val Pro 30 Gly Pro Ser Leu Ala Leu Arg Leu Thr 80 Trp Arg Val 95 His Arg Met 110 Val Lys Ala Giu Giu Val Ala Ala Lys 160 Gin Giu Giu 175 le. Ala Thr 190 Asp Leu Thr Arg Val Pro Lys Thr Gly Ile Pro Gly Gly Gin 145 Ala Arg Ala His Gil Val Giu Gly Asn Ala Gly Arg 130 Gly Val1 Giu Xaa Lys 210 ILys Arg Ala Gly Arg Pro Gly 115 Xaa Phe Ser Xaa Met 195 Gly Pro Arg Thr Tyr Ser Phe 100 Lys Xaa Leu Arg Asn 180 Leu Lys 2 a00u e Xaa Trp Gly Lys Ph 215 <210> 100 <211> 77 <212>

PRT

<213> Homo sapiens <220> <221>

SIGNAL

<222> -30. l <300> <400> 100 Met Leu Arg Leu Asj Leu Ile Val Ser Va.

-i1 Val Gly Gly Giy Va.

Asp Gly Ala Leu 11~ Tyr Gin Lys Lys Pr e Tyr Met p Ile Ile Asn Ser Leu -25 1. Leu Ala Leu Ile Pro 0 -5 1 Phe Ala Leu Val Thr 10 Tyr Arg Lys Leu Leu 25 Val His Giu Lys Lys 40 Val Thr Thr Val Phe Met -20 GlU Thr Thr Thr Leu Thr 1 Ala Val Cys Cys Leu Ala Phe Asn Pro Ser Gly Pro Giu Val Leu WO 99/25825 WO 9925825PCT/IB98/01862 <210> 101 <211> 81 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -31. l <300> <400> 101 Met Ser Asn Thr is Thr Ala Leu Thr Cyo Cys His -10 Arg Pro Leu Pro Arg Val Glu Leu Arg Tyr Pro Gin Ser Pro Cys Arg Thr Leu Leu <210> 102 <211> 126 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -20. 1 <300> <400> 102 Met Lys Val Hit Met His -15 Phe Ile Phe His His Cys 1 Pro Glu Ala Leu His Arg Ser Lys Phe Ser Lys Gin Glu Lys Leu Phe Glu Arg 50 Gly Leu Glu Lys Leu Leu Val Glu Ile Asn His Glu Arg Tyr Phe Gly Ser Ser 1 <210> 103 <211> 273 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -45. 1 <300> <400> 103 Met Asn Trp Ser Ile Phe C -40 Ser Thr Ala Phe Gly Arg I Val Leu Val Tyr Leu Val. I Lys Asp Phe Asp Cys Asn '1 1 Val Leu Val Ser Leu -25 Leu Gly Leu Pro His -5 Glu Pro Trp, Asp Pro 10 Ala met Asn Ser Phe 25 Arg Gin Giu Ala Ser 40 Pro His Pro His Pro Pro Val Arg Ala Pro 1 Arg Trp Gin Asp Ser Leu Asn Glu Arg Ser Ala Asp Arg Cys Asp Thr Gln 35 ['yr rhr ksp ~rg Lys His His 20 Ala Gly Asn Leu Gly 100 Phe Cys 5 Arg Glu Glu Leu Gly Lys Cys His G2.y Asn Asn Gly 70 His Ala Leu -10 Glu Met Glu Gly 55 Leu Asp Phe Ile Glu Thr Lys Arg Gly His Ser Cys His Giu Lys Leu Glu Val1 Leu 105 Leu Leu ASP His Leu Glu Tyr Tyr Ser Phe Arg Lys Ser His Thr Thr Gly Pro Ile Phe Val Leu ;iu Gly Leu Leu Ser Gly Val Asn Lys Tyr -35 lie Trp Leu Ser Leu Val Phe Ile Phe Arg -20 ~hr Ala Glu Arg Val Trp Ser Asp Asp His -5 2.

~hr Arg Gin Pro Gly Cys Ser Asn Val. Cys .0 WO 99/25825 PCT/IB98/01862 Phe. Asp Glu Phe- Leu Ile Leu Val T 4 Tyr Arg Glu Val G Ser Gly Arg Leu T Trp Thr Tyr Val C Pha Leu Tyr Val P1 100 Val Val Lys Cys Hi 1: Ile Ser Lys Pro Se 135 Thr Ala Ala lie C 150 Val Ser Lys Arg Cy 165 Met Xaa Thr Gly Hi 180 Xaa Asp Xaa Xaa Se Xaa Pro Xaa Leu Pr 215 Leu <210> 104 <211> 158 <212> PRT <213> Homo sapiens <220> <221> SIGNAL hh 0 Ir yr ys he is r r 0 0 Pro 25 r Cys 1 Glu Leu Ser His 105 Ala Glu Ile His His I 185 Gly Asp A Val Pro Lys Asn Leu 90 Ser Asp Lys Leu ;lu 170 Iro sp Lrg Se Se; Ar Prc 75 Val Phe Pro Asn Leu 155 Cys Xaa Xaa Pro r His r Leu I His 60 Gly Phe Tyr Cys Ile 140 Asn Leu Asp Ile I Arg 220 Le 45 Arg Lys Lys Pro Pro 125 Phe Leu 1 la 'hr ?he !05 sp Val Arg Leu Trp Ala Leu Gin 30 Val Val Met His Val Ala Glu Ala His Gly Glu Asn Lys Arg Gly Gly Leu Trp 80 Ala Ser Val Asp lie Ala Lys Tyr Ile Leu Pro Pro 110 115 Asn Ile Val Asp Cys Phe 130 Thr Leu Phe Met Val Ala 145 Val Giu Leu lie Tyr Leu 160 Ala Arg Lys Ala Gn Ala 175 Thr Phe Ser Xaa Lys Gin 190 195 Leu Gly Ser Asp Ser His 210 His Val Lys Lys Thr Ile 225 <222> -37.*-l <300> <400> 104 Met Ala Ser Ly Ile Cys Leu GI Ser Leu Cys Ar Ser Met Gly GI: Phe Glu His Lei Leu Lys Giu Va Cys Asp His Hil Lys Val Ile Cy! His Thr Gly Pr Pro Gly Ser Pre 110 <210> 105 <211> 51 s u

U

Y

1

S

0 Ile Leu Ala Lys Gin Lys Gly Trp His Leu Leu Cys 1 Ser Ala Leu Clu 65 Leu Gly Leu Thr -15 Ile Ser Asn Ser 50 Lys Cys Gly Asn -30 Glu Thr Cys Gin 35 Pro Leu Glu Ser Glu 115 Val Pro Va1 Pro 20 His Asp Leu Arg Ile 100 Gin Leu Ser 5 Va1 Arg Asn Leu Ser 85 Gin Glu Ser Asn Cys Ala Gly Phe 70 Gin Gly Glu Leu Lys Gly Asn Lys Cys Glu Met Va1 Asp Glu Ile Ile Lys Lys His Ser Gly 120 Thr Cys Ala Ser Val Arg Clu Arg Gly 105 Ser Cys Gly Val Tyr Glu Asp Asp Gly Glu Pro His Thr Ser Arg Leu Arg His Thr 0 Gin Giu Ala Glu Gly Arg Gly <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -19..-1 WO 99/25825 PCT/IB98/01862 <300> 110 <400> 105 Met Arg Thr Leu Phe Asn Leu Leu Trp Leu Ala Leu Ala Cys Ser Pro -10 Val His Thr Thr Leu Ser Lys Ser Asp Ala Xaa Lys Pro Pro Gin Arg 1 5 Arg Cys Trp Arg Arg Val Ser Phe Gin Ile Ser Arg Cys Lys Thr Gly 20 Val Trp Trp <210> 106 <211> 359 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -34..-l <300> <400> 106 Met Leu Leu Ser Ile Glv Met IA uu Met. Leu Ser Ali Thr Glu Asp Lys Va1 Ile Arg Leu Ile Glu Ser Ile Asp 175 Lys Ala Pro Lys Gin 255 Va1 Pro His Il( Ala Asp Gly Pro Xaa Ala Ile Pro Phe Leu Cys 160 Arg Leu lie Cys Thr 240 Gly Thr Xaa Asp Leu Asp 1 Leu Phe Pro Xaa Gly Ser Ser Thr Pro 145 Leu His Pro Cys Ser 225 Lys Asp Glu Xaa Arg 305 Thi Ile Pro Leu Pro Leu Tyr Met Va1 Xaa 130 Leu Ile Arg al Leu 210 -is Lys Ser His Phe 290 Ile -30 Val Leu Ala Ile Val Asp Lys Gly Phe 115 Glu Glu Leu Ala His 195 Asp Ala Thr Asp Thr 275 Gly lie Gir Ala Xaa 20 Asn Lys Cys Ala Ser 100 lie Lys Tyr Ile Arg 180 Lys Glu Tyr Cys Ser 260 Pro kla Gln 1 Leu Tyr 5 Phe Ser Asp Asn Ala 85 Asn Gly Gly 4 Tyr Val 165 Arg Phe i Tyr His Pro 245 Asp Leu I Leu Phe Asn Gly Lys Asn Phe 70 Ile Asp Glu Gly Leu 150 Ile Asn Lys ;lu :ys 230 /al rhr eu (aa Ala -10 Phe Tyr Pro Ser 55 Asp Va1 Ile Ser His 135 Ile Phe Arg Lys Asp 215 Lys Cys Asp Arg Gly 295 -25 Phe Glu Arg Glu 40 Ser Ile His Glu Ser 120 Leu Pro Met Leu Gly 200 Gly Cys Arg Ser Pro I 280 Xaa Leu Asn Leu 25 Asn Gly Lys Asn Va1 105 Ala Ile Phe lie Arg 185 Asp Asp Val 2 Glm Ser 265 Leu Pro E Asr Ala Pro Ala Thr Val Va1 Leu Ser Leu Leu rhr 170 Lys lu Lys ksp -ys 250 .,n (aa kla Thr Leu Ser Ala Cys Phe Leu Asp Lys Ser Val Ile 155 Lys I Asp Tyr Leu I Pro 1 235 Val Glu C Phe C His 3 Asn C 315 Gi Leu Gin Glu Glu lie Asn Ser Lys Leu Pro 140 Kaa .eu ;ln ksp krg rp Tal flu :ys :aa 100 ;lu Val Pro Thr Gly Pro Va1 Ala Asp Ile Lys 125 Glu Val Ser Leu Val 205 Ile I Leu Pro Asn Gin 285 Gin Asp 1 Tyr Va1 Phe Leu Ile Leu Gin Asp Asp 110 Asp Phe Gly Arg Lys eu rhr 3er ;lu :ys Caa rhr Thr Xaa Giu Glu Asp Asp 310 WO 99/25825 WO 9925825PCTIIB98/01862 Asp Ser Ser Asp'Ala Glu Glu 320 325 <210> 107 <211> 291 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -42. l <300> <400> 107 Mac Asp Ser Arg Val Ser Ser Pro Glu Lys Gin Asp Lys Glu Asn Phe Val Gly Ser Lau met Cys I Leu Gly Val Leu P Thr Cys A Thr Gin V Ala Val A Gin Thr T 1 Leu Ala G 120 Asp Ala T 135 Asp Val A Glu Ala T Met Ser A.

Ser Pro 1, 200 Ala Thr G; 215 Leu Giu G ASn Lys A: <210> 108 <211> 67 <212> PRT lai Asn Asn 3er Phe Leu ~eu Lys Ile ~rg Ile Gin ~ro Cys Ile ~sn Ile Pro al Asp Gly la Asn Val hr Leu Arg 05 ly Arg Giu hr Giu Leu rg Ile Pro 155 hr Arg Giu 170 la Ser Lys le Ala Leu lu Lys Asn, ly Ile Gly 235 la Lys Lau~ -5 Ile Ala Asp Pro 60 Val1 As n Asn Giu I'rp 140 Val1 Ser Gin Ser Arg -20 Val Lys Asp Val1 45 Gin Val1 Asp Val1 Ile 125 Gly Gin Arg Leu Leu 205 Thr Leu Ile Glu Lys 30 Phe Glu Tyr Val1 Leu 110 Ala Ile Lau Al a Lys 190 A.rg I le Gil' Ile Tyr i5 Ala Val1 Ile Tyr His 95 Gly His Arg Gin Lys 175 Se r Tyr Val1 Val Thr Glu Lys Lys Lau Arg 80 Gin Thr Ser Val1 Arg 160 Val1 Ala Leu Phe Asp 240 Cys Phe 1 Arg Gly Val1 Thr 65 Ile Al a Gin Ile Ala 145 Ser Leu Set Gin Pro 225 Asn G1y Pro Ala Pro Asp Arg Tyr Thr Thr Gin 130 Arg Met Ala 'let rhr 210 H{is -30 *Trp Val1 Gly Leu Asp Ser Phe Leu 115 Thr Val1 Ala Ala Val1 195 Leu Pro Lys I1e Ser Val1 Lau Arg Set Ala Leu 100 Ser Leu Glu Ala Glu 180 Leu Ser Met Lys Lau Ile Phe Ile Thr Val1 Val1 Leu Gin Leu Ile Glu 165 Gly Ala Thr Asn Leu 245 Phe Trp Arg Leu Val Thr Set Ala Ile Asp Lys 150 Al a Glu Glu la 1 I le 230 Pro ;ly Val Set Tyr <,213> Homno sapiens <220> <221>

SIGNAL

<222> -26. l <300> <400> 108 Met Set Thr Trp Leu Leu Leu Ile Ala Leu Lys Thr Leu Ile Thr Trp -20 Val Ser Leu Phe Ile Asp Cys Val Met Thr Arg Lys Leu Thr Asn Cys -5 1 Asn Ala Arg Giu Thr Ile Lys Gly Ile Gin Lys Arg Giu Ala Set Asn 15 WO 99/25825 PCT/IB98/01862 112 Cys Phe Ala le Arg His Phe Glu Asn Lys Phe Ala Val Glu Thr Leu 30 Ile Cys Ser <210> 109 <211> 127 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -63. l <300> <400> 109 Met Ser Ala Ala Gly Ala Arg Gly Leu Arg Ala Thr Tyr His Arg Leu -55 Leu Asp Lys Val Glu Leu Met Leu Pro Glu Lys Leu Arg Pro Leu Tyr -40 Asn His Pro Ala Gly Pro Arg Thr Val Phe Phe Trp Ala Pro Ile Met -25 Lys Trp Gly Leu Val Cys Ala Gly Leu Ala Asp Met Ala Arg Pro Ala -10 -5 1 Glu Lys Leu Ser Thr Ala Gin Ser Ala Val Leu Met Ala Thr Gly Phe 10 Ile Trp Ser Arg Tyr Ser Leu Val Ile Ile Pro Lys Asn Trp Ser Leu 25 Phe Ala Val Asn Phe Phe Val Gly Ala Ala Gly Ala Ser Gin Leu Phe 40 Arg Ile Trp Arg Tyr Asn Gin Glu Leu Lys Ala Lys Ala His Lys 55 <210> 110 <211> 97 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 110 Met Lys Gly Trp Gly Trp Leu Ala Leu Leu Leu Gly Ala Leu Leu Gly -15 10 Thr Ala Trp Ala Arg Arg Ser Arg Asp Leu His Cys Gly Ala Cys Arg 1 5 Ala Leu Val Asp Glu Leu Glu Trp Giu Ile Ala Gin Val Asp Pro Lys 20 Lys Thr Ile Gin Met Gly Ser Phe Arg Ile Asn Pro Asp Gly Ser Gin 35 Ser Val Val Glu Vai Thr Val Thr Xaa Ser Pro Lys Thr Lys Val Ala 50 55 His Ser Gly Phe Trp Met Lys Ile Arg Leu Leu Lys Lys Gly Pro Trp, 70 Ser <210> III <211> 86 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -20.,1- <300> <400> 111 Met Lys Gly Trp, Gly Trp, Leu Ala Leu Leu Leu Gly Ala Leu Leu Giy -15 -10 Thr Ala Trp Ala Arg Arg Ser Gin Asp Leu His Cys Gly Ala Cys Arg WO 99/25825 PCT/IB98/01862 ''3 1 5 Ala Leu Val Asp Giu Thr Arg Met Gly Asn Cys Pro Gly Gly Pro 20 25 Giu Asp His Ser Asp Gly Ile Phe Pro Asp Gin Ser Arg Trp Gin 35 40 Val Ser Gly Gly Gly Ala Leu Cys Pro Leu Arg Gly Pro Pro His so 55 Ala Ala Gly Giy Asp Met <210> 112 <211> 71 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 112 Met Pro Ala Gly Val Pro Met Ser Thr Tyr Leu Lys Met Phe Ala -20 -15 Ser Leu Leu Ala Met Cys Ala Giy Ala Giu Val Val His Arg Tyr 1 5 Arg Pro Asp Leu Thr Ile Pro Giu Ile Pro Pro Lys Arg Gly Giu 15 20 Lys Thr Giu Leu Leu Gly Leu Lys Giu Arg Lys His Lys Pro Gin 30 35 Ser Gin Gin Giu Giu Leu Lys <210> 113 <211> <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -42. l <300> <400> 113 Met Asp Giy His Trp Ser Ala Ala Phe Ser Ala Leu Thr Val Thr -35 -30 Met Ser Ser Trp Ala Arg Arg Arg Ser Ser Ser Ser Arg Arg Ile 1 -20 -15 Ser Leu Pro Gly Ser Pro Val Cys Trp Ala Trp Pro Trp Tyr ProI -5 1 5 Thr TZhr Ser Phe Pro Leu Arg Cys Arg Giy Arg Val <210> 114 <211> 118 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -83. l <300> <400> 114 Met Leu Pro Val Gin Ser Phe Thr Leu Val Ala Gin Ala Gly Val C -7 5 -70 Trp Arg His Leu Ser Ser Leu Gin Leu Leu Pro Pro Glu Phe Lys C -60 -55 Phe Ser Cys Leu Ser Leu Pro Ser Ser Trp Asp Tyr Arg Arg Pro -45 -40 Pro Cys Pro Ala Gly Phe Phe Vai Phe Leu Val Glu Thr Gly Leu 1 -30 -25 His Val Gly Gin Ala Gly Leu Giu Leu Leu Thr Ser Cys Ser Pro Gin Pro Arg Ala Tyr Leu Val1 1i a ?ro ~sp 1ln ;iy ?ro 'is ?ro WO 99/25825 PCT/IB98/01862 114 10 Ala Ser Ala Ser Gin Ser Ala Ala Ile Thr Gly Val Ser His Val Pro 1 5 Gly Lys Lys Lys Leu LeU Lys Val Giu Lys Lys Asn Leu Arg Xaa Leu 20 Leu Thr Xaa Ile Lys Thr <210> 115 <211> 76 <212> PR? <213> Homo sapiens <220> <-221> SIGNAL <222> -22..-l <300> <400> 115 Met Giu Leu Ile Ser Pro Thr Val Ile Ile Ile Leu Gly Cys Leu Ala -15 Leu Phe Leu Leu Leu Gin Arg Lys Asn Leu Arg Arg Pro Pro Cys Ile 1 5 Lys Gly Trp Ile Pro Trp Ile Giy Val Gly Phe Xaa Phe Gly Lys Ala 20 Pro Leu Giu Phe Ile Giu Lys Ala Arg Ile Lys Val Cys Gly Arg Gly 35 Xaa Arg Gly Leu Gin Arg Arg Gin Cys Phe Leu Phe <210> 116 <211> <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -52. l <300> <400> 116 Met Ala Giu Thr Lys Asp Ala Ala Gin Met Leu Vai Thr Phe Lys Asp -45 Val Ala Vai Thr Phe Thr Arg Giu Giu Trp Arg Gin Leu Asp Leu Ala -30 Gin Arg Thr Leu Tyr Arg Giu Val Met Leu Glu Thr Cys Gly Leu Leu -15 -10 Val Ser Leu Giy Gin Ser Ile Trp Leu His Ile Thr Giu Asn Gin Ile 1 5 Lys Leu Ala Ser Pro Gly Arg Lys Phe Thr Asn Ser Pro Asp Glu Lys 20 Pro Giu Val Trp Leu Ala Pro Gly Leu Phe Gly Ala Ala Ala Gin 35 <210> 117 <211> 82 <212> PR? <213> Homo sapiens <220> <221> SIGNAL <222> -1 <300> <400> 117 Met Giu Leu Ile Ser Pro Thr Val Ile Ile Ile Leu Gly Cys Leu Ala -15 Leu Phe Leu Leu Leu Gin Arg Lys Asn Leu Arg Arg Pro Pro Cys Ile 1 5 Lys Gly Trp Ile Pro Trp Ile Gly Val Giy Phe Glu Phe Gly Lys Ala 20 Pro Leu Glu Phe Ile Giu Lys Ala Arg Ile Lys Tyr Giy Pro le Phe WO 99/25825 PCT/IB98/01862 115 35 Thr Val Phe Ala Met Gly Asri Arg Met Thr Phe Val Thr Glu Giu Gly 50 <210> 118 <211> 89 <212> PR? <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 118 Met Ilie Ile Ser Leu Phe Ile Tyr Ile Phe Leu Thr Cys Ser Asn Thr -10 Ser Pro Ser Tyr Gin Gly Thr Gin Leu Gly Leu Gly Leu Pro Ser Ala 1 5 10 Gin Trp Trp Pro Leu Thr Gly Arg Arg Met Gin Cys Cys Arg Leu Phe 25 Cys Phe Leu Leu Gin Asn Cys Leu Phe Pro Phe Pro Leu His Leu Ile 40 Gin His Asp Pro Cys Giu Leu Val Leu Thr Ile Ser Trp Asp Trp Aia 55 Glu Ala Gly Ala Ser Leu Tyr Ser Pro <210> 119 <211> <2i2> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -l <300> <400> 119 Met Thr Met Ala Giu Cys Pro Thr Leu Cys Val Ser Ser Ser Pro Ala -10 Leu Trp Ala Ala Ser Glu Thr Thr Asp Asp Val Cys Arg Giu 1 5 <210> 120 <211> 115 <212> PR? <213> Homno sapiens <220> <221> SIGNAL <222> -103. 1 <300> <400> 120 Met Val Ile Arg Val Tyr Ile Ala Ser Ser Ser Giy Ser Thr Ala Ile -100 -95 Lys Lys Lys Gin Gin Asp Val Leu Gly Phe Leu Giu Ala Asn Lys Ile -80 Gly Phe Giu Glu Lys Asp Ile Ala Ala Asn Giu Giu Asn Arg Lys Trp -65 Met Arg Glu Asn Val Pro Glu Asn Ser Arg Pro Ala Thr Gly Asn Pro -50 -45 Leu Pro Pro Gin Ile Phe Asn Glu Ser Gin Tyr Arg Gly Asp Tyr Asp -30 Ala Phe Phe Glu Ala Arg Glu Asn Asn Ala Val Tyr Ala Phe Leu Giy -15 Leu Thr Ala Pro Ser Gly Ser Lys Glu Ala Gly Arg Cys Lys Gin Ser 1 Ser Lys Pro WO 99/25825 PCT/IB98/01862 <210> 121 <211> 105 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -76..-1 <300> <400> 121 Met Pro Leu Leu Cys Gin Met Thr Met Leu Leu Leu Pro Leu Gin Ile Lys Val Ser Ser Ile Val Tyr Leu Trp Thr Gly Leu Ile Val Glu Leu Arg Gin Val <210> 122 <211> 93 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -22..-1 <300> <400> 122 Met Lys Pro Val Leu Leu Gin Leu Val Pro Leu Glu Pro Pro Pro Cys Thr Met Gin Glu Phe Cys Gly Ile Val Ile Lys His Lys Gly <210> 123 <211> 109 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -42..-1 <300> <400> 123 Met His Ile Leu Gin Ile Val His Cys Pro Asp Leu Val Cys His Leu Gin Glu Gin Lys Ala Ile Tyr Ala Ser Gin Gin -55 Gin Val Val Ser 10 Val Pro Gly Cys Asp Cys Ser SIle -70 Ser Thr Thr Val Leu Glu Leu Ser 1 Ile Cys Ser Glu 65 Clu SMet SLys Ile Ser -5 Glu Val Gin -15 Pro Ser Glu Ser 50 Val Met Leu Glu Thr -20 Phe Lys Ser Phe Lys Ala Lys 35 3lu Ile Glu Cys Ala -35 Pro Ile Glu Leu Gin Pro 20 Gly Thr Met Tyr Asp -50 Asn Thr Ala Leu 15 Val Arg 5 ;lu Phe Phe Pro SLeu Leu Leu Arg Ser Ala Val Val Asn Gin Gin Ala Leu Val Asp Trp Ser 1 Pro Leu Ser Phe Phe Ser Leu Lys Trp Tyr Pro Pro Lys Asn Leu Ala Asn Phe Glu Phe Cys Leu Ala Leu Lys Tyr Ile Cys Thr His Leu Cys Cys Ser Ser Lys Arg Asn Arg Asn Leu Leu Thr Thr Val Asp Asp Gly Ile Gin Ala -35 Asp Thr Gly Lys Asp Ile Trp Asn Leu Leu Phe -20 Glu Phe Cys Gin Ser Asp Asp Pro Ala Ile Ile -5 1 Thr Val Leu Ala Ser Val Phe Ser Val Leu Ser 15 G1n Thr Glu Gin Glu Tyr Leu Lys Ile Glu Lys WO 99/25825 PCT/IB98/01862 117 30 Val Asp Leu Pro Leu Ile Asp Ser Leu Ile Arg Val Leu Gin Asn Met 45 Glu Gin Cys Gin Lys Lys Pro Giu Asn Ser Ala Gly Val 60 <210> 124 <211> 51 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> <300> <400> 124 Met Arg Leu Val Pro Leu Gly Gin Ser Phe Pro Leu Ser Glu Pro Arg -10 -5 -1 Cys Leu Gin Pro Val Lys Trp Asp His Asn His Cys Leu Thr Ser Leu 10 Thr Val Val Val Arg Thr Giu Cys Val Glu Val Phe His Lys Leu Trp, 25 Met Leu Val <210> 125 <211> 56 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -27. l <300> <400> 125 Met Asn Arg Val Pro Ala Asp Ser Pro Asn Met Cys Leu Ile Cys Leu -20 Leu Ser Tyr Ilie Ala Leu Gly Ala Ile His Ala Lys Ile Cys Arg Arg -5 1 Ala Phe Gin Gilu Glu Gly Arg Ala Asn Ala Lys Thr Gly Val Arg Ala 15 Trp Cys Ile Gin Pro Trp Ala Lys <210> 126 <211> 162 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222>

-I

<300> <400> 126 Met Leu Gin Thr Ser Asn Tyr Ser Leu Val Leu Ser Leu Gin Phe Leu -15 Leu Leu Ser Tyr Asp Leu Phe Val Asn Ser Phe Ser Giu Leu Leu Gin 1 5 Lys Thr Pro Val Ile Gin Leu Val Leu Phe Ile Ile Gin Asp Ile Ala 20 Val Leu Phe Asn Ile Ile Ile Ile Phe Leu Met Phe Phe Asn Thr Ser 35 Val Phe Gin Ala Gly Leu Val Asn Leu Leu Phe His Lys Phe Lys Gly 45 s0 Thr Ile Ile Leu Thr Ala Val Tyr Phe Ala Leu Ser Ile Ser Leu His 65 70 Val Trp Val Met Asn Leu Arg Trp Lys Asn Ser Asn Ser Phe Ile Trp 85 Thr Asp Giy Leu Gin Met Leu Phe Val Phe Gin Arg Leu Ala Ala Val WO 99/25825 PCT/IB98/01862 118 100 105 Leu Tyr Cys Tyr Phe Tyr Lys Arg Thr Ala Val Arg Leu Gly Asp Pro 110 115 120 His Phe Tyr Gin Asp Ser Leu Trp Leu Arg Lys Glu Phe Met Gin Val 125 130 135 Arg Arg 140 <210> 127 <211> 126 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -68..-1 <300> <400> 127 Met Ala Ser Ala Ser Ala Arg Gly Asn Gin Asp Lys Asp Ala His Phe -60 Pro Pro Pro Ser Lys Gin Ser Leu Leu Phe Cys Pro Lys Ser Lys Leu -45 His Ile His Arg Ala Glu Ile Ser Lys Ile Met Arg Glu Cys Gin Glu -30 Glu Ser Phe Trp Lys Arg Ala Leu Pro Phe Ser Leu Val Ser Met Leu -15 -10 Val Thr Gin Gly Leu Val Tyr Gin Gly Tyr Leu Ala Ala Asn Ser Arg 1 5 Phe Gly Ser Leu Pro Lys Val Ala Leu Ala Gly Leu Leu Gly Phe Gly 20 Leu Gly Lys Val Ser Tyr Ile Gly Val Cys Gin Ser Lys Phe His Phe 35 Phe Glu Asp Gin Leu Arg Gly Ala Gly Phe Gly Pro Thr Ala 50 <210> 128 <211> 140 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -40..-1 <300> <400> 128 Met Thr Ser Met Thr Gin Ser Leu Arg Glu Val Ile Lys Ala Met Thr -35 -30 Lys Ala Arg Asn Phe Glu Arg Val Leu Gly Lys Ile Thr Leu Val Ser -15 Ala Ala Pro Gly Lys Val Ile Cys Glu Met Lys Val Glu Glu Glu His 1 Thr Asn Ala Ile Gly Thr Leu His Gly Gly Leu Thr Ala Thr Leu Val 15 Asp Asn Ile Ser Thr Met Ala Leu Leu Cys Thr Glu Arg Gly Ala Pro 30 35 Gly Val Ser Val Asp Met Asn Ile Thr Tyr Met Ser Pro Ala Lys Leu 50 Gly Glu Asp Ile Val Ile Thr Ala His Val Leu Lys Gin Gly Lys Thr 65 Leu Ala Phe Thr Ser Val Gly Leu Thr Asn Lys Ala Thr Gly Lys Leu 80 Ile Ala Gin Gly Arg His Thr Lys His Leu Gly Asn 95 100 <210> 129 <211> 43 <212> PRT <213> Homo sapiens WO 99/25825 PCT/IB98/01862 <220>19 <221> SIGNAL <222> -1 <300> <400> 129 Met Gin Cys Phe Ser Phe Ile Lys Thr Met Met Ile Leu Phe Asn Leu -15 Leu Ile Phe Leu Cys Gly Phe Thr Asn Tyr Thr Asp Phe Giu Asp Ser 1 Pro Tyr Phe Lys Met His Lys Pro Val Thr Met <210> 130 <211> 69 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -21. 1 <300> <400> 130 Met Trp Trp Phe Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu Val -15 Ile Trp Thr Ser Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala Val Thr 1 5 Leu His His Ile Asp Pro Ala Leu Pro Tyr Ile Ser Asp Thr Gly Thr 20 Val Ala Pro Glu Lys Cys Leu Phe Gly Ala Met Leu Asn Ile Ala Ala 35 Val Leu Cys Gin Lys <210> 131 <211> 78 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -19.,1- <300> <400> 131 met Ser Pro Gly Ser Ala Leu Ala Leu Leu Trp Ser Leu Pro Ala Ser -10 Asp Leu Gly Arg Ser Val Ile Ala Gly Leu Trp Pro His Thr Gly Val 1 5 Leu Ile His Leu Giu Thr Ser Gin Ser Phe Leu Gin Gly Gin Leu Thr 20 Lys Ser Ile Phe Pro Leu Cys Cys Thr Ser Leu Phe Cys Val Cys Val 35 40 Val Thr Val Gly Gly Gly Arg Val Gly Ser Thr Phe Val Ala.

<210> 132 <211> <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -47. i <300> <400> 132 met Arg Leu Pro Pro Ala Leu Pro Ser Gly Tyr Thr Asp Ser Thr Ala -40 Leu Giu Gly Leu Val Tyr Tyr Leu Asn Gin Lys Leu Leu Phe Ser Ser -25 Pro Ala Ser Ala Leu Lcu Phe Phe Ala Arg Pro Cys Val Phe Cys Phe WO 99/25825 PCT/IB98/01862 -10 10-5 1 Lys Ala Ser Lys Met Gly Pro Gin Phe Glu Asn Tyr Pro Thr Phe Pro 10 Thr Tyr Ser Pro Leu Pro Ile Ile Pro Phe Gin Leu His Gly Arg Phe 25 3 <210> 133 <211> 53 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -42. i <300> <400> 133 Met Asp Ser Arg Val Ser Val Gly Val Asn Asn Lys Ser Leu Ser Phe Leu Leu -5 Met Cys Leu Lys Ile Se r Giu Lys Gin Asp Lys Trp Giu Asn Phe Ile Leu Phe Gly Val Cys Gly Pro Ile Ile Thr Ile Ser Ile Trp <210> <211> <212> <213> <220> <221> <222> <223> 134 1053

DNA

Homo sapiens sig.,.peptide 131. .169 Von Heijne matrix score 4.19999980926514 seq MLAVSLTVPLLGA/MM <221> PolYA-site <222> 1042. .1053 <300> <400> 134 gagcgagtcg gacgggctgc gacagcgccg gcccctgcgg ccgcaggtcg tcacagacga tgatggccag gccccggagg ctaaggacgg cagctccttt agcggcagag ttttccgagt gaccrttcttg atg ct~g gct gt~t tct ct~c acc gtt ccc ctg ct~t gga gcc Met Leu Ala Val Ser Leu Thr Val Pro Leu Leu Gly Ala atg Met 1 gaa Glu cag Gin atg ctg ctg Met Leu Leu ccc ccg ctc Pro Pro Leu gca gaa agg Ala Giu Arg gaa Glu 5 tct cct ata gat Ser Pro Ile Asp cca Pro 10 cat His cag cct ct~c agc ttc aaa Gin Pro Leu Ser Phe Lys ttg Ct~t ggt gtt Leu Leu Gly Val cca aat acg Pro Asn Thr ctg ttt gaa Leu Phe Glu ctt gtt gga Leu Vai Gly ccg Pro ga t Asp aag ctg cga Lys Leu Arg gag tcc ata Giu Ser Ile ggc cgg gtc Gly Arg Val 120 169 217 265 313 361 409 457 505 553 gca cat Ala His gta aaa Val Lys att Ile ggg gat gtg Gly Asp Val act ggg aca Thr Gly Thr gca Ala ct~t gaa aat Leu Giu Asn ggC Gly ggt Gly '70 cga Arg gaa ata gag acc Giu Ile Giu Thr att Ile 75 gtg Val1 gcc cgg ttt ggt Ala Arg Phe Giy tcg Ser cct tgc aaa Pro Cys Lys ac c Thr ggg Gly ggt gat gag Giy Asp Giu tgt ggg aga Cys Gly Arg ggt at~c cgt Gly Ile Arg aag gga cta gca Ala 100 ttt ccc aat ggg Pro Asn Gly ttt gtg gcc Phe Vai Ala ccc ctg Pro Leu gca tac Ala Tyr ctg ctg gaa gta aat ccc aaa cgt gaa gtg WO 99/25825 PCTJIB98/01862 Lays Gly Leu 115 ctg tee tc Lou Ser Ser Phe Glu Vai Asn Pro Trp Lys Arg Giu 125 aac atg tcc Asn Met Ser Val Lys Leu Lou gag aca ccc Giu Thr Pro ggg aag Gly Lys ttt gtg aat Phe Val Asn 130 gat ctt Asp Lou aca gtc act Thr Val Thr 145 agc Se r eag Gin 150 aga Arg ggg agg aag Gly Arg Lys att Ile 155 ctt Lou 140 tat Tyr ttc ace gat Phe Thr Asp te t Se r age aaa tgg Ser Lys Trp caa Gin 165 cgc Arg ega gac tac Arg Asp Tyr ctg LeU, 170 ga t Asp ctg gtg atg LOU Val Met gag ggc Glu Gly 175 aca gat gac Thr Asp Asp aaa gtt tta Lys Val Lou 195 ect gca gaa Pro Ala Glu ggg Gly 180 ttg Lou ctg ctg gag Leu Lou Giu tat Tyr 185 ttc Phe act gtg ace Thr Val Thr gac cag etg Asp Gin Lou egg Arg 200 gtg Val ceg aat gga Pro Asn Giy gte Val1 205 a tg Met &gg gaa gta Arg Glu Val 190 cag cg tet Gin Lou Ser gee agg ata Ala Arg Ile gac ttt gte Asp Phe Val 210 ega aga Arg Arg ctg Leu 215 9gc Gly gea gaa aca Ala Glu Thr ace Thr 220 ggg Gly gte tac gtt Val Tyr Val ctg atg aag Lou Met Lys 225 gcg Val1 gg e Gly 235 ate Ile get gat ctg Ala Asp Lou ttt Phe 240 745 793 841 889 937 985 1033 1053 gag aac atg Glu Asn Met ect Pro 245 ttt eca gac Phe Pro Asp aac Asn 250 ate Ile egg ccc age Arg Pro Ser age tet Ser Ser 255 ggg ggg tac Gly Gly Tyr tee atg etg Ser Met Lou 275 ttt aag gta Phe Lys Val 290 <210> 135 <211> 675 <212> DNA tgg gtg gge Trp Val Gly 260 gat tte tta Asp Phe Lou aaaaaaaaaa a atg teg met Ser tet. gag Ser Giu 280 ace Thr 265 aga Arg cge cet aae Arg Pro Asri ect ggg ttt Pro Gly Phe 270 agg atg att Arg Met Ile ccc tgg att Pro Trp Ile aaa Lys 285 <213> Homo sapiens <220> <221> poiyA signal <222> 638. .643 <221> polyA site <222> 662. .675 <300> <400> 135 aeegaaeagg aacageaeaa ectgggacec agaeatgcag taeetetaeg caaagtaaaa gtageagtgg ttcageacac tttggtatgt tgaetgtta atg atg tac gtt tet Met Met Tyr Val. Ser ata gaa atg tea Ile Glu met Ser tgt tac att Cys Tyr Ile aaa tet. tgg Lys Ser Trp tat Tyr tet Ser ggt eca ace att tee cat ttg ttc gac tat gtg gte Gly Pro Thr Ile Ser His Leu Phe Asp Tyr Vai Val 15 ggc tta aag tee ttt tet ctt aaa cag tta aaa aaa Gly Lou Lys Ser Phe Ser Leu Lys Gin Leu Lys Lys 30 aag tat tta ttt gaa tee tgt tge tat agg agt ttg Lys Tyr Leu Phe Glu Ser Cys Cys Tyr Arg Ser Leu 114 162 210 258 306 tat gtg tgt gte tte att taaacataee tgeatacaaa gatggtttat Tyr Val Cys Val Phe Ile ttetatttaa tatgtgaeat ttgttteetg gatatagtcc 5tgaaeeaea agatttatea WO 99/25825 PCTJIB98/OI 862 122 Ccgtaaattg ttaaccattt tatgttcaga tatttttcaa taataitgaga agaaaatggg 426 tatttctcta gtttttacct agtttgcttt cataacctta tatgttgaca caataattca cagagaagaa catttaaagg gttaatattt ttattgtggc tctatttga aatgtgtcta aaaaaaaaa <210> 136 <211> 1112 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 111.-194 <223> Von Heijne matrix score 4.80000019073486 seq GVLLEPFVHQVGG/HS aaca tagaga gaataatttg t tgaaacgt t aaataaatgc ccagcaagtg ttaaagataa ttcagataat tgtttattta aatatatatg actaattttt atctatttga aaatgaaaaa <221> polyA-signal <222> 1080. .1085 <221> polyA-site <222> 1101.-1112 <300> <400> 136 ccgagagaga ctacacggta ctgggacaca cggacaaaca ccgctggact ccgctgcctc ccccatctcc ccgccatctg acagacagaa gacgtactgg cgcccggagg atg agc Met Ser cca gcc Pro Ala ctg gag Leu Glu ttc agg gcc atg Phe Arg Ala Met ccc tt 't gtc cac Pro Phe Val His ga t Asp -20 cag Gln gtg gag ccc cgc Val Glu Pro Arg gtc ggg ggg cac Val Gly Gly His gcc aaa Ala Lys tca- tgc Ser Cys ggc gtc ctt Gly Val Leu gtg ctc cgc Val Leu Arg 486 546 606 666 675 116 164 212 260 308 356 401 461 521 581 641 701 761 821 881 941 1.001 1061 1112 aat gag aca Asn Glu Thr tac gag acc Tyr Glu Thr acc ctg Thr Leu ctc cct Leu Pro tgc aag ccc Cys Lys Pro 15 gct gag atg Ala Glu Met ctg gtc cca agg Leu Val Pro Arg gaa Glu cat cag His Gin ttc Phe cgc aaa ttc Arg Lys Phe aaa gga Lys Gly ttt ttc Phe Phe caa Gin agc caa agg Ser Gin Arg ccc Pro 45 ccc Pro 30 ctt Leu tct Ser tcc Ser ccc cag tac Pro Gin Tyr ctg ccc cat Leu Pro His gtt agc tgg Val Ser Trp cca Pro gga Gly ccc tgg tcc Pro Trp Ser ctg tgg cca Leu Trp Pro agt gtg gcc Ser Val Ala tgaatacccc accccggctc tctctctgag cacgcattcc ggcagagagg cctgagagga ctgctgaaac gcccccacct aggttctaga gacttaaggg aaccagctct atctgccttg aataaaaaca tattttataa gggaagcctt tgctagggtg gccttgctta cccagtcttc gttccccctg tattcaggct tagggcatta aaaattcccg aagaattaca taaaaattaa <210> 137 <211> 547 <212> DNA <213> Homo sapiens <220> <221> sig~peptide <222> 359. .454 ctctgcaccc cctgcagcag aaggtgttca gacagcccca gaaaagc tgc tgttcatttt gaacaaaagg tcttgtgctg ccttactctt Ctgctttaaa caaactataa taaacatttt agagctgggg tcgaggactg gccagtcgtt tcctcaaaga tttcaaggcc gttattttgt cctgggtgcc tgtggtttgt ggatgcttct gcaagc -catg agagcaatgt caatgatgga gccacctcag agcagattga tgtaaggcgc Ctgtcttaat accaca tgtc gacgtgagac tacccgtgtg tttgtttgcc taaccc tcag aggctgttgg tttcagtctt aaaaaaaaaa aagtgtcatc gtgatgctgg tcgtcggcac tactcatggc tgtgctcccc agcaaagacc ggggcactgt cctttatttt gcaaacctgt agtttctgtt ttaggattag WO 99/25825 WO 9925825PCT/IB98/OI 862 <223> Von H-eijnd matrix score 4 seq FSFMLLGMGGCLP/GF <221> poiyA..site <222> 536. .547 <300> <400> 137 ctggggagcc ctgcctaaga ctcatgctac cagctagcct ctcatccctt ttctactgag ggttttattg tgagctggcc ttggaattao aaggtggaag gcigtgcaaaa atgtcattcc atcctttatg acgcctttcc tgaatcacag CtcccaCCCa actttgCgaa cacaacccac atg ttg ggg acc acg ggc ctc ggg a Met Leu Gly Thr Thr Gly Leu Gly 1 -25 ctg ggc ttt ttc tcc ttt atg tta c Leu Gly Phe Phe Ser Phe Met Leu I aagaagt taa aggaagtgga accaccacca catgcttgtc gtgcattggg ttagaggagt Lca cag ggt ~hr Gin Gly ataagtttcc atgcactccg acacactttt tgccaggcaa gtgct tcctc tatctcagca cgaag tcaca acaagga taa ggattatcag Cctggtgtcc ctccccagga cat tatga cct tcc cag cag gct Pro Ser Gin Gin Ala 120 180 240 300 358 406 454 502 547 :tt gga atg ggc ggg tgc ctg cet -10 -s ae r gga tte ctg cta cag ect ccc aat cga tct ect act ttg cet gca tcc Gly Phe Leu Leu Gin Pro Pro Asn Arg Ser Pro Thr Leu Pro Ala Ser 1 5 10 acc ttt gcc cat taaagtcaat tctccaccca taaaaaaaaa aaa Thr ?he Ala His <210> <211> <212> <213> <220> <221> <222> <223> 138 1198

DN'A

Homo sapiens sig...peptide 26. .316 Von Heijne matrix score 4 seq RLPLWVSFIASSS/AN <221> polyA-signal <222> 1164. .1169 <221> polyA-.site <222> 1187. .1198 <300> <400> 138 atcctgcgaa agaagggggt tcatc a tg Met gcg gat Ala Asp -95 ggg ctc Gly Leu gac cta aag cga Asp Leu Lys Arg ttc ttg Phe Leu gtg tca Val Ser tat aaa aag Tyr Lys Lys gat aga gat Asp Arg Asp gag cat gct Glu His Ala eca agt gtt gaa Pro Ser Val Glu gte cet gtt gtt Val Pro Val Val cat gee at His Ala Ile aaa Lys gtg gca eat Val Ala Asn -65 ga c Asp gee Ala eat gct eca Asn Ala Pro ctt gca aca Leu Ala Thr ttg cga cct Leu Arg Pro gac ca Asp Gin gge age aaa Gly Ser Lys ett Leu cag Gin ggt tte tta tcc Gly Phe Leu Ser -50 gga ctt tcc aae Gly Leu Ser Lys gtg gtt caa ttt Val Val Gin Phe act Thr aat Asn ttt Phe aaa Lys 52 100 148 196 244 292 340 agt etc etc Ser Ile Ile tgt Cys tat eec ecc Tyr Asn Thr gtg egt ttc ate Val Ser Phe Ile eta gaa aag gag agc age agt Ser Ser Ser -15 gcc aet Ala Asn 1 aat cgt tta. cct Asn Arg Leu Pro ace gga cta ett Thr Gly Leu Ile ttg gtg Leu Val gtc agc Val Ser ctt gct cca ttg ttt gaa gee ctg aga caa gtt gtg WO 99/25825 WO 9925825PCTJIB98/01862 124 Leu Glu Lys G1W*Leu Ala Pro Leu Phe Giu Glu Leu Arg 15 20 gaa gtt tct taatctgaca gtggtttcag tgtgtacctt atcttcatta Ciu Val Ser Gin Val Val taacaacaca atatcaatcc caagaaaggg cccctttttc gatagatcag tgctatatt taccacagaa atggttcagt ggatgagaga ttctattcag togtgctg ccaattgtac egaataataa taaggacata ggtacataaa atggcttggt acatattatt ctgcagtata ctgctcttta atgaaaatga Caaccaatca gtgrttttaat tctgtttcca acgttagtat agtaaaaata aaatagtatt <210> 139 <211> 1400 <212> DNA <213> Homo sapiens agcaatcttt caacttatac ttctggtgta ctatcacagc tgga ttagaa aatatgccca ttttcttca aaaagtaata gatgaatatt aaactatagcc gttgtgtgc gtatgtaaac tttaaaagta agactacaat taaagagc ta gggtctrttct tcccatggag tcaaactggt ggcttgcaga gaatgttt 888 tcagtac accaatcagt taatgttttt tcttcataaa atgatagtac aaaaaaaaaa aatgctttta gcatatagat tatttagtga ttagtccggt acattgatcc ataaagccaa tatttctttg aatcactaac tgattattc tcctcaaact atttaaatac agecat ttt t a tccatgtgct gtaatttata gatctaggga caccagatat act tgagccg ctttttateg cattgagtga tcctttgt tcagagggtg ctgctttctg aattcgttat ttcatatgtg <220> <221> <222> sigpeptide 36. .107 <223> Von Heijne matrix score 5.69999980926514 seq ILGLLGLLGTLVA/ML <221> polyA...signal <222> 1302. .1307 <221> PolyAsite <222> 1389. .1400 <300> <400> 139 cagtccctga agacgcttct actgagaggt'ctgcc atg gcc tct 497 557 617 677 737 797 857 917 977 1037 1097 1157 1198 53 101 149 197 245 293 341 389 437 485 ctt ggc ctc Met Ala Ser caa ctt gtg Gin Leu Val gtt gcc atg Val Ala Met ggc Gly ctg Leu aca Thr tac atc cta ggc Tyr Ile Leu Gly ctc ccc agc _tgg Leu Pro Ser Trp 5 gca gtt ggc ttc Ala Val Gly Phe ctt Leu -10 aaa Lys tcc Ser ctg ggg ctt ttg Leu Gly Leu Leu aca agt tct tat Thr Ser-Ser Tyr aag ggc ctc tgg Lys Gly Leu Trp Leu Gly Leu ggc aca ctg Gly Thr Leu gtc ggt gcc Val Gly Ala agc Ser gcc Ala 1 att gtg Ile Val atg gaa Met Glu aca cac Thr His 20 agc aca ggc Ser Thr Gly atc acc cag Ile Thr Gin tgt Cys 40 gcc Ala 25 gac Asp tgt Cys atc tat agc Ile Tyr Ser acc ctt Thr Leu ctg ggc ctg Leu Gly Leu tcc agt gca Ser Ser Ala aga tgc aca Arg Cys Thr ccc Pro atc Ile gc t Ala gac atc cag Asp Ile Gin gc t Ala 55 tgc Cys cag gcc atg Gin Ala Met tcc tcc ctg Ser Ser Leu gcc Ala 70 gaa G lu att atc tct Ile Ile Ser gtg Val1 gac Asp atg gtg aca Met Vai Thr gtg ggc atg Val Gly Met aga gtg gcg Arg Val Aia gta gca Val Ala ggt.

Gly gtc ttc tgc Val Phe Cys gga gtc ttt Gly Vai Phe cag Gin 85 ttc Phe tcc cga gcc Ser Arg Ala aaa Lys atc ctt gga ggc ctc ctg gga ttc att Ile Leu Giy Gly Leu Leu Gly Phe Ile cct Pro gtt gcc tgg aat Val Ala Trp Asn cat ggg atc His Gly Ile cta cgg gac ttc Leu Arg Asp Phe 110 tac tca cca Tyr Ser Pro WO 99/25825 PCT/1B98/OI 862 otg gtg cot Leu Val Pro ggc att att Gly Ile Ile 145 ttt too tgc Phe Ser Cys gao Asp 130 tot Se r ago atg aaa ttt Ser Met Lys Phe gag Giu 135 C tg Lou att gga gag got Ile Gly Glu Ala too ctg ttc Ser Leu Phe ata gct gga Ile Ala Gly ato Ile 155 tao Tyr 125 ott tao ttg Leu Tyr Leu 140 atc oto tgc Ile Leu Cys gat gco tao Asp Ala Tyr 160 oaa gc Gin Ala coa Gin toa too cag Ser Ser Gin cot ott cc Pro Lou Ala aga Arg 165 aca Thr cgo too aao Arg Ser Asn tao Tyr 170 agg Arg agg ago tot Arg Ser Ser 175 000 Pro 008 Pro 185 cot ggt oaa Pro Gly Gin add gto aag agt gag tto aat Lys Val Lys Ser Glu Phe Asn tgaagaaooa ggggooagag Oocgagggoo aoaggtgagg tagaotgaot ttggooattg goaggttgaa ttgooaagga otooootgoo ooaagtoooo agaggatooo tttgoootot oooaotgaoo gaooototgt oattgotggg gatgggaagg toaagottoo otooaaagaa ctcoaoagtg tooagaotaa gggaaoagaa agoaggatgc aataaaaaaa aaaaa <210> 140 <211> 538 <212> DNA <213> Homo sapiens <220> <221> sig-.peptjde <222> 35. .130 otggggggtg gaoaotacoa gattgagcaa tgotogccat aacootcaao ggtttaooog gatoaaagao agaagoagtg aotgattggo tttgtgoatg agga tgggag too tao ago Ser Tyr Ser 200 gotgggtotg ctggatogtg aggoagaaat gocagootot t tgaaaoooo ggao tooato OctOOctotg gottttgtgg 00 tggaaoo t aactgaaata, gacaggaagg otg aoa ggj Leu Thr Gl: tgaaaaaoag tcagaaggtg gggggotagt Otgttttcot attooottaa Oooaaaoooa gotgaggttg goattgctot ooatoooao t aaaooatcot oagcctggga atat gtg y' Tyr Val 205 tggaoagcao otgctgaggg gtaaoagcat Caocttgctg gooaggaoto otaa toacat gotottagot aacctactto ottgttatga aoggtatooa catttaaaaa 533 581 629 677 725 785 845 905 965 1025 1085 1145 1205 1265 1325 1385 1400 103 151 199 247 300 <223> Von Heijne matrix score 8 seq VPMLLLIAGGSFG/LR <221> PoiyA-signai <222> 505. .510 <221> PolyA site <222> 526. .538 <300> <400> 140 gottggagtt otgagoogat ggaggagtto acto atg ttt goa oto gog gtg atg Met Phe Ala Lou Ala Val met ogt got ttt ogo aag aao aag aot oto ggo tat gga gto 000 atg ttg Arg Ala Phe Arg Lys Asn Lys Thr Leu Gly Tyr Gly Val Pro Met Lou ttg Lo u otg att got Leu Ile Ala gga ggt tot ttt ggt ott ogt gag Gly Gly Ser Phe Gly Leu Arg Glu 1 ttt tot oaa Phe Ser Gin oga tat gat got gtg aag agt aaa atg gat cot gag ott. gaa aaa aaa Arg Tyr Asp Ala Val Lys Ser Lys met Asp Pro Giu Leu Glu Lys Lys 15 oog aaa gag aat aaa ata tot tta gag tog gaa tat gag gga ag Pro Lys Glu Asn Lys Ile Ser Lou Glu Ser Glu Tyr Glu Gly Se~ 30 35 tgt tgaagggota otatotttoo ttggoootto tcoottgttg ggaotcaatc t ato r Ile %.-yb tooagaotat otooooagag aatottgtoa aggottggct ttaagotttg ttgggaaaat WO 99/25825 126 caaagactcc aagtttgatg actggaagaa tat 420 tgacctcctc caaggaagaa atccagaaag ccttaagact attctttttt cctttttttt tttaaataaa aatactatta <210> 141 <211> 1167 <212> DNA <213> Homno sapiens <220> <221> sig..peptide <222> 169. .267 <223> Von Heine matrix score 7.80000019073486 seq LTFLFLHLPPSTS/LF <221> polYA-Signal <222> 1132. .1137 <221> polyA..site <222> 1155. .1167 <300> <400> 141 gtaggaacta ctgtcccaga gctgaggcaa tgctttagta gtagtttaaa gtagtaactg gaaatttgaa gaccagatca tgggtggtct ggggatttct ctactgtatt gcatgtgaat aca Thr gcc Ala gcc tgg ctg tca Ala Trp Leu Ser ttg Leu -25 Ctt tct tcc tcc Leu Ser Ser Ser cca Pro -20 PCTIIB98/OI 862 :tcgagga CCCaggcctt gggaagatcc aagacaactt gactctgctg 480 actggaaaaa aaaaaaaa 538 caggtcattt ggagaacaag tagtggggtg gaattcagaa 120 gaacagga atg agc cag 177 Met Ser Gin ttt gga ccc ttc tct 225 Phe Gly Pro Phe Ser tcc acc agt cta ttt 273 Ser Thr Ser Leu Phe 1 ctt ggc ttg att ttg 321 Leu Gly Leu Ile Leu gac ttt gcc cta tcc 369 Asp Phe Ala Leu Ser att atg gat cca aaa 417 Ile Met Asp Pro Lys gtcacatgc caataaacaa 472 agtagagctt atgaaatggt 532 ttgttttttt aaagacggag 592 ttggctcact gtaacctccg 652 gtagctggga ttgcaggtgc 712 acagggtttc accacgttgg 772cttggcctcc caaagtgatg 832 atttttaaag tatgttccag 892 aaaaggtcat ggggaagcag 952 ttattgtcta ggccacttgt 1012 tctctagctt acaatggacc 1072 tgtcaaaact aatttttata 1132 1167 ca Leu Thr att aac tta Ile Asn Leu ctt ctt tct Leu Leu Ser t~vL tg ttctc cat cta cca cca Phe Leu Phe Leu His Leu Pro Pro -s gca aga gga caa ata aag ggc cct Ala Arg Gly Gin Ile Lys Gly Pro 10 ttc tgt gga gga tat act aag tgc Phe Cys Gly Gly Tyr Thr Lys Cys 25 atc cct aac aga att gag ttt tct Ile Pro Asn Arg Ile Glu Phe Ser 40 45 tat ttg Tyr Leu gaa Glu aga Arg aaa aca aaa Lys Thr Lys taatgaagcc atcagtcaag g~ taaattttcc agaagaaatg tcagtaagga tgagcttget tCtcgCtctg tcactcaggc cctcccgggt tcaagccatt gtgccaccat gcctggctaa tcgggctggt ctcgggctcc ggattacaga tgtgagccac ttctgtgtca tggttggaag aggtgattca tggctctgtg gaagaatatg agtcagttat ttttgaactg ggaaacacct ataaatgttt attttcacat <210> 142 <211> 730 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 143. .238 aaatccaacz agacaaataa tggagtgcag CtCctgcctc tttttgtgtt tgacctcttg cgtgcctagc acagagtagg aatttgaggt tgccagcctt tgtctgcatt cgaaaaaaaa tggtatgatc agtctcctga tttggtagag atccgcctgc caaggatgag aaggatatgg gaatggttcc ggaatttact cactttaaaa aaaaa <223> Von Heijne matrix score 8.80000019073486 WO 99/25825 WO 9925825PCTIIB98/01862 seq V <221> poly; <222> 697. <221> polyP <222> 721. <300> <400> 142 nctttgcctt cttggtgaga gagccgatgg cgc aag aac Arg Lys Asn gtt gga ggt Val Gly Gly

PMLLLIVGGSFG/LR

-signal 702 site 730 tctntccaca ggtgtccnct cccaggtcca actgcagact tngaattcgt gcgtgagctg ctgagatttg ggagtctgcg ctaggcccgc tggagrttct aagagrttcac t~c atg ttt gca ccc gcg gtg acg cgt gct ttt Met Phe Ala Pro Ala Val. Thr Arg Ala Phe aag act ctc ggc tat gga gtc ccc atg ttg ttg ctg att Lys Thr Leu Gly Tyr Gly Val Pro Met Leu Leu Leu Ile tct ttt ggt ctt Ser Phe Gly Leu gag Ctt tct Giu Phe Ser 5 gct gtg Ala Val caa atc Gin Ile aaa aaa Lys Lys aag Lys agt aaa Ser Lys tct tta Ser Leu 1 atg gat Met Asp gag tcg Glu Ser cct gag ctt Pro Giu Leu 20 gaa tat gag Giu Tyr Glu gaa Glu aat aaa ata Asn Lys Ile ttt gat gac Phe Asp Asp gac ctc ctc Asp Leu Leu aaa atc aaa Lys Ile Lys tgg Trp aag aat att Lys Asn Ile 35 gga Gly ccc agg cct Pro Arg Pro tgg act Thr cga tat gat Arg Tyr Asp ctg aaa gag Leu Lys Giu gac tcc aag Asp Ser Lys gaa gat cct GlU Asp Pro aag aca act Lys Thr Thr .tt aactggactt aa ctggatatg ta tacttcccag ca cctcgaaata caa gga aga Gin Gly Arg aa t Asn 65 tgactctgct gattctcttt tccttttttt cctaatatat acttctatca agtggaaagg ggtaatttga tgacaaataa tcttcactaa ctattccatc tgtggatgaa agtaacaatg aaaaatgtga atactgctcc aaaaaaaaaa <210> 143 <211> 1174 <212> DNA <213> Homo sapiens <220> <221> sigpeptide <222> 108. .170 <223> Von Heijne matrix score seq SFLPSALVIWTSA/AF gaa agc ctt Giu Ser Leu ttttaaataa aaattccagg aggtcatgta t tggccacgt aag Lys aaatacta cccatgga caggtttt atatttta 120 172 220 268 316 364 412 460 520 580 640 700 730 116 164 212 260 <221> poiyA..signai <222> 1141. .1146 <221> polYA-site <222> 1161. .1174 <300> <400> 143 cacgttcctg ttgagtacac gttcctgttg atttacaaaa tgaagactaa cattttgtga agttgtaaaa cagaaaacct ttt cag caa ggc ctc agt ttc ctt cct tca gcc Phe Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala -10 tct gct gct ttc ata ttt tca tac att act gca Ser Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala 1 ggtgcaggta tgagcaggtc gttagaa atg tgg tgg Met Trp Trp ctt gta att tgg aca Leu Val Ile Trp Thr gta aca ctc cac cat Val Thr Leu His His ata. gac ccg gct tta cct tat atc agt gac act ggt aca gta gct, cca Ile Asp Pro Ala Leu Pro Tyr Ile Ser Asp Thr Gly Thr Val Ala Pro WO 99/25825 WO 9925825PCTIIB98/01862 25 att aaa tgc tta Lys Cys Leu ttt Phe tat Tyr ggg gca atg cta Gly Ala Met Leu aat Asn 40 gcg gca gtt tta tgc Ile Ala Ala Val Leu att gct acc Ile Ala Thr gaa gag aac Giu Giu Asn at& ctg agt Ilie Leu Ser gtt cgt tat Val Arg Tyr aag Lys aac Asn caa gtt cat gct Gin Val His Ala atc atc aaa Ile Ile Ljys aag gct ggc Lys Ala Gly ctt Leu ttc Phe ctg agt cct Leu Ser Pro gta ctt gga Val Leu Gly cog aaa aca Gin Lys Thr tgt tta gga Cys Leu Gly 8CC ctt Thr Leu ctt Lieu gta Val1 tct att gtg gca Ser Ile Val Ala ttt gct gca Phe Ala Ala agt gga gct Ser Giy Ala ggc Gly gtg Val1 105 ctt Lieu acc ttt ggt Thr Phe Gly tca tta tat Ser Leu Tyr a tg Met 115 ggc Giy gtt cag acc Val Gin Thr atc Ile 120 tgg Trp tcc tac caa Ser TDyr Gin atg cag Met Gin 125 ccc aaa atc Pro Lys Ile atc tgg tgt Ile Trp Cys 145 ttg cac agt Leu His Ser aaa caa gtc Lys Gin Val ttc Phe 135 agc Ser atc aga ctg Ile Arg Leu gta agt gca Val Ser Ala atg ctg act Met Leu Thr tgc Cys 155 aaa Lys ttg ttg gtt Leu Leu Val 140 tca tca gtt Ser Ser Vai ctc cat tgg Leu His Trp, ggc aat ttt Gly Asn Phe 160 aac ccc Asn Pro gag gac aaa Giu Asp Lys 175 gaa Glu ggt Gly 180 ttt Phe ggg act Gly Thr 165 tat gcg Tyr Ala tcc ttc Ser Phe gat tta gaa Asp Leu Giu ctt cac atg Leu His Met 185 ttt ggt ttt Phe Gly Phe atc act act gca Ile Thr Thr Ala cag Gin 170 tgg tct atg Trp Ser Met t tc cgt gat ttt Arg Asp Phe tta acc ctc Leu Thr Leu 225 cgg cta ctt Arg Leu Leu 240 cag Gin 210 tat Tyr act tcc tta Ile Ser Leu cgg Arg 215 tgc Cys 200 gtg gaa gcc Val Giu Ala cct att aac Pro Ile Asn ctg act Leu Thr aac tta Asn Lau tac at Tyr Ile 205 cat gga His Giy 308 356 404 452 500 548 596 644 692 740 788 836 884 938 998 1058 1118 1174 120 180 232 gac act gca Asp Thr Ala aat Asn 235 220 gaa cga aca Giu Arg Thr tcc aga gat Ser Arg Asp att Ile 245 tgaaaggata aaatatttct gtaatgatta tgattctcag ggattgggga aaggttcaca gaagttgctt, attcttctct gaaattttca accacttaat caaggctgac agtaacactg atgaatgctg ataatcagga aacatgaaag aagccatttg atagattatt ctaaaggata tcatcaagaa gactattaaa aacacctatg cctatacttt tttatctcag aaaataaagt caaaagacta tgaaaaaaaa aaaaaa <210> 144 <211> 1158 <212> DNA <213> Homo sapiens <220> <221> poiyA..Signai <222> 1i33. .1138 <22i> poiyASite <222> 1146. .1158 <300> <400> i44 aarttgagct tggggactgc agctgtgggg agatttcagt gcattgcctc ccctgggtgc tcttcatctt ggatttgaaa gttgagagca gcatgttttg cccactgaaa ctcatcctgs tgrsagtgta mtggattatt ccttgggcct gaatgacttg aatgtttccc cgcctgagct aacagtccat gtgggtgatt cagctctg atg gga tgt gtt ttc cag agc aca Met Gly Cys Val Phe Gin Ser Thr WO 99/25825 WO 9925825PCTIIB98/01 862 gaa gac aaa tgt ata ttc aag ata gac tgg act Giu Asp Lys cac gcc aag His Ala Lys Cys Ile Phe Ile Asp Trp Thr ctg tca cca gga gag Leu Ser Pro Gly Glu gac gaa Asp Giu tat Tyr cag Gin ota tao tat Leu Tyr Tyr tac Tyr t tg Leu too aat ctc agt Ser Asn Leu Ser gtg Val1 att ggg cgc Ile Gly Arg aac ogo gta Asn Arg Val cac His 50 ga t Asp atg ggg gac Met Gly Asp atc tta Ile Leu gao cag Asp Gin tgc aat gat Cys Asn ASP gga aCC tat Gly Thr Tyr aag aag 9cg Lys Lys Ala 9gc Giy a to Ile gtg Val1 oto ctg ctc Lou Lou Leu caa Gin gtg caa gag Val Gin Giu tgt gaa atc cgc Cys Giu Ile Arg gta ctg cat geg Val Leu His Val cto aaa ggg gag Leu Lys Gly Giu ago Ser cag gtg tcc Gin Val Phe ott cca gag gag 000 aaa ggt acg Leu Pro Giu Giu Pro Lys Gly Thr caa atg Ott act Gin Met Leu Thr taaagagggg ccaaggggoa agagotttca tgtgcaagag 105 gcaaggaaac tgattat tgaatctca aagaaat tttagttaak aataaaa oaoaaaagoo ctgtgaa atatttata aatggaa gttyttttta ctatgcc ttttataggt accctat toagtggcc atgcotg gooooagggt toaagac oagagcaaga ctytgtt <210> 145 <211> 754 <2i2> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 5. .142 Ott gan tat gga taa o ta gt t taa tgc taa gagtaaacgc toattaaatt tgataattat tgttttgtcc atagtamttg ataoctttsa aggtgttttg tcctagcact agtgagctat aataaaaaaa oagcottcgg atttcagrtc tgtattatta acatataatg aacocggca tcagttacc ggggatagaa ttgggaggoc gawggcacca gagaaaaaaa gotaagtct agaataaaaa Ctttaaacac tccaaatatg ccthtgaoaa acattgatgc aagaaa taag gaggcagcag ctgcat tyta aaaaaa tacoacagag takgagttat acttccccct ttttggacac caaagcyat tacatytgta cagkycaggc aamtgcctga gcctgggwga 280 328 376 424 472 520 572 632 692 752 812 872 932 992 1052 1112 1158 49 97 145 193 241 <223> Von Heijne matrix score 6.59999990463257 seq VCCYLFWLIAILA/QL <221> poiyA-signai <222> 716. .721 <221> poiyA-site <222> 742. .754 <300> <400> 145 tgtg acg ago gtg tc tgg ggc ttc gtc ggo ttc ttg gtg Oct tgg tto Met Ser Val Phe Trp Gly Phe Val Gly Phe Leu Vai Pro Trp Phe -40 ato 000 Ilie Pro tgt tca Cys Ser aag ggt cot aac Lys Giy Pro Asn gtt tgo tgc tat Val Cys Cys Tyr cgg gga gtt ac att ace atg ttg gtg aco Arg Giy Val Ile Ile Thr Met Leu Val Thr -25 etc ttt tgg ct~g att gca att ctg goc caa Leu Phe Trp Leu Ile Ala Ile Leu Ala Gin oto Leu -10 -5 1 aao oct ctc ttt gga cog caa ttg aaa aat gaa aco ato tgg tat Asn Pro Leu Phe Giy Pro Gin Leu Lys Asn Giu Thr Ile Trp Tyr ctg aag tat cat tgg cct tgaggaagaa gaoatgocct acagtgctoa Leu Lys Tyr H-is Trp Pro WO 99/25825 PCTJIB98/O1 862 130 ctagatgcaa aatcacctct aaaccagacc gtctttgagg tcacgagaag agaatgcctt 301 acttttcttg acttgcctgt tttggccatt atgccttatt ctacaatgca gcgtgttttc gtgcctccat aacctgaact gtgccgactc gaagatgctg ttcttctgag agatacgtta tggctcctgc cttctcacgt gggaatcagt aagactccag tggggtggtc agtaggagag aatcgcacca aactatactt tcaggatgaa tattttcctc ctttctatgt aaaaaaaaaa <210> 146 <211> 1073 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 98. .181 <223> Von Heijne matrix score 3 .59999990463257 seq PLSDSWALLPASA/GV agctgcctta ctttgccttt cacaaaacga CtCtCtCCtt gaagtgttta cacgt tcaga tttctccttt aaa aacgttaaca tttgcacttt ttatgtactc ggaatctgtg gaaac tgctg gggaagagcc ctgccatctt gcacatttga ggtgaattac ttctgagata gatttgaaga caagacaaac atctcaacag ttggaataaa <221> PolyA..signal <222> 1035. .1040 <221> PolYA-.site <222> 1060. .1073 <300> <400> 146 ccgattacag ctaggtagtg gagcgccgct gcttacctgg gtgcaggaga cagccggagt cgctggggga gctccgcgcc gccggacgcc cgtgacc atg tgg agg ctg ctg gct Met Trp Arg Leu Leu Ala cgc gct agt Arg Ala Ser ctc ctc ccc Leu Leu Pro gog ccg ctc ctg Ala Pro Leu Leu gcc agt gct ggc Ala Ser Ala Gly Cgg Arg -15 gta Val1 gaa Giu gtg CCC ttg tca.

Val Pro Leu Ser aag aca ctg ctc Lys Thr Leu Leu ga t Asp cca Pro tcc tgg gca Ser Trp Ala gta cca agt Val Pro Ser 361 421 481 541 601 661 721 754 115 163 211 259 307 355 403 451 499 547 595 643 ttt gaa Phe Giu agg gca Arg Ala gat gtt tcc Asp Val Ser cca ctt gtg Pro Leu Val

I

att cct.

Ile Pro cca aaa Pro Lys aaa ccc aag Lys Pro Lys 20 gta aga aga Val Arg Arg gaa Glu ctt aga. ttt att gaa Leu Arg Phe Ile Glu cct. aaa aat tta agt Pro Lys Asn Leu Ser ttt aca. gaa ggc aat Phe Thr Glu Gly Asn gac ata cgg Asp Ile Arg ttt gca. atc Phe Ala Ile cct tcc act Pro Ser Thr gaa Giu 50 gg t Gly acg gag Thr Glu ttg gca ttg Leu Ala Leu ggc tac ctg Gly Tyr Leu gaa Giu ttt Phe atg Met tgg ggc cac Trp Gly His ccc aag aac Pro Lys Asn tt t Phe atg cgc ctg Met Arg Leu gcc ata tgg Ala Ile Trp cga Arg cgc Arg aac cgc tct Asn Arg Ser gcc cct ttc Ala Pro Phe 100 gga ggc aaa Gly Gly Lys atg Met aag Lys ccc atc act Pro Ile Thr cgc aaa Arg Lys agt gtt ggg Ser Val Gly gtg aca cct Val Thr Pro 125 tgt gaa ttt Cys Giu Phe 140 atg ggg Met Gly ggt gct att, Gly Ala Ile aag gct ggc Lys Ala Gly 115 ctt Leu ttc Phe gaa gaa gtg Glu Glu Val caa Gin 145 gtt gta gag Val Val Glu ctt gac cag Leu Asp Gin 150 gac cac tac Asp His Tyr 120 ggt ggg cgt Gly Gly Arg gcc cac aag Ala His Lys WO 99/25825 WO 9925825PCTIIB98/O1 862 ttg Leu 155 cga Arg ccc ttc gca gca Pro Phe Ala Ala aag Lys 160 gaa Giu gct gtg agc Ala Val Ser aga gaa cgt Arg Giu Arg cgc ggg act cta gag aag atg Arg Gly Thr Leu Giu Lys Met 165 170 aaa gat caa Lys Asp Gin gag Glu 175 gcc Ala aac cag aac ccc Asn Gin Asn Pro tgg aca Trp Thr 185 ttt gag cga Phe Giu Arg agc cca tat Ser Pro Tyr 205 atg ccc aaa Met Pro Lys act gcc aac Thr Ala Asn a tg Met 195 9gg Gly ggc ata cgg Gly Ile Arg aaa gta ctg Lys Val Leu 200 aag ttc tac Lys Phe Tyr ttg acc cac Leu Thr His sag Lys 210 a tac tgg Lys Tyr Trp ggc Gly cgt gtg tagtgagtgt aggagotaac tgtatatagg ctactgaaag Arg Val 220 aaggattctg catttctatt cccctcagcc tacccactga gccataacta aggagcagca tttgagtaga tttctgaaaa aaaaagaaaa ctgtattttt attaaataaa atttaaacat aaa <210> 147 <211> 413 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 46. .189 <223> Von Heijne matrix score 4.09999990463257 seq VFMLIVSVLALIP/ET agtctttggg acgatgttat cacttcagga tagctcttaa ttgttgattt aaaaaaaaaa <221> POIYA-signa1 <222> 377. .382 <221> polyA...site <222> 402. .413 <300> <400> 147 tgagaagagt tgagggaaag tgctgctgct 739 787 835 890 950 1010 1070 1073 57 105 153 201 249 297 342 402 413 gggtctgcag acgcg atg gat aac gtg Met Asp Asn Val cag ccg aaa ata Gin Pro Lys Ile aaa Lys Cgg Arg cat cgc ccc ttc His Arg Pro Phe tgc Cys -35 aac Asn ttc agt gtg aaa Phe Ser Val Lys ggc cac Gly His gtg aag atg Val Lys Met ttc atg ctc Phe met Leu ttg aca gtt Leu Thr Val ctg gat att Leu Asp Ile atc Ile gca Ala tca-ctg gta Ser Leu Val aca aca gta Thr Thr Val acc aca aca Thr Thr Thr gta tct Val Ser gtg ttg Val Leu gtg ttt Val Phe ctg ata. cca gaa.

Leu Ile Pro Giu ctt Leu ggt gga ggg Gly Gly Gly 10 ggg gcc ctt Gly Ala Leu gca ctt gtg Ala Leu Val 15 cgg aag ctt Arg Lys Leu aca gca gta tgc Thr Ala Val Cys ctg ttc aat ccc Leu Phe Asn Pro tgc Cys agc Ser gcc gac Ala Asp att tac Ile Tyr ggt Cct tac Gly Pro Tyr cag Gin aag cct gtg cat Lys Pro Val His ga Glu aaa as gaa.

Lys Lys Glu gtt ttg Val Leu taattttata ttacttttta gtttgatact aagtattaaa catatttctg tattcttccs aaaaaaaaaa a <210> 148 <211> 609 <212> DNA <213> Homo sapiens <220> WO 99/25825 WO 9925825PCTIIB98/OI 862 <221> <222> <223> <221> <222> <221> <222> <300> <400> Cgtcgg tC88tg sig-peptide 139. .231 Von Heijne matrix score 4.40000009536743 seq TCCHLGLPH-PVRA/PR polyA..signai 579. .584 polyA site 598. .609 148 'agtt aga t ggaaagggac gcctggtttc Cceccaagcg aaccgggatg ggaagtgact tgaacctag ctggattgaa agagaggcta gaagttcegc ttgccagcag Crf~r~tt- tAfY9 cctccrrA P.

v v W ww- -WdCx aat acc cac aeg 9t9 ett gtc tca ctt Met Ser Asn Thr His Thr Val Leu Val Ser Leu ccc Pro ccg Pro cat ceg eac ccg His Pro His Pro gee Ala -15

'U

etc Leu aec tge tgt Thr Cys Cys eac His etc Leu 9gC etCceca eac Gly Leu Pro His ceg tgg gat ect Pro Trp Asp Pro gtc cge get eec cgc ect ett ect ege gta gaa Val Arg Ala Pro Arg Pro Leu Pro Arg Val Giu agg tgg cag Arg Trp Gin cta aat gag Leu Asn Glu 1 gac tca Asp Ser gag eta agg Glu LeU Arg 20 tat Tyr eca cag gee Pro Gin Ala a tg Met aat tee: ttc Asn Ser Phe cgg tea teg ceg tgc agg aec tta agg eaa gaa gea teg Arg Ser Ser Pro Cys Arg Thr Leu Arg Gin Glu Ala Ser 35 get gac aga tgt gatcetc tgaaeetgat agattgetga ttttatctta Ala Asp Arg Cys Asp Leu ttttateett gaettggtac aagttttggg atttetgaaa agaccataca gataaceaca aatateaaga aagtegtctt eagtattaag tagaatttag atttaggttt eetteetgct teccacctec ttegaataag gaaaegtctt tgggaecaac tttatggaat aaataagetg agctgcaaaa aaaaaaaa <210> 149 <211> 522 <212> DNA <213> Homo sapiens <220> <221> polyAsite <222> 512. .522 <300> <400> 149 ceaactgcag nttegaattt acegagcgga gaggagatgc acacggcact egagtgtgag gaaaaataga a atg aag gta eat atg eac aca aaa ttt tgc etc att tgt Met Lys Val His Met His Thr Lys Phe Cys Leu Ile Cys 120 171 219 267 315 363 411 471 531 591 609 110 158 206 254 302 350 ttg etg Leu Leu gac cat Asp -His aca ttt Thr Phe gge ect Gly Pro att ttt cat Ile Phe His 20 gaa gcg ett Glu Ala Leu tgc aae cat Cys Asn His tgc Cys cgt Arg cat gaa gaa cat His Glu Giu His cac aga eag His Arg Gin gag eca age Glu Pro Ser ttt Phe cat His get Ala gga atg aca Gly Met Thr gaa Glu tea aag caa Ser Lys Gin get Ala 55 tat Tyr gaa aat gaa Giu Asn Giu aaa aaa Lys Lys aga tta Arg Leu tac tat att Tyr Tyr Ile tee ttt ttt Ser Phe Phe gaa Glu ggt Gly ett ttt gag Leu Phe Glu egt Arg 70 t ta Leu ggt gaa aat Gly Giu Asn ttg gag aaa Leu Giu Lys aca aac ttg Thr Asn Leu ggC ctt gga gag Gly Leu Gly Giu WO 99/25825 WO 9925825PCTJIB98/OI 862 aga aaa gta gtt gag att Arg Lys Val Val Glu Ile tct cat tta ggt att ttg Ser His Leu Gly Ile Leu 110 115 cat aac cac cag cat tcc His Asn His Gin His Ser aat cat gag Asn His Glu 100 gca gtt caa Ala Val Gin cat aat cat His Asn His eaaaaaaaaa gat ctt ggc cac gat cat gtt Asp Leu Gly His Asp His Val 105 gag gga aeg cat ttt cac tca Giu Gly Lys His Phe His Ser 398 t ta Leu 135 120 eat Asn tca gee eat Ser Glu Asn 125 act Thr 446 494 gtg acc agt Val Thr Ser 130 tcc ace 5cr Thr <:210> <211> <212> <213> <220> <221> <222> <223> 150 1322

DNA

Homo sapiens s igpept ide 126. .260 Von Heijne matrix score 4.59999990463257 seq VLVYLVTAERVWS/DD <221> polyAsignal.

<222> 1283. .1288 <221> poiyA..site <222> 1309. .1322 <300> <400> 150 ccgaeacct tccccgcttc tggatatgaa attcaagctg cttgctgagt cctettgccg gctgctggge gccaggegag ccctgaggag tagtcectca gtagcagctg acgcgtgggt ccacc etg aec tgg agt etc ttt gag gge ctc ctg egt ggg gtc aac aag Met Asn ?rp Ser Ile Phe Glu Gly Leu Leu Ser Gly Val Asn Lys tac Tyr cgc Arg tcc ace gcc ttt Ser Thr Ala Phe gtg ctg gtg tac Val Leu Val Tyr ggg Gly *-25 ctg cgc etc Arg Ile gtg ecg Val Thr tgg ctg tct Trp Leu Ser -20 gcc gag cgt Ala Glu Arg -5 ctg gtc Leu Val gtg tgg Val Trp ceo aeg gec His Lys Asp tgc ttt get Cys Phe Asp ttc gac Phe Asp Leu ttc etc ttc Phe Ile Phe egt gat gao Ser Asp Asp 1 tcc eec gtc 5cr Asn Val tgg gcc ctg Trp Ala Leu tgo eat act Cys Asn Thr 10 ttc cct gtg Phe Pro Val cgc cag ccc ggc Arg Gin Pro Gly tgc Cys ctc Leu gag ttc Glu Phe toc cat gtg Ser His Val ceg ott.

Gin Leu etc ctg gtg Ile Leu Val gcc Ala ace Thr 40 ceg Gin 25 tgc Cys cgc Arg gtg Val1 ccc tce ctg Pro Ser Leu c tc Leu cga Arg gtc atg cec Val Met His 120 170 218 266 314 362 410 458 506 554 602 650 tac cgg gag Tyr Arg Glu gtt Val1 ctc Leu gag aeg egg Glu Lys Arg cac His 60 ggc Gly gee gcc: cat Glu Ala His ggg gag Gly Glu eec egt ggg Asn Ser Gly tgg tgg ace Trp Trp Thr goc ttt ctc Ala Phe Leu cgc Arg tat Tyr tac ctg aec Tyr Leu Asn eag eeg cgg Lys Lys Arg ggt ggg ctc Gly Gly Leu gtg gac etc Val Asp Ile gtc tgc egc Val Cys Ser c ta Leu toe Ser ttc eag gcg egc: Phe Lys Ala Ser cct.

Pro 115 100 gtg Val1 gtc Val1 tat gtg tto Tyr Val Phe aeg tgc ceo Lys Cys His cec His 105 gca Ala ttc tac Phe Tyr ccc aee Pro Lys 110 ccc eat Pro Asn tat etc ctc cct Tyr Ile Leu Pro get cce tgt Asp Pro Cys ate gtg gec Ile Val Asp tgc Cys 130 WO 99/25825 WO 9925825PCTIIB98/OI 862 ttc a r-c tcc aag .ccc Phe Ile Ser Lys Pro 135 atc Ile tca gag aag aac Ser Giu Lys Asn Ile gcc aca gct Ala Thr Ala ctg gtg agc Leu Val Ser 165 gcc atg tgc Ala Met Cys tgc atc ctg Cys Ile Leu 140 Ctc aac Leu Asn att ttc acc Phe Thr Leu ctc gtg gag Leu Val Giu ctc ttc atg gtg Phe Met Val 145 ctc atc tac Leu Ile Tyr 160 aaa gct caa Lys Ala Gin tcc tgc aaa Ser Cys Lys aga tgc cac Arg Cys His gag Glu 170 ccc Pro ctg gca gca Leu Ala Ala agg Arg 175 Cct Ser aca ggt cat Thr Gly His 180 caa gac Gin Asp cac His 185 gg t Gly cac gat acc His Asp Thr acc Thr 190 gac ctc ctt Asp Leu Leu gac ctc atc Asp Leu Ile 195 catC His ttt Phe 205 gac Asp ctg ggc tca gac Leu Gly Ser Asp agt Ser 210 acc Thr cct cct ctc Pro Pro Leu tta Leu 215 gac cgc ccc Asp Arg Pro cga Arg 220 cat gtg aag His Val Lys 888 Lys atc ttg tgaggggctg Ile Leu ctagcatctC tcatagg gcaagagaga ggattca gccCcagctc gacggca cagaatggaa atagtgai aacacacatg cgggcaci ctcatttgct ggttaaa <210> 151 <211> 1290 <212> DNA <213> Homno sapiens <220> <221> sig..peptide <222> 50. .160 <223> Von Heijne mn cctggactgg tctggcaggt tgggcctgga tggggaggct t gc gac ctg ggg ctt ~aaa aacctgagag gctctgggag ggccagttcc ccaatgccca catcgtgtgt aaaaaaaa tgggggagct ccagttccta ccctctgctc gggttggagg ggcccactgt aagccaggga aagccatgag gtcctcaact tgcagc tcgg gaggagggcg cagaacttaa gtaggggcag ccagccacct tttccttttc ttcatagaag taaaagtcaa 746 794 842 890 938 994 1054 1114 1174 1234 1294 1322 58 atrix score 4 seq PLSLDCGHSLCRA/CI <221> poiyA_site <222> 1280. .1290 <300> <400> 151 gaggagagcc tcaggagtta ggaccagaag agcagtgca atg gct tca Met Ala Ser aaa atc ttg ctt Lys Ile Leu Leu aac Asn gaa Glu gta caa gag Val Gin Giu ccc ttg agt Pro Leu Ser gag gtg Giu Val -25 cta gac Leu Asp acc tgt ccc atc Thr Cys Pro Ile tgc ctg Cys Leu gag ctg ttg Giu Leu Leu cga gcc tgc Arg Ala Cys tgt ggc Cys Gly act gtg agc TIhr Val Ser 5 aac aag Asn Lys tgt ggt Cys Gly cac agc ctc tgc His Ser Leu Cys aec agc atg gga Thr Ser Met Gly gga Gly cta Leu 1 aaa agc Lys Ser cag gct Gin Ala gag gca gtg Glu Ala Val atc agt tac Ile Ser Tyr agc tgt cct Ser Cys Pro 20 aat cag cat Asn Gin His gtg Val1 tca ttt gaa Ser Phe Glu cat His 106 154 202 250 298 346 394 ctg gcc aac Leu Ala Asn 25 gtg Val1 gag aga ctc Glu Arg Leu aag gag Lys Glu gtc aag ttg agc Val Lys Leu Ser cat gga gag aaa His Gly Giu Lys cca ga~c aat ggg aag aag aga Pro Asp Asn Gly Lys Lys Arg 55 etc cta ctc tte tgt aag gag Leu Leu Leu Phe Cys. Lys Giu gat ctc tgt gat cat Asp Leu Cys Asp His gat agg aaa gtc att Asp Arg Lys Val Ile WO 99/25825 WO 9925825PCT/IB98/01 862 tgc tgg Cys Trp ctc acg Leu Thr ec Leu tge gag cgg Cys Giu Arg tcC cag Ser Gin 85 aag gaa Lys Giu gag cac cgt Giu His Arg ggt cac Gly His cac aca gtc His Thr Val gag gaa gta Giu Giu Val ctc Leu ttc Phe 100 aag Lys tgt cag Cys Gin aaa ctc cag gca Lys Leu Gin Ala gtc Val1 442 490 538 aag agg ctg Lys Arg Leu aag Lys 115 gag Giu gaa gag gag Giu Giu Giu gaa Giu 120 aag Lys gag aag ctg Glu Lys Leu gaa gct Giu Ala gac atc aga Asp Ile Arg aga caa agg Arg Gin Arg 145 aat gag gag Asn Glu Giu gaa Glu 130 aca Ile aaa act tcc Lys Thr Ser egg Trp 135 ga t Asp tat cag gta Tyr Gin Val caa aca gaa Gin Thr Giu ttt Phe 150 caa Gin cag cet aga Gin Leu Arg agc Ser 155 gaa Giu caa act gag Gin Thr Glu 140 tc cta aae Ile Leu Asn gaa. aag aag Giu Lys Lys cag aga gag Gin Arg Glu 160 acg ctg Thr Leu ctg Leu 165 gag Giu aga teg gaa, Arg Leu Glu gat aag tc Asp Lys Phe 175 cag Gin gca, Ala 180 ctc Leu gce gag gat Ala Glu Asp gag Giu 185 gag Giu gtt cag cag Val Gin Gin ttg gtg aga Leu Val Arg gag Giu 195 ctg Leu atc tca gat Ile Ser Asp geg Val1 200 age Ser tgt cgg agt Cys Arg Ser cag egg Gin Trp tca aca aeg Ser Thr Met gag atc egg Glu Ilie Trp 225 act gea tec Thr Val Phe gag Giu 210 agg Arg etg eag gac Leu Gin Asp gga atc atg Gly Ile Met ctg aaa aag Leu Lys Lys cca.

Pro 230 ctg Leu atg gte tcc Met Val Ser aag Lys 235 caa Gin aaa tgg age Lys Trp, Ser 220 aaa ceg aag Lys Leu Lys aeg tet aga Met Phe Arg cat gee eca His Ala Pro 240 gaa ceg Giu Leu ga t Asp 245 tgc Cys age agg atg Ser Arg Met 586 634 6-82 730 778 826 874 922 970 1018 1066 1114 1162 1210 1258 1290 aca. gce gtc Thr Ala Val 255 gte Val1 cgg Arg 260 aa t Asn tac tgg gtg Tyr Trp Val ga t Asp 265 gaa Giu aca ctg aat Thr Leu Asn aac cta aae Asn Leu Asn t tg Leu 275 att Ile ctt gee ctt Leu Val Leu tca Ser 280 egt Cys gatceag aga Asp Gin Arg caa geg Gin Val aca Cct gtg Ile Ser Val gga tcc caa Gly Ser Gin 305 tcc aag aaa Ser Lys Lys cca Pro 290 tat Tyr egg ccc eec Trp Pro Phe tat aat eat Tyr Asn Tyr etc ccc tc Phe Ser Ser ggg Gly 310 e eg Leu cat cac cgg His Tyr Trp gaa Giu 315 aga Arg ggt gec teg Gly Val Leu 300 geg gac geg Val Asp Vai aca tat tcc Thr Tyr Ser ace gcc egg Thr Ala Trp 320 cgc cat Arg His atc Ile 325 get Val1 ggg gea tac Gly Val Tyr tgt Cys 330 aat Asn aeg aag tat Met Lys Tyr gt Val1 340 aga aga ege Arg Arg Cys 335 eac Ty r gca Ala 345 egg Trp cgt caa aae Arg Gin Asn etc Leu 350 cag Gin acc aaa eac Thr Lys Tyr aga cet cea Arg Pro Leu 355 tat gge gc Tyr Gly Ala ttt ggc Phe Gly gee ata ggg Val Ile Gly aae aaa ege Asn Lys Cys <210> 152 <211> 1364 <212> DNA aag Lys 370 aaaaaaaaaa a <213> Homo sapiens <220> WO 99/25825 PCTIIB98/OI 862 <221> <222> <223> sig-.peptide 83. .139 Von Heijrie matrix score 8.60000038146973 seq LLWLALACSPVHT/TL <221> polyk..site <222> 1356. .1354 <300> <400> 152 gccrgggagc Cgaggcagcc accgtctca~g cctggccagc ccrcrggacc ccgaggrrgg acccrtactgt gacacaccta cc atg cgg aca ctc ttc aac ctc ccc tgg crr Met Arg Thr Leu Phe Asn Leu Leu Trp Leu gcc crg gcc Ala Leu Ala aaa aaa gcc Lys Lys Ala aag ccg gtg Lys Pro Val rgc agc ccr Cys Ser Pro gcc tca aag Ala Ser Lys grr cac act acc crg tca aag tca Val His Thr Thr Lou Ser Lys Ser gat gcc Asp Ala acg ctg Thr Leu 15 ggr trg Gly Leu ctg gag aag agt Leu Glu Lys Ser 112 160 208 256 304 cag Gln crc Leu trr rca gar Phe Ser Asp aaa gct gag Lys Ala Glu caa gac cg Gin Asp Arg gtg grg acg Val Val Thr agr gtg Ser Val grr crr gag Val Leu Glu aga Arg car His 45 ga t Asp 30 c gc Arg agc tac tgc Ser Tyr Cys gac Asp gca Ala aag gcc cgg Lys Ala Arg cac rrr gct His Phe Ala ggg Gly gtc Val1 gra crg ggc Val Leu Gly tat Tyr ggg Gly grc act cca tgg Val Thr Pro Trp aac agc Asn Ser car ggc tac His Gly Tyr rca ccc grc Ser Pro Val grc acg ggc Val Thr Gly ga t Asp tgg Trp acc aag grc Thr Lys Val Phe 80 aga Arg agc aag ttc Ser Lys Phe crg cag rrg Leu Gin Leu cgr ggc cgr Arg Gly Arg gag Giu 100 cga Arg aca cag arc Thr Gin Ile arg ttt gag Met Pho Glu gcr grc agg Ala Vai Arg crc cac gac Lou His Asp 105 aag cat Lys His grg Val1 110 cac His caa ggg rgg Gin Gly Trp a rg Met 115 crc Lou gcc aag ggc Ala Lys Gly 120 tgg Trp crg Leu 125 t tc Phe ata grg cct Ile Val Pro ctg trr gag Lou Phe Giu gac Asp act tac gar Thr Tyr Asp cgg aac grc Arg Asn Val ta Lou 145 gtg Val1 agr gag gar Sor Giu Asp 135at gag are Gluli gag gag ctg Glu Giu Lou gar ggc ttc Asp Gly Pho 170 grg ggc crc Val Gly Leu agc Ser 155 gtg Val1 acc gtg grc Thr Val Val gca aag aac Ala Lys Asn gtg gag grc Val Glu Val cag ctg cta Gin Lou Lou agc Ser 180 gcc Ala cag cat ttc Gin His Phe 165 cag aag cgc Gin Lys Arg crg cac cag Lou His Gin 352 400 448 496 544 592 640 688 736 784 832 880 928 arc cac atg Ile His Met cac ttg gcc His Leu Ala gcc Ala 200 acc Thr 185 cgg Arg gag Giu 195 gcc Ala ctg ctg gcc Leu Lou Ala crc Leu 205 arg Met gtc arc ccg Val Ile Pro ccr Pro 210 gag Glu arc acc ccc Ile Thr Pro ggg Gly gac cag crg Asp Gin Leu ggc Gly 220 ggr Gly rrc acg cac Phe Thr His aag Lys 225 acc Thr ttt gag cag Phe Giu Gin 215gc crg gcc LouAi ccc gtg crg Pro Vai Leu car cag ccr His Gin Pro gatr Asp 235 ggc Gly trc agc crc Phe Ser Lou a tg Met 240 c tg Leu rac gac tac Tyr Asp Tyr tct aca gcg Ser Thr Ala 245 gcc tgc gtc Ala Cys Val cct aat gca ccc Pro Asn Ala Pro tcc tgg gtt cga Ser Trp Val Arg WO 99/25825 WO 9925825PCTIIB98/01862 Cag gtc Gin Val 265 ctc aac Leu Asn 250 ctg Leu gac ccg aag Asp Pro Lys tcc Ser 270 gac Asp 255 260 aag t99 cga agc Lys Trp Arg Ser aaa L~ys 275 aag Lys atc ctc ctg ggg Ile Leu Leu Gly ttc tat ggt Phe Tyr Gly 280 cct Pro a tg MetC 285 agg Arg tac gcg acc Tyr Ala Thr tcc Ser 290 ctg Leu gat gcc cgt Asp Ala Arg gag Glu 295 gee gtc ggg Val Val Gly gcc Ala 300 gac Asp tac atc cag Tyr Ile Gin aca Thr 305 gag Giu aag gac cac Lys Asp His agg ccc Arg Pro 310 cgg atg gtg Arg Met Val aag agc cgc Lys Ser Arg 330 ceg cag gcg Leu Gin Val egg Trp 315 age Ser agc cag gcc Ser Gin Ala tca Ser 320 gtc Val2 cac ttc tcc His Phe Phe gag tac aag Glu Tyr Lys 325 ceg aag tcc Leu Lys Ser ggg agg cac Gly Arg His gtc Val1 335 gcc Ala etc tac cca acc Phe Tyr Pro Thr cgg ctg gag Arg Leu Glu 345 atc egg Ile Trp c tg Leu 350 ggc Gly cgg gag ctg Arg Glu Leu ggc Gly 355 tac Tyr get ggg gtc tc Val Gly Val Ser gac ctg cc Asp Leu Leu gag ctg ggc Glu Leu Gly cag Gin 365 ctg gac tac Leu Asp Tyr 976 1024 1072 1120 1168 1216 1.261 i32 1 1.364 59 taggtgggca ttgcggcctc cgcggtggac gcaggtgtga aatacaggcc tccactccgt.

<210> 153 <211> 1470 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 57. <223> Von Heijne matrix score 3 .90000009536743 seq MLLSIGMLMLSAT/QV gtgttctttt ctaagccatg gagegagega ttgcaaaaaa aaa <221> polyA..signal <222> 1438. .1443 <221> polyA-.site <222> 1458. .1470 <300> <400> 153 gctggcaaga ctgtttgtgt tgcgggggcc ggacttcaag gtgattttac aacgag atg Met ctg ctc tcc Leu Leu Ser gec ttg act Val Leu Thr ata ggg atg ctc Ile Gly Met Leu gtc cag ctc ttt Val Gin Leu Phe 10 tta gca tat aac Leu Ala Tyr Asn atg Met gca Ala ttt Phe ctg tca gcc aca caa gtc tac acc Leu Ser Ala Thr Gin Vai Tyr Thr gca Ala ttc tta aac Phe Leu Asn 15 gaa aat gca Giu Asn Ala ctg cct gta Leu Pro Val gaa Glu gac at Asp Ile gac ccc cct Asp Leu Pro ggt Ctt tea Gly Phe Leu cct cca cca Pro Pro Pro gca Ala at Ile aga Arg Ctt gge tat aga Phe Gly Tyr Arg 30 ccc Leu cag aca tct gat Gin Thr Phe Asp cca gct Pro Ala aac tca aaa Asn Ser Lys cca Pro 60 tca Ser 45 gag Glu aat gcc tgt Asn Ala Cys gaa Glu gaa Clu atc gge eta aag Gly Leu Lys ccc ata gcg Pro Ile Val geg eta act Vai Leu Ile 107 155 203 251 299 347 395 gta aaa gac Val Lys Asp aat Asn ttt Phe tct ggc act Ser Giy Thr t tc Phe tea Leu aga Arg aga ccc. gat Cgt Arg Leu Asp Cys aat Asn 90 gat ata aag Asp Ile Lys aat gca cag Asn Ala Gin aga Arg 100 WO 99/25825 WO 9925825PCT/IB98/01862 gca gga tac aag- gca gcc ata gtt cac Ala Gly Tyr Lys Ala Ala 105 Ile aat gtt Val att agc atg Ile Ser met cca tct gtc Pro Ser Val 135 ttc ace tat Phe Thr Tyr His Asn Val Asp 110 gag gta Ctaaag Glu Val Leu Lys gat tct gat gac ctc Ser Asp Asp Leu 115 443 gga Gly 120 ttt Phe tcc aac gec att Ser Asn Asp Ile aaa Lys att ggt gaa Ile Gly Giu tca Ser 140 cac His 125 tca Ser 115gc t Ile Acp Ile 1i3Ap0 i gct agt tct Ala Ser Ser gee aaa ggg Glu Lys Gly 150 ctt cct Leu Pro ggc Gly 155 C ta Leu ctt atc tta Leu Ile Leu gtt Val1 160 .e tc Ile ctg aaa get Leu Lys Asp 145 cca gaa ttt Pro Giu Phe eta gtg ggc Ile Val Gly gee Glu ag t Ser atc Ile ttg gae tac Leu Glu Tyr 165 tgt Cys tac Tyr 170 gtc Val1 att Ccc ttc Ile Pro Phe cc etc ttg Leu Ile Leu ate Ile 185 age Arg att ttc atg Ile Phe Met ace aa ttt gtc Thr Lys Phe Val 180ga cgl Aet 195As age cat ega Arg His Arg cet cct gte Leu Pro Vai 215 ate tgt teg Ile Cys Leu gc t Ala 200 cat His age eec age Arg Asn Arg ct t Leu 205 gga Gly eea get cae Lys Asp Gin eea ttc eag Lys Phe Lys aee Lys 220 get Asp get gag tat Asp Glu Tyr get Asp 225 age Arg ctt aeg aa Leu Lys Lys 210 gte tgt gcc Vel Cys Ala etc ctt ccc Ile Leu Pro get gag tat Asp Giu Tyr 230 egt tcc Cys Ser gee Glu 235 tgc Cys gge gac eee Gly Asp Lys ctc Leu 240 cc t Pro cat gct tat His Ale Tyr eag tgt gte Lys Cys Vel 245 ecc Thr gec Asp 255 aaa Lys tgg cte act Trp, Leu Thr ae Lys aee aee ecc Lys Lys Thr tgt Cys 265 gtg tgc egg Val Cys Arg 491 539 587 635 683 731 779 827 875 923 971 1019 1067 1115 1163 1209 1269 1329 1389 1449 1470 caa Gin 270 agt Ser gtt gtt cct Val Vai Pro 260ca tct caa SerGi ggc get Gly Asp ace gee Thr Giu tce ttt Ser Phe 310 tct tca Ser Ser tce gec tct gec Ser Asp Ser Asp 280 cat acc cct tta His Thr Pro Leu 295 ggg gct tte tcg Gly Ale Leu Ser gec tat gag gee Asp Tyr Giu Giu ace gac Thr Asp ctg age Leu Arg 300 gea tcc Giu _Ser 315 gec gec Asp Asp agt Ser 285 cc t Pro cee gee gaa Gin Glu Giu tta gct tct Leu Ala Ser gtc Val1 305 eec Asn eat gee geg Asn Giu Val 290 age gcc ceg Ser Ale Gin atg ace gee Met Thr Giu cgc tca cat Arg Ser His cag Gin 320 act Thr eat gee Asn Glu 325 gca Ala gee eat gee Glu Asn Glu att Ile 345 get Asp 330 at Asn get Asp gee cat gat Giu His Asp gac egt agt get Asp Ser Ser Asp gtc gtg Val Val 350 eat act Asn Thr gtc cag ttg cag Val Gin Leu Gin 355 gtt tgactttcag Val1 340 cct Pro eat ggt gee Asn Giy Giu cgg Arg 360 tac eec eta Tyr Asn Ile gce Ala 365 eegetgettg gtttettl ctcccttae agatttci ttectgaeac eggectti teeeegectg gtgctget gcceeeacaa aeeeeeaa <210> 154 <211> 982 <212> DN~A <213> Homo sapiens <220> <221> sig...peptide <222> 72. .197 :cc ctttaeatg :gt agaaataact ttg etctggeate aac tcaegcatca aaa a attaggtate tettttttag tetctgccae attcagctct tectgtaatt tgattttttg tactcteceg tttaatcaae geatateCtt cattcectaa tcttteggaa tgaeegtata WO 99/25825 WO 9925825PCTIIB98/01862 <,223> Von Heijine matrix score 7.19999980926514 seq ILFSLSFLLVI IT/ FP <221> poly <222> 970.

<300> <400> 154 gctgccegtt tcaagaatga gag aat ttc Giu Asn Phe atc ceg tt Ile Leu Phe A-sit 982 e cttcacactt agctccaaac ccatgaaaaa ttgccaageta taaaagcttc g atg gat tcc agg gtg tct tca cct gag aag caa gat aaa Met Asp Ser Arg Val Ser Ser Pro Glu Lys Gin Asp Lys -35 gtg gge gtc aac aat aaa cgg ctt ggt gca egc 9gc egg Val Gly Val Asn Asn Lys Arg Leu Gly Vai Cys Giy Trp -20 tcc ccc tct ttc ceg teg gtg atc att acC ttc ccc atc Ser Leu Ser Phe Leu.Leu Val Ile Ile Thr Phe Pro Ile tcc ata Ser Ile gta ttc Val Phe tgg atg Trp Met cgt ctg Arg Leu tgc ttg aag Cys Leu Lys 10 gga cgc atc Gly Arg Ile atc act Ile Ile agg gag tat Arg Giu Tyr gaa, cgt gc gOtt Giu Arg Ala Val caa Oct gac Gin Ala Asp tt g Leu atc ctg gtc Ile Leu Val c tg Leu tgc Cys 25 cca Pro aaa Lys 30 tee Phe gcc aag ggg cca Ala Lys Giy Pro ggt Gly tgc ata gat Cys Ile Asp gtg Val1 45 caa Gin gec aag gtt Val Lys Vai Acp cc AspLe cga aca gte Arg Thr Val tcc gta act Ser Vai Thr gct gtc tca Ala Vai Ser act Thr act Thr aac act ccc Asn Ile Pro cca Pro 60 gt Val1 gag atc Ctc Giu Ile Lieu cag gea gat Gin Val Asp gga Gly 75 gec Val gtc tat tac Val Tyr Tyr aga Arg caa Gin acc aga gac Thr Arg Asp atc tat age Ile Tyr Ser gca aca ttt Ala Thr Phe 110 158 206 254 302 350 398 446 494 542 590 638 686 734 782 gca geg gct Aia Val Ala ceg ctg Leu Leu 88 C As n 90 ceg Leu aac gat gec Asn Asp Val cat His ggg Gly Oct caa acc Ala Gin Thr 100 tcc Ser act Thr 105 gga Gly aga aae gec Arg Asn Val tta Leu 110 gcc Ala aca cag acc Thr Gin Thr t tg Leu cag atc eta Gin Ile Leu cga gaa gag Arg Giu Giu tc Ile 125 ggg Gly cat agc aec His Ser Ile 115ac cgl ace GinTh tta ctt gat Leu Leu Asp gaa atc aaa Giu Ile Lys 150 gcc gag gct Ala Giu Ala gatC Asp 135 gate Asp acc gaa ctg Thr Giu Leu tgg Trp 140 gtg Val1 atc cgg gtg Ile Arg Vai gte cgg at Val Arg Ile ccc Pro 155 gaa Giu cag teg cag Gin Leu Gin aga Arg 160 gee Val1 gcc cga geg Ala Arg Val 145 Ccc aeg gca Ser Met Ala ccc gca gce Leu Ala Ala gag gcc acc Giu Ala Thr 165 gaa gga Giu Gly cgg Arg 170 tcc Ser gcg aga gcc Ala Arg Ala aag Lys 175 tca Ser gaa atg age Glu Met Ser aaa tcc ceg Lys Ser Leu 180 ceg Leu aag Lys 190 cgc Arg gcc Ccc atg Ala Ser Met gct gag tc Ala Giu Ser ccc Pro 200 acc Thr get ccc cag Ala Leu Gin ctg Leu 205 acg Thr Cac ceg Cag Tyr Leu Gin acc etg Thr Leu agc acg gta Ser Thr Val aeg aac ata met Asn Ile 230 aag ccc cca gag aag aae Giu Lys Asn tcC Ser 220 ggc Gly act gtg CCC Ile Val Phe 210cg c Pco ceg ccc Pr2Lu5 r 225cc a aac cac Lag gag ggc at Glu Giy Ile g9t Gly gte agc Cat Val Ser Tyr gatc Asp 2350 aat aaa. gcc tgaggtcetc Ctgcggtagt cagctaaaaa aaaaaaaa WO 99/25825 WO 9925825PCT/1B98/01862 Lys Leu Pro Asn'Lys Ala 245 <210> 155 <211> 455 <212> DNA <213> Homo sapiens <220> <221> pOlyAsignai <222> 425. .430 <221> pOiyAsite <222> 443. .455 <300> <400> 155 gtt atg Met 1 gga acc Gly Thr cca ccc aga Pro Pro Arg tat ttg cct Tyr Leu Pro aac cta Ctg gag tta ctt Asfl 5 cag Gin £Leu Leu Giu Leu Leu att aac atc aag gct Ile Asn Ile Lys Ala gag cac atg gtt att Glu His Met Val Ile tcc tat ctg Ser Tyr Leu att cat act gat cgc '1hr Asp Arg ctg tgt cat Leu Cys His s0 aaa ggt att Lys Gly Ile atc gaa Ile Glu aac att gat Asn Ile Asp gac Asp cac His 40 aaa Lys 25 ctg ggt ttc Leu Gly Phe Ctg caa cgc Leu Gin Arg ttt Phe aga aag gaa act Lys Giu Thr tac Tyr cag aaa cgt Gin Lys Arg gaa Glu gcc agc aat tgt Ala Ser Asn Cys ttt gaa Phe Glu ttc gca Phe Ala tgt tct Cys Ser att tat cga Ile Tyr Akrg gaa act att Glu Thr Ile att C99 cat Ile Arg His tgaacagtca aac aaa ttt Asn Lys Phe gcc gtg gaa act tta att Ala Val Glu Thr Leu Ile agaaaaacat tattgaggaa aattaatatc acagcataac cagtgattat tttttaaagt cttctttcat gtaagtagca tcatctcatt aattcaatta aaaccattac cccaaaaaaa <210> 156 <211> 738 <212> DNA <213> Homno sapiens <220> <221> sig-peptide <222> 90. .278 <223> Von Heijne matrix score seq GLVCAGLADMARP/AE <221> poiyAsignai <222> 704. .709 <221> polyA_site <222> 724. .738 <300> <400> 156 gggaaaagtg actagctccc cttcgttgtc agccagggac acccggctgc caacgatccc tcggcggcg atg tcg gcc Met Ser Ala cccacccttt acattttgtg aacagggctt tactatcttt aaaaaa gagaacacag ccacgctccc gcc ggt gcc cga ggc Ala Gly Ala Arg Gly 48 96 144 192 240 289 349 409 455 113 161 209 257 c tg Leu ccc Pro gtt Val1 C99 gcc acc tac Arg Ala Thr Tyr cac His -50 ccg Pro Cgg ctc ccc gat Arg Leu Pro Asp aaa Lys cca Pro gtg gag ctg atg Val Glu Leu Met c tg Leu gag aaa ttg Glu Lys Leu ttc ttc tgg Phe Phe Trp agg Arg -35 gc t Ala ttg tac aac Leu Tyr Asn cat His -30 tgg Trp gca ggt ccc Ala Gly Pro aga aca Arg Thr gct gga Ala Gly cca att atg Pro Ile Met aaa Lys -15 ggg ttg gtg Gly Leu Val tgt Cys WO 99/25825 WO 9925825PCT/1B98/01862 ttg get gat atggcc Leu Ala Asp Met Ala 141 aga ct gca gaa aaa ctt age aca gct caa tct Arg Pro Ala Giu Lys Leu Ser Thr Ala Gin Ser 305 gct Ala att Ile gtt ttg atg gct Val Leu Met Ala att ccg aaa aat Ilie Pro Lys Asn aca Thr 15 t gg Trp ggg ttt att Gly Phe Ile agt etg ttt Ser Leu Phe tgg tca aga Trp Ser Arg 20 gct gtt aat Ala Val Asn 35 att tgg aga 110 Trp Arg tac tca cet Tyr Ser Leu tte ttt gtg Phe Phe Val gta Val1 ggg Gly gca gca gga gcc tct cag ett ttt Ala Ala Gly Ala Ser Gin Leu Phe eg t Arg tat aac caa gaa Tyr Asn Gin Giu cta aaa gct aaa gca cac aaa taaaagagtt cctgatcace tgaacaatct Leu Lys Ala Lys Ala His Lys agatgtggac aaaaccattg ggaeetagtt tattatttgg ttattgataa agcaaj actgtgtgtt tagaaggcac tgtaactggt agetagttct tgactcaata gaaaaj geaaactttt aataacagtc tctctaeatg acttaaggaa cttatetatg gatati acatttttct accatttgtc cgtaataaac cataettgct cgtaaaaaaa aaaaa <210> 157 <211> 649 <212> DNA <213> Homo sapiens <220> <221> sig~peptide <222> 88. .147 <223> Von Heijne matrix score 12.3999996185303 seq ALLLGALLGTAWA/

RR

agcta atgca tagta xaa <221> polYA-signal <222> 619. .624 <221> P0iYA..site <222> 637. .649 <300> <400> 157 ceaaagtgag agtccagcgg tcttccageg cttgggccac ggeggcggcc ctgggagcag aggaggageg accecattac gctaaag atg aaa ggc tgg ggt tgg ctg gce ctg Met Lys Gly Trp Gly Trp Leu Ala Leu 353 401 449 500 560 620 680 738 114 162 210 258 306 359 419 479 539 599 649 ctt ctg Leu Leu etc cac Leu His ggg gec ctg Gly Ala Leu tgt gga gca Cys Giy Ala ctg gga Leu Giy -5 tgc agg Cys Arg 8CC gcc Thr Ala tgg gct cgg agg age cag Trp Ala Arg Arg Ser Gin ga t Asp gct ctg gtg gat Ala Leu Val Asp gaa eta. gaa tgg gaa Giu Leu Glu Trp Giu att, gcc cag Ile Ala Gin atc aat cca Ile Asn Pro ccc cca aac Pro Pro Asn gtg Val1 gat Asp gac ccc aag Asp Pro Lys ggc agc cag Gly Ser Gin aag acc LYS Thr 30 tca gtg Ser Val att cag atg gga tee ttc cgg Ile Gin Met Gly Ser Phe Arg gtg gag gta act gtt act gtt Val Glu Val Thr Val Thr Val aaa gta gct Lys Val Ala cac His tc Ser ggC ttt gga Giy Phe Gly aggaccttgg tctaatagaa atgaagaaaa eagactcaga catttggaag aagcgcagg ettattcccc atgcacttgc tactttgttt ctgctgtaga atttgttagc aaaeagggag cacatccaca tgactggttt ttaatgcagc actgtgat aatctgagtc ggagctaaaa ataaaaaatg aaaaaaeaaa <210> 158 <211> 714 <212> DNA <213> Homo sapiens tgaaattega ctgcttaaaa aaaaagattt ggctctgtct ttcetggctg eaaaeettaa tcctgatcag cacccttet aeatgcaaac atecgtteaa aaaaaaaaaa WO 99/25825 WO 9925825PCT/1B98/01862 <220> <221> <222> <223> <221> <222> <300> <400> sig..peptide 33..92 Von Heijne matrix score 12.3999996185303 seq ALLLGALLGTAWA/RR poiyA.site 703. .714 158 agcagaggcg gagcgacccc attacgctaa ag atg Met -20 aCC Thr aaa ggc tgg ggt Lys Gly Trp Gly gcc tgg get egg Ala Trp Ala Arg tgg ctg Trp Leu agg agc gcc ctg ett Ala Leu Leu cag gat ctc Gin Asp Leu tgg gaa att Trp Gu Ile ctg ggg gcc Leu Gly Ala cac tgt gga His Cys Gly ctg etv gga Leu Leu Gly gca Ala 10 gac Asp tgC agg gct ctg Cys Arg Ala Leu gtg Val1 att Ile 1 gat gaa cta gaa Asp Glu Leu Glu gcc cag Ala Gin ccc aag aag Pro Lys Lys cag atg gga Gin Met Gly ttc Phe cgg atc aat Arg Ile Asn cca Pro gcc Ala ggc agc cag Gly Ser Gin tca Ser 45 grtg gag gtg Val Glu Val gcc cgc tca Ala Arg Ser egg atg aag Arg Met Lys aac tac gta Asn Tyr Val gag Giu gag Glu cac ctc aca His Leu Thr tat ggg gaa Tyr Gly Giu cag Gin 75 cgg Arg gag ctg ctg Glu Leu Leu 60 att gat cct Ile Asp Pro aat gga gaa Asn Gly Giu gag gag Giu Giu tcc acc Ser Thr tCc agt Ser Ser ata Ile cat His cet tat Pro Tyr tgt gac Cys Asp cgc aag Arg Lys 53 101 149 197 245 293 341 389 437 cgt gta gtg Arg Val Val gaa ctg gac Giu Leu Asp cta Leu 100 gcg Ala caa ggc atc Gin Gly Ile tgt ggg-age Cys Gly Ser cga atc Arg Ile 105 att gtg Ile Val tca gat att Ser Asp Ile agc Ser 110 ga t Asp ggc acc ctc aag Gly Thr Leu Lys gag gaa tac Glu Giu Tyr tte tcc cga Phe Ser Arg aca gat ctt Thr Asp Leu 150 gag Glu 135 tgt Cys 120 gc t Ala gac aat gtt Asp Asn Val gag Glu 125 gac Asp ata Ile gaa ctc att Giu Leu Ile gaa ttc Giu Phe 130 aag cga Lys Arg gac cat gcc Asp His Ala aaa ctt tgc Lys Leu Cys tcg cat gat Ser His Asp 160 145 gag cta Glu Leu ag t Ser tgaaceactg gagcagcc gcaatgcett ttatatal gtacaaaaaa aaaaaa <210> 159 <211> 596 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 33. .107 <223> Von Heine m cca cactggcttg atggatcacc tta tgtttttact gaaattaact cccaggaggg gaaaatggtg gaaaaaatat gaaaccaaaa <221> <222> <221> <222> score seq MFAASLLAMCAGA/EV poiYA-.s ignal 546. .551 polyA_site 584. .596 WO 99/25825 PC'T/IB98/01862 <300> 143 <400> 159 cacagttcct ctcctcctag agcctgccga cc atg ccc gcg ggc gtg ccc atg Met Pro Ala Gly Val Pro Met tcc acc tac Ser Thr Tyr ggg gca gad Gly Ala Glu ctg aaa atg Lou Lys Met gtg gtg cac Val. Val His ttc gca gcc Phe Ala Ala -25 ag t Ser ctc ctg Leu Leu agg tac Arg Tyr -10 tac Tyr gcc atg tgc gca Ala Met Cys Ala ctg aca ata cct Leu Thr Ile Pro cga ccg gac Arg Pro Asp gaa Glu dad Lys att cc& cca eag cgt gga gad ctc Ile Pro Pro Lys Arg Gly Glu Lou 20 gad aga aaa cac aad cct caa gtt Glu Arg Lys His Lys Pro Gin Val.

taactatgcc aagaattctg tgaataatat gcatcaaact acttgtcctt aagcacttag gtggatgctt~ agccgatacg ttgaaattta tgccaaagca catatcatca aaccatttca aatataacgc gaaatagaat atttgtaagt aattaagaaa ttatttaaaa ctatgaacta <210> 160 <211> 403 <212> DNA Homo sapiens <220> <221> poiyA..signai <222> 375. .380 <221> poiyAsite <222> 390. .403 <300> <400> 160 tgaagagaat ggctgttgca gtcggcgtca agagcgcgag gactcggcgg ctgagcgcgc gact, atg cgc att cgc atg aca gat Met Arg Ile Arg Met Thr Asp aag tc t att tga.

cta gtt dad acg Lys Thr 25 tct cad Ser Gin 40 tc ttaaa *aatgcta acggttt atatggt ctatatg tcattaa cag gag ga 4 Gin Giu Gl tatgtatttc ac tgcaagag gattgatatt ttggaagatg ggttgtcttt aaaaaaaaga ctt Lou gag ctt ttg gga ctg Glu Lou Leu Gly Leu aaa ttaatttatt gaggtgctca tcttgaaaac tttagtcttg atttcatata a 53 101 149 197 245 305 365 425 485 545 596 120 169 217 265 313 363 403 ctc tgc Leu Cys ttc ctc Phe Leu ggc ctg Gly Leu agg gag Arg Glu act gac Thr Asp aag ccg Lys Pro cgt Arg gac tgc aat Asp Cys Asn gagcagctcc agtgccgggg attcggacgg ccgacagcag ctagaggcgc tgctcaacaa gga Cgg aca ctg gtc ggc tgc ttt Gly Arg Thr Leu Val Gly Cys Phe 10 ~tc atc ctg ggc tcg gcg cag gag Fal Ile Leu Gly Ser Ala Gin Glu 25 ;ct gcc ggg gag ccc cgt gtg ctg er Ala Giy Giu Pro Arg Val Leu tcg gat tcc ttc t Ser Asp Ser Phe S gcc Ala agt Ser atg Met 40 gta ccc gga cac cac atc gtt tcc att gag gtg cog Val Pro Gly His His Ile Vol Ser Ile Glu Vol Gin 55 ctg acc ggg Leu Thr Gly cc t Pro ccg tat ctc tgaccacgat ggcgcttacc Pro Tyr Leu tttcogactt cattaooctt otgaccaaaa aaaaaa <210> 161 <211> 727 <212> DNA <213> Homno sapiens <220> <221> sig...peptide <222> 126. .575 <223> Von Heijne matrix score 8.60000038146973 seq LELLTSCSPPASAISQ <221> POIyA-signa.

WO 99/25825 PCT/IB98/01862 <222> 670. .67514 <221> poiyA site <222> 721. .727 <300> <400> 161 ctcagaactg tgctgggaag gatggtaggg cgactggggc tcacctccgc aCCgttgtag gacccggggt agggttttga gcccgtggga gCtgccccac gcggcctcgt cctgccaacg gtcgg atg gcg gag acg aag gac aca gCg cag atg ttg gtg acc ttc aag Met Ala Glu Thr Lys Asp Thr Ala Gin Met Leu Val Thr Phe Lys -150 -145 1 AtA gat gtg Asp Val -135 gcc gtg acc ttt acc cgg gag Ala Val Thr Phe Thr Arg Glu -130 gcc Ala tg Leu cag agg acc ctg tac Gin Arg Thr Leu Tyr -115 gtc cac ctg cta gag Val His Leu Leu Glu gag tgg aga Giu Trp Arg -125 atc ggg ttc Ile Gly Phe -110 cga gag ggc Arg Giu Giy cat ggg cag His Gly Gin -95 tgt gca gag CYS Ala Glu cag ctg gac ctg Gin Lou Asp Lau -120 ccn aaa cca gag Pro Lys Pro Glu -105 ata gtg aag aga ggc ctc tca Gly Leu Ser tgg agt gca Trp Ser Ala -100 cat His gct acc Ala Thr gag ctg Glu Leu ttt cac Phe His tct tgt tgc cca ggc Ser Cys Cys Pro Gly tg gtg gnn cgc Val Xaa Arg gag ttc Giu Phe cat His -65 tgc Cys -80 C tC Leu agc tca ctg caa Ser Ser Leu Gin ctt Leu ctg cct ccc Leu Pro Pro aag gga ttc Lys Gly Phe agg Arg tcc Ser -50 tgc Cys Ctc agc Ctc Leu Ser Leu ccg Pro -45 ttt Phe agt agc tgg gat Ser Ser Trp Asp tac Tyr cgc cca cca Arg Pro Pro cca Pro cat His CCg gct ggt Pro Ala Gly gta ttt tta Val Phe Leu Val gag Va25 l acg ggg ctt Thr Gly Leu tgt agt cca Cys Ser Pro agc cac cgt Ser His Arg cac His ccc Pro gtt ggc cag Val Gly Gin gc t Ala -15 caa Gin ctt gaa ctc Leu Giu Leu ttg acc tca Leu Thr Ser aca ggc gtg Thr Gly Val 120 170 218 266 314 362 410 458 506 554 602 652 712 727 113 gcc tct gcc tcc Ala Ser Ala Ser agt gct gcg att.

Ser Ala Ala Ile gcc cgg cag Ala Arg Gin aga aaa act gct taaggttgaa aagagaaatt Arg Lys Thr Ala taagaaattg ctgacggaat aaacataac tagaactaca agcaaaaaaa aac <210> 162 <211> 944 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 90. .155 <223> Von Heijne matrix score 5.900000095367-43 seq IILGCLALFLLLO/RK acaccgaagg aaatgaaaga <221> P0iyA-signal <222> 913. .918 <221> P01YA-site <222> 932. .944 <300> <400> 162 gaatcaggtt ccgtagccca cagaaaagaa gcaagggacg gcaggactgt ttcacacttt tctgcttctg gaaggtgctg gacaaaac atg gaa cta. att tcc cca aca. gtg Met Glu Leu Ile Ser Pro Thr Val -20 att. ate, atc ct9 ggt tgc ctt gct Ctg ttc tta ctc ctt, cag cgg aag WO 99/25825 Ile Ile Ile PCT/IB98/OI 862 Leu*Giy Cys Leu Ala Leu Phe Leu Leu Leu Gin Arg Lys aat ttg cgt aga ccc Ceg tgc atc aag ggc tgg att cct tgg att gga Asn Leu Arg Arg Pro Pro Cys Ile Lys Gly Trp Ile Pro Trp Ile Gly 10 gtt gga Val Giy ttt gag ttt ggg Phe Giu Phe Gly aaa Lys gcc cCt cta gaa ttt ata Ala Pro Leu Giu Phe Ile gag aaa gca Giu Lys Ala 209 257 305 aga atc aag gta t Arg Ilie Lys Val C Cys Phe Leu Phe acggggaaca tacctgc aacaattaac aacaata gtgcatcgac atctttg agttccaaat gtgtatg tccaatttaa gtttgtg ttgtacaaat tgcttgc gacgttgtta tttgatg cctccttgta gtagatg gaacagaccc ttattaa ggtatttgat tgtctaa <210> 163 <211> 598 <212> DNA <213> Homo sapiens <220> <221> sig-peptide <222> 126. .287 <223> Von Heiine m~ ~t 99t cgt 99c aga cgg 99t ctc cag agg aga 'S Gly Arg Gly Arg Arg Gly Leu Gin Arg Arg 40 45 aactetct ttcattgact cttaagtgca 9ggctagaac caa Gin t tg tcc gaa tgt aaa aca a tg ttt tgt taa CCtcaactaa aggdtctagt tgtgcaaaat gtagagatta taagtaaatg atgagtaatt gt tggtccgt atgtac tcca ttgtcttgaa cttttaagtt atttgtatga t ttgcgaaag aCttttcgta ttttcagtaa cgta tccaaa acacaataga ttcactac agtatctttt tattcaatt tattaaaaaa catttctgaa aaatgaaata tttttacttc t tgggaaaga t tggagttaa caggctctgt gCccgaaga aaatgtctga tccagtcaca aaaaaaa tcctctact Caattgcagc atcgaagtta taaagtgtaa caccaaagta att t ttagc t gaCtagtaat %cactttaag aatattttat ~tri v score 3 .90000009536743 seq LETCGLLVSLVES/ 1W <221> PoiYA.signal <222> 561. .566 <22i> PoiyA-site <222> 587. .598 <300> <400> 163 ctcagaactg tgctgggaag gatggtaggg cgactggggc tcacctccgc accgttgtag gacccggggt agggttttga gcccgtggga gctgccccac gcggcctcgt cctgccaacg gtcgg atg gcg gag acg aag gac gca gcg cag atg ttg gtg aCC ttc aag Met Ala Giu Thr Lys Asp Ala Ala Gin Met Leu Val Thr Phe Lys -50 -45 gat gtg gct gtg acc ttt acc cgg gag gag tgg aga cag ctg gac ctg Asp Val Ala Val Thr Phe Thr Arg Giu Giu Trp Arg Gin Leu Asp Leu 417 477 537 597 657 717 777 837 897 944 120 170 218 266 314 362 410 470 530 590 598 gcc cag agg Ala Gin Arg ctg gtt tca Leu Vai Ser atc aaa ctg Ile Lys Leu acc Thr cta Leu ctg Leu tac cga gag Tyr Arg Giu gtg Val1 -i5 tgg Trp ctg gag acc tgt Leu GlU Thr Cys ggg ctt gtg gaa agc att Val Glu Ser Ile ctg cat ata aca Leu His Ile Thr gaa aac cag Giu Asn Gin aag Lys gct tca cct Ala Ser Pro is gtg tgg ttg Vai Tro Leu gga agg aaa ttc Gly Arg Lys Phe act Thr 20 ttc aac tcg cct gat Asn Ser Pro Asp cct gag Pro Glu gag Giu cag gct cca ggc Ala Pro Gly Cgacgccatc aaggatgtct tggttctctg ttccttcttc tcctcaggct ggctcctcat agggatgctg ggtgctgcag ccccatgttc aatccatcct cccacCttgg aataaatgct aaaaaaaa ggt gcc gca Gly Ala Ala ttggttcagg ccttgactgg ttcttttcac gcc Al a cttctgattg ggcagcaggc aatgagaaaa WO 99/25825 WO 9925825PCT/IB98/OI 862 <210> <211> <212> <213> <220> <221> <222> <223> 164 360

DNA

Homo sapiens s ig-peptide 85. .150 Von H-eijne matrix score 5.90000009536743 seq IILGCLALFLLLQ/RK <221> poiyA-site <222> 349. .360 <300> <400> 164 caggtccgt agccacagaa aagjaagcaag ggaeggcagg actgtttcac acttttctgc ttctggaagg tgctggacaa aaac atg gaa cta att tee cca aca gtg att Met Giu Leu Ile Ser Pro Thr Vai Ile ata ate ctg Ile Ile Leu ttg cgt aga Leu Arg Arg gg t Gly ccc Pro tgc ctt gct ctg Cys Leu Ala Leu ceg tgc atc aag Pro Cys Ile Lys 10 ggg aaa gcc cct Gly Lys Ala Pro ttc Phe tta etc ctt eag cgg aag aat Leu Leu Leu Gin Arg Lys Asn ggC tgg att Gly Trp Ile Ce t Pro ata Ile tgg att gga gtt Trp Ile Gly Val ill 159 207 255 303 348 gga Gly ate Ile ttt gag ttt Phe Glu Phe eta gaa Leu Glu ttt Phe gag aaa gca Glu Lys Ala aag tat gga Ly's Tyr Gly cca Pro gaa Giu 25 ata Ile aga Arg a tg Met ttt aca gte Phe Thr Val ttt get Phe Ala 45 aat gtg Asn Val ace ttt gtt act Thr Phe Val Thr aaaaaaaaaa aa <210> 165 <211> 490 <212> DNA gaa gaa gga Giu Giu Giy atg gga aae Met Giy Asn ttt cta aaa Phe Leu Lys ega Arg tee Ser <213> <220> <221> <222> <223> Homo sapiens sig...peptide 77. .124 Von Heijne matrix score 4.80000019073486 seq SLFIYIFLTCSN'T/SP <221> PolYA-signal <222> 461. .466 <221> P0lYA-site <222> 477. .490 <300> <400> 165 atgagcttee ageeeeaaga gtggaggetg ceacatceca aeatagtatc tattgaaaag gaageagtgt gtatct atg att ata tet ctg ttc ate tat ata ttt ttg aca Met Ile Ile Ser Leu Phe Ile Tyr Ile Phe Leu Thr -15 -10 tgt age aac ace tet eca tet tat caa gga act caa etc ggt etg ggt Cys Ser Asn Thr Ser Pro Ser Tyr Gin Gly Thr Gin Leu Gly Leu Gly 1 5 1 A 112 160 208 256 etc ccc agt gee cag tgg Leu Pro Ser Ala Gin Trp tgc agg eta ttt tgt ttt Cys Arg Leu Phe Cys Phe tgg ct ttg aca ggt agg agg atg cag tge Trp Pro Leu Thr Gly Arg Arg Met Gin Cys 20 ttg tta eaa aac tgt ctt ttc cet ttt ccc Leu Leu Gin Asn Cys Leu Phe Pro Phe Pro 35 WO 99/25825 PCT/IB98/01862 ctc cac ctg att cag cat gat ccc tgt 17gag ctg gtt ctc aca atc tcc Leu His Leu Ile Gin His Asp Pro Cys Glu Leu Val Leu Thr Ile Ser 50 55 tgg gac tgg gct gag gca ggg gct tog ctc tat tCt ccc taaccatact Trp Asp Trp Ala Giu Ala Gly Ala Ser Leu Tyr Ser Pro gtcttccttt cccccttgcc acttagcagt tatcccocca gctatgcctt Ccctcoot cccttgccct ggcatatatt gtgccttatt tatgctgcaa atataacatt aaactatcaa gtgaaaaaaa aaaaaaa <210> 166 <211> 488 <212> DNA <213> Homno sapiens <220> <221> poiyA_.Signai <222> 458. .463 <221> poiyA site <222> 475. .488 <300> <400> 166 cogcttcoga aaagagaoag acaatgcagc catcata atg aag gtg gac aaa gao Met Lys Val Asp Lys Asp cgg cag atg gtg gtg ctg gag gaa Arg Gin Met Val Val Leu Glu Giu gaa Giu 15 aga Arg ttt cgg aac att Phe Arg Asn Ile too Ser cca gag Pro Glu gag Ctc aaa Giu Leu Lys agc tao aag Ser Tyr Lys atg gag ttg ccg Met Giu Leu Pro cag coo agg Gin Pro Arg ttc Phe tac Tyr gtg gtt tac Val Val Tyr cot ttg tgt Pro Leu Cys tac gtg cgt Tyr Val Arg tto ato Phe Ile gao Asp 45 gtg Val1 ggo cga gtg GlY Arg Val too Ser gaa, GJlu ttc too ago Phe Ser Ser ggC tgo aag Gly Cys Lys tat Tyr cog Pro aoa Thr oaa oag atg Gin Gin Met atg Met 353 413 473 490 103 151 199 247 295 343 394 454 488 gca ggg agt Aia Gly Ser aaa Lys aao agg otg gtg Asn Arg Leu Val cag Gin 80 oto Leu gca gag oto Ala Giu Leu aca aag Thr Lys cto oaa Leu Gin gtg tto gaa Val Phe Giu atc Ile cgc aco act gat Arg Thr Thr Asp gao Asp act gag gcc Thr Giu Ala tgg Trp 95 100( gaa aag ttg tot tto ttt cgt tgatotctgg gctggggaot gaattcotga Giu Lys Leu Ser Phe Phe Arg tgtctgagtc ctoaaggtga otggggaott ggaaoooota ttaaataaat tttaaaatgo aaaaaaaaaa aaaa <210> 167 <211> 771 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 48. .356 <223> Von Heijne matrix score 4.90000009536743 seq VYAFLGLTAPSGS/KE ggaootgaac aaocaagact <221> P01YA-signal <222> 742. .747 <221> PoiyA-site <222> 760. .771 <300> <400> 167 ocacagooct tttoaggaoo caaacaaoog oagoogotgt tccagg atg gtg ato WO 99/25825 PT19/16 PCT/IB98/01862 Met Val Ile cgt gta Arg Vai -100 caa caa Gin Gin tat att gca Tyr Ile Ala tct Ser -95 gg t Giy tcc tct ggc tct Ser Ser Gly Ser ac a Thr gcg att aag aag Aia Ile Lys Lys aaa Lys gat gtg Asp Vai ctt Leu gca Ala ttc cta gaa Phe Leu Giu aac aaa ata gga Asn Lys Ile Gly Ptt gaa PheG0 gaa aaa gat Glu Lys Asp aat gta cct Asn Val Pro cag att ttc Gin Ile Phe aCt Ile gag Giu gcc aat gaa Ala Asn Giu gag Giu aat cgg aag tgg Asn Arg Lys Trp aat agtc ga Asn Ser Arg cca Pro -45 tat Tyr gcc aca ggt aac Ala Thr Giy Asn ccc Pro gatc Asp atg aga gaa Met Arg Giu ctg cca cct Leu Pro Pro gcc ttc tt Ala Phe Phe 104 152 200 248 296 344 aat gaa agc Asn Giu Ser gaa gcc Giu Ala cag Gin -30 gcd Ala cgc ggg gac Arg Gly Asp tat Tyr tta Leu aga gaa aat Arg Giu Asn aat As n -15 gaa Glu 9t9 tat Val Tyr gcc ttc Ala Phe caa gca Gin Ala ggc ttg aca Gly Leu Thr tc ggt tca aag Ser Gly Ser Lys gca gaa gtg Ala Glu Val aag cag caa gca Lys Gin Gin Ala tgaaccttga gcactgC atagcagaat tagcttt gggtrtcccac atgcaaa ttatggtgag gagaacg atagtgttgt tacctgc cttccctCcc tctgcca ggagaaagag aaaaaaa, <210> 168 <211> 959 <2i2>

DNA

<213> Homo sapiens <220> <221> sig..peptide <c222> 69. .359 gc c gc t cac gga caa t ta aaa ttaagcatcc tgaaaaatga tcaaaagaaa taggcttaat tcaaaatgaa tacaaaatta tattaacata aaattatatt gccatcctgc. atacaccaat ctatggcaac ctaagtgcat aa gtcccattg gttgaaataa aaattztgaac aataagtzaga gattttacaa ctgcagcc cttttacaaa tagarttagtt atatggtga tatcgtagaa agaaaacacc acattaaaaa <223 VonHeije mtrix score 4 seq RLPLVVSFIASSS/AN <221> polyA-signai <222> 927. .932 <221> poiyA_site <222> 947. .959 <300> <400> 168 cggagagaac caggcagccc agaaacccca ggcgtggaga ttgatcctgc gagagaaggg ggrtccatc atg gcg gat gac cta aag cga ttc ttg tat aaa. aag tta cca Met Ala Asp Asp Leu Lys Arg Phe Leu Tyr Lys Lys Leu Pro -6A* 449 509 569 629 689 749 771 110 158 206 254 302 350 agt gtt gaa Ser Val Giu cct gtt att Pro Val Ile cct ggt tcc Pro Gly Phe ggg Gly aaa Lys ctc cat gcc att Leu His Ala Ile gtg tca gat aga Vai Ser Asp Arg ga t Asp gc t gga gta Gly Val ttg cga Leu Arg.

gtg gca aat Val Ala Asn gac Asp -60 gcc Al a aat gct cca gag Asn Ala Pro Giu tta tcc act Leu Ser Thr ctt gga Leu Gly ttt Phe -45 aaa Lys ctt gca aca Leu Ala Thr gga agc aaa Gly Ser Lys ctt tcc aaa Leu Ser Lys agt atc atc Ser Ile Ile cag Gin tgt Cys -25 gtg Val1 tac tat aac acc Tyr Tyr Asn Thr gtg agt ttc ata Val. Ser Phe Ile gtg gtt caa. ttt Val Val Gin Phe tac Tyr gcc Ala cgt tta cct ttg Arg Leu Pro Leu WO 99/25825 WO 9925825PCTIIB98/OI 862 15 agc agc agt gcc aat aca gga cta att gtc agc Ser Ser Ser Ala Asri Thr Gly Leu Ile Val Ser 1 5 gct cca ttg ttt gaa gaa ctg aga caa gtt gtg Ala Pro Leu Phe Glu Glu Leu Arg Gin Val Val 20 cta gaa aag gaa ctt Leu Glu Lys Glu Leu gaa gtt tct Giu Val Ser taatctgaca gtggtttcag tgtgtacctt agcaatcttt agactac caacttatac taaagag ttctggtgta gggtct CtatCacagc tcccatg tggatcagaa tcaaact aatatgccca ggcttgc tttttcttca gattatg aaaagtaata aaatcag <210> 169 <211> 464 <212> DNA <213> Homo sapiens <220> <221> Sig..peptide <222> 33. .98 aat 'cta tt gag gg t aga ttt tac aatactttta gcatatagat tat ttagtga ttagtctggt acattgatcc ataaagccaa tatttctttg aatcactaaa atcttcatta ta tCCatgtgct ca gatctaggga tac caccagatac gg~ aCttgagccg tt~ cattgagtga ggd aaaaaaa :c atg aag cct Met Lys Pro :a ctg cag ctg "a Leu Gin Leu acaacaca agaaaggg taga tcag ccacagaa atgagaga xagtgctg ~ataataa iaca taaa atatcaatcc CCCCtttttc tgctatatt atggttcagt ttctattcag ccaattgtac taaggaca ta atggcttggt <223 VonHeine mtri score 9.80000019073486 seq LWVFCLALQLVPG/SP <221> PolyA-signal <222> 437. .442 <221> PolYA-site <222> 455. .464 <300> <400> 169 gccagaactt actcacccat cccactgaca 398 440 500 560 620 680 740 800 860 920 9 59 53 101 149 197 245 293 gtg ctg ect ctc Val Leu Pro Leu cag Gin -is ccc Pro ttc ctg gtg Phe Leu Val aag cag cgt Lys Gin Arg gtg ttc Val. Phe -10 gtt ctg Val Leu tgc Cta gc Cys Leu Al gtg aag Lys tat atc Tyr Ile 10 cac ctg His Leu -5 ttg gaa Leu Glu Val1 cc-t cca Pro Pro Cet ggg agt Pro Gly Ser

I

ccc tgc ata Pro Cys Ile gaa gat tgc Glu Asp Cys tca gca cct Ser Ala Pro gag aaa gga Glu Lys Gly gaa aac tgt act Giu Asn Cys Thr ttt cag tgc tgt Phe Gin Cys Cys tgt aca atg Cys Thr Met cag Gin tcc ttc tgt Ser Phe Cys tca gaa Ser Giu aca ttt caa Thr Phe Gin 40 cgc Arg ggg9 Gly ata gtc .tgt tca Ile Val Cys Ser aac aga atc Asn Arg Ile cac aag ggc tca His Lys Gly Ser gtc Val1 gaa Glu atc atg cct Ile Met Pro gc c Ala tgaggcatat ttcctagatc attttgcctc tacgatgttt tttcttggtc cacctttagg aaggtattga gaagcaagaa actggaggcc caatatctaa cctgcaaatc gtttttgagt ttggcaataa aggctaatct accaaaaaaa aaa <210> 170 <211> 799 <212> DNA <213> Homo sapiens <220> <221> sig-.peptide <222> 110..235 WO 99/25825 WO 9925825PCT/IB98/O 1862 <223> Von Heijne matrix score 5.19999980926514 seq LLFDLVCHEFCQS/DD <221> polyAsignai <222> 764. .769 <221> PoiyA..site <222> 787. .799 <300> <400> 170 ccaaccccag gaagagtcg aagagcagcc agtgtttcgg agctgccaaa caagtacgtt ctgaaaatcc agaatggctt cttgtgccct gtatacttgj gatgtttac atg cac at Met His Ile tta caa ctg ctt Leu Gin Leu Leu act Thr gga Gly aca gtg gat gat Thr Val Asp Asp gga Gly -30 aa t As n act caa gca at Ile Gin Ala Ile gta cat Vai His tgt cct gac Cys Pro Asp tgc cat gaa Cys His Giu act Thr t tc Phe aaa gac act Lys Asp Ile t gg Trp -15 ga t Asp cta ctt Leu Leu ttt gac ctg gtc Phe Asp Leu Vai atc ctt caa gaa Ile Leu Gin Giu tgc cag tct Cys Gin Ser gatc Asp cca ccc atc Pro Pro Ile cag Gin gcc Ala aaa aca gtg cta Lys Thr Val Leu gcc Ala 15 caa Gin tct gct ttt tca Ser Val Phe Ser g cg Val 20 ata Ile ttg tc gcc atc Leu Ser Ala Ile tat Tyr 118 166 214 262 310 358 406 454 502 550 tca cag act Ser Gin Thr gag Giu cct cta at Pro Leu Ile cag aaa aaa Gin Lys Lys agg act gat Arg Thr Asp gac Asp cca Pro agc ctc Ser Leu gag aac Glu Asn gag tat cta Giu Tyr Leu atc cgg gtc Ile Arg Val 50 tog gca gag Ser Ala Giu aag Lys 35 tta Leu gaa aaa gta Giu Lys Val caa aac acg Gin Asn met gat ctt Asp Leu cag tgt Gin Cys act aaa Thr Lys tot aac aca Ser Asn Thr tta acc caa Leu Thr Gin 65 gat gat Asp Asp gag Giu atc Ile ccc cac ttg Phe His Leu att tta Ilie Leu cgt gaa tcc Cys Giu Phe acg Thr ctt Leu 95 gca Val 80 tc c Ser aaa Lys gca Ala aat atc ccc Asn Ile Phe cag Gin 100 t tg Leu tta aca aag Leu Thr Lys tta aag gac Leu Lys Asp gcg gct cag Vai Ala Gin gga Gly 110 caa Gin aag gaa ggc Lys Giu Gly cag Gin 115 tcc Phe agc aaa cag Ser Lys Gin ccc tct gca Ser Ser Ala gat tct att Asp Phe Ile 140 tcg gaa aaa Leu Giu Lys 155 aag cgc Lys Cys 120 gtg gaa Val Giu aac ctt ccc Asn Leu Leu tat agc ccc Tyr Ser Pro gcg Val1 598 646 694 acc cta cgt Ile Leu Arg gaa Giu 145 t tg Leu gat aag gog Asp Lys Ala ccc gct ga Leu Ala As 150 taaaacctga t gac p Asp aac ccc oca Asn Phe Pro aag gtc cag Lys Val Gin act Tht atcggaatta cttccgaca agaaataaac tttattttc tcactgaaaa aaaaaaaaa <210> 171 <211> 320 <212> DNA <213> Homo sapiens <220> <221> POlyA site <222> 308. .320 <300> <400> 171 tcaccatcca gagoagccag tgtccgggag gtcagaag acg ccc cac tc aag ccc WO 99/25925 WO 9925825PCT/IB98/01862 Met Pro His Ser Lys Pro etg gac tgg Leu Asp Trp ggg etc Gly Leu tet, tca gtg get gaa tgt cca gca Ser Ser Val Ala Glu Cys Pro Ala gag eta ttt Glu Leu Phe cct tcc aca ggg ggc etc. gca Pro Ser Thr Gly Gly Leu Ala aga ege gte teg tee ect egg Arg Cys Val Leu Ser Pro Trp

A

ggg aag ggt eca gga ctt gae ate tea Gly Lys Gly Pro Gly Leu Asp Ile Leu 30 gee age cat tec ctt ceg age Ccc Ala Ser His Phe Pro Ser Leu Ser Leu ggc gee tee aac ctg tgaaatggga teataatcae egeccttaeet eceetaegge Gl1y Val Phe Asn Leu cgtgejgagjg acegagegeg cggaageeee tcataaaeet eggaegetag tgtaaaaaaa aaaaaa <210> 172 <211> 331 <212> DNA <213> Homo sapiens <220> <221> sig-peptide <222> 129. .209 <223> Von Heijne matrix score 4.90000009536743 seq CLLSYIALGAIA/KI 103 151 199 254 314 320 120 170 218 266 316 <221> P0lyAsite <222> 318. .331 <300> <400> 172 aeggaaacca gatggggcaa eggggcggtt cacccage ctgeecactt ecagceaga aaaaggaa aeg aac agg gee cct get Met Asn Arg Val Pro Ala -25 tgt tea ctg agt tac ata gea etc 5 Cys Leu Leu Ser Tyr Ile Ala Leu etagtgeaga cgtagctg agetccc aattetacta atggcgeeee ttcttcccga gat tee eca aac aeg tgt eta ate Asp Ser Pro Asn Met Cys Leu Ile -20 ga gee ate eat gea aaa ate tgt ly Ala Ile His Ala Lys Ile Cys agg aga gea Arg Arg Ala -10 tee Phe cag gaa gag Gin Glu Glu gga aga gca Gly Arg Ala aat gca aag aeg ggc gtg Asn Ala Lys Thr Gly Val taaageeccc ttggaatagc aga get tgg tge at Arg Ala Trp Cys 11 eaaaaaaaaa aaaaa <210> 173 <211> 1075 <212>

DNA

<213> Homo sapiens <220> <221> sig-peptide <222> 78.-.359 a e eca egg gee aaa Pro Trp Ala Lys score 4.19999980926514 seq IILTAVYFALSIS/LH <221> P01YA-signal <222> 1042.-.1047 <221> polyA site <222> 1063. .1075 <300> <400> 173 gtggcaggga geagecagga gcggtttec ggaactgtg ggacggcc ccgggggccc gagaaaaeag aaggaag atg etc eag acC age aac tac age ctg gtg etc WO 99/25825 WO 9925825PCT/1B98/O1 862 Met Leu Gin Thr Ser Asn Tyr Ser Leu Vai Leu tct ctg eag Ser Leu Gin tea gaa etg Ser Glu Leu ate cag gat Ile Gin Asp ttc Phe ctc Leu ctg ctg etg tcc Leu Leu Leu Ser tat Ty r gac etc ttt gtc Asp Leu.phe Val.

eaa aag act Gin Lys Thr cc t Pro -60 ttc Phe gtc ate cag ett Val Ile Gin Leu gtg Val att Ile aat tcc ttc Asn Ser Phe etc tte ate Leu Phe Ile ttc etc atg Phe Lau Met att gca gte Ile Ala Val tte tc Phe Phe etc Lou -45 ttc Phe aac atc ate Asn Ile Ile ate Ile 9te Val1 aac ace tte Asn Thr Phe eat His gte ValI -30 ace Thr eag get 9gC Gin Ala Gly ctg Leu get Ala ~ad etc eta Asn Leu Leu aag tte aaa Lys Phe Lys ate ate Ile Ile etg aca Leu Thr 10 atg aae Met Asn gtg tac Val. Tyr_ age ate Ser Ile tee cct Ser Leu 1 ttc ata Phe Ile ttt gee etc Phe Ala Leu aaa aae tee Lys Asn Ser gte tgg gte Val Trp Val 5 tta ege tgg Leu Arg Trp aae age Asn Ser aga eta Arg Leu tgg aca gat Trp Thr Asp 20 gtg ttg tac Val Leu Tyr gga Gly ctt caa atg Leu Gin Met e tg Leu ttt gta tte cag Phe Val Phe Gin gea gca Ala Ala tgc tac tte Cys Tyr Phe aga Arg tat Tyr aaa egg aca gee Lys Arg Thr Ala eta ggc gat ect Leu Gly Asp Pro 35 cac His gta Val1 aag Lys ttc tac eag Phe Tyr Gin gac Asp tet ttg tgg etg Ser Leu Trp Leu cgc Arg gag tte atc Giu Phe Met cctteetgat aaeagetgga tcccctet cettetacct aceggeettt tgggacagaa tgatagttac actttaaaaa aa caa Gin 550 gtt ega agg tgaeetcttg teaeaetgat ggatactttt Val Arg Arg agaageeaea etttccaagg tgeaeaatta etgttccacc St tgeegagg ggaee tccg gtgetectga aatgttttat tttgetgctt aaggtteaga gagtgtcccc cetteette gttetgtggc geeeeeaaag etgatcaeac aaatagagaa tgeagggaga ctagetgtgt ateggtctcc ctcEcctctc tcttaccctt gateteccag egcagacatt taaattgaat gttggcccta teageat tea agtgcggeat tgtaceatte gtgaagettt tgaccaaagg tagattttta tcttgttcca 158 206 254 302 350 398 446 494 542 593 653 713 773 833 893 953 1013 1073 1075 109 157 tgeatgggca agaaggaaga ceettccttg attctccctg teetttagcc a tgegaagag taeeeaaggc aaaaaaaaaa <210> <211> <212> <213> <220> <221> <222> <223> 174 632

DNA

Homo sapiens sig...peptide 62. .265 Von Heijne matrix score 4.59999990463257 seq LPFSLVSMLVTQG/LV <221> polyp, signal <222> 602. .607 <221> polyA site <222> 621. .632 <300> <400> 174 caetgggtea aggagtaagc agaggataaa caaetggaag gagagcaagc aeaaagteat e atg get tea geg tet get cgt gga aae caa gat aaa gat gee cat ttt met Ala Ser Ala Ser Ala Arg Gly Asn Gin Asp Lys Asp Ala His Phe -60 eca eca eca age aag cag agc ctg ttg ttt tgt eca aaa tea aaa ctg Pro Pro Pro Ser Lys Gln Ser Leu Leu Phe Cys Pro Lys Ser Lys Leu WO 99/25825 PCTJIB98/01862 cac aec His lie gaa age Glu Ser cac His -45 aga gca gag atc tca Arg Ala Giu Ile Ser 153 0 aag ate a tg Cga Lys Ile Met Arg gaa egt cag gaa Giu Cys Gin Giu tcC egg aag Phe Trp Lys gtc Val aga Arg -15 gc e Ala ace cag Thr Gin Ceg Leu caa Gin gga eCa gee eac Gly Leu Val Tyr 1 Ctg ccc aaa gtt Leu Pro Lys Val ccC CCC tCC Pro Phe Ser gge tat Ceg Gly Tyr Leu 5 CCC gct g ce Lou Ala Gly Leu gea agc aeg Vai Ser Met CCC gga tca Phe Gly Set cct gga aag Leu Gly Lys gca Ala 20 99a Gly gca gct aat tCC aga Ala Ala Asn.Ser Arg ctc teg gga CCC ggc Lou Lou Gly Phe Gly age aaa tCC cat CCC gea eca Cac Val Ser Tyr CCC gaa Phe Glu at& Ile 35 ggg Gly gta Cgc cag Val Cys Gin gat cag cc Asp Gin Leu cge Arg gec ggt

CCC

Ala Gly Phe cac His 99te Gly eca cag cae aac Pro Gin His Asn agg Arg ege CtC CCC ace CgC gag gaa tgc Cys Leu Leu Thr Cys Glu Glu Cys aaa Lys 55 agctgg t Ile Lys His Gly Leu Ser Caaaeectge gtctgtgact gag aag gga Glu Lys Gly gac tcC cag CCC eca gee, tCC Asp Ser Gin Pro Ser Ala Ser tcegaageCC Cteaaaeeec Cgaaeeegta cacatteaaa ateecaageg Cactttaaaa Caaaaeaeee ctaatgtaaa aaaaaaaaa <210> 175 <211> 430 <212> DNA <213> Homo sapiens <220> <221> polyA signal <222> 402. .407 <221> polyAsie <222> 419. .430 <300> <400> 175 gtaeegggaa agegattegt gaa aeg aaa gta gaa gaa gag cat aec aat gca Met Lys Val Glu Giu Glu His Thr Asn Ala 205 253 301 349 397 445 493 543 603 632 53 101 149 197 245 293 ata ggc ace ctc Ile Gly Thr Leu cac His 1s ctg Leu ggc gge Ceg aca Gly Gly Leu Thr gee Al a 20 agg Arg acg eea gea gae Thr Leu Val Asp aae ata Asn Ile eca aea aeg Ser Thr Met gtc gae aeg Val Asp Met ata gtg ate Ile Val Ile gc C Ala aac Asn cta Cgc acg Leu Cys Thr gaa Giu ata acg Cac Ile Thr Tyr atg Met 50 ctg Leu tCa ccC Ser Pro aag caa Lys Gin gga gca c cc Gly Ala Pro gea aaa eca Ala Lys Leu gga aaa aca Gly Lys Thr gga gec age.

Gly Val Ser gga gag gat Gly Glu Asp cCC gca eec Leu Ala Phe aca gea cat Thr Ala His acc tCCC Thr Ser geg gge ceg Val Gly Leu gga Gly ace Thr 80 cac His aac aag gcc aca Asn Lys Ala Thr ctg gga aac Cga Leu Gly Asn gga Gly aaa tta ata gca Lys Leu Ile Ala caa Gin aga cac aca Arg His Thr gagaaea gcagaatgac ctaaagaaac ccaacaatga aeaeeaagta tagaeeegac tcaaacaaeC gtaaeeeeeg aaataaaeea gcaaaaccaa aaaaaaaaaa <210> 176 <211> 185 340 400 430 WO 99/25825 WO 9925825PCTIIB98/01862 <212> DNA <213> Homo sapiens <220> <221> <222> <223> sig-peptide 42. .113 Von Heijne matrix score 3.70000004768372 seq ILFNLLIFLCGFT/UY <221> polyA~sice <222> 172. .185 <300> <400> 176 ctttcagaac tcactgccaa gagccctgaa Caggavccac tcc *att aag 8CC Phe Ile Lys Thr ggc ttc acc aac Gly Phe Thr Asn atg atc ctc ttc aat ttg Met Ile Leu Phe Asn Leu acg gat ttt gag gac tca Thr Asp Phe Glu Asp Ser c atg cog tgc ttc agc Met Gin Cys Phe Ser ctc atc ttt ctg tgt Leu Ile Phe Leu Cys ccc tac ttC aaa atg Pro Tyr Phe Lys Met cat aaa cct gtt aca atg His Lys Pro Val Thr Met <210> 177 <211> 585 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 108. .170 <223> Von Heilne matrix taaaaaaaaa aaaaa score seq SFLPSALVIWTSA/AF <221> poiyA..signal <222> 550. .555 <221> polyA-site <222> 574. .585 <300> <400> 177 cacgttcctg ttgagtacac gttcctgttg tgaegactaa cattttgtga agttgtaaaa atttacaaaa cagaaaacct ttt cag caa Phe Gin Gin tct gct gct Ser Ala Ala eta Ile ga a Glu

I

gac ccg Asp Pro aaa tgc Lys Cys ggc ctc agt ttc ctt cct tca. gcc Gly Leu Ser Phe Leu Pro Ser Ala -10 ttc ate ttt tca tac att act gca Phe Ile Phe Ser Tyr Ile Thr Ala

S

gct tta cct tat atc agt gac act Ala Leu Pro Tyr Ile Ser Asp Thr 20 tta ttt ggg gca atg cta aat att Leu Phe Gly Ala Met Leu Asn Ile ggtgcaggta tgagcaggtc gttagaa atg tgg tgg Met Trp Trp ctt gta att tgg aca Leu Val Ile Trp Thr gta aca ctc cac cat Val Thr Leu His His ggt aca gta gct Gly Thr Val Ala gcg gca. gtc Ala Ala Val tta Leu caa aaa tagaaatcag gaagataatt caacttaaag aagttcattt catgaccaaa Gin Lys ctcttcagaa acatgtcttt acaagcatat ctcttgtatt gctttctaca ctgttgaatt gtctggcaat atttctgcag tggaaaattt gatttagcta gttcttgact gataaatatg gtaaggtggg cttttccccc tgtgtaattg gctactatgt cttactgagc caagttgtaa tttgaaataa aatgatatga gagtgacaca aaaaaaaaaa. a <210> 178 WO 99/25825 WO 9925825PCTIIB98/OI 862 <211> <212> <213> <220> <221> <222> <223> 613

DNA

Homo sapiens sig~peptide 118. .171 Von Heijne matrix score 5.90000009536743 seq ALALLWStLPASDL/GR <221> polyA.signai <222> 583. .588 <221> poiyA-.site <222> 602. .613 4300> <400> 178 ggggtgggtg gactagaagc atr.tgggagt agtggccagg gagctgccgc acagagcctg gtgtccacaa gcttccaggt ggccctggac gctagccacg tggggt tgga gcctggg atg agc ccc Met Ser Pro gac ctg ggc Asp Leu Gly ggc Gly cgg Arg agc gcc ttg Ser Ala Leu tca gtc att Ser Val Ile 5 gcc crtt Ala Leu -10 gct gga.

Ala Gly ctg tgg tcc ctg cca gcc tct Leu Trp Ser ctc tgg cca Leu Trp Pro Leu Pro Ala Ser cac act ggc gtt His Thr Gly Val ctc Leu aag Lys 1 atc cac Ile His agc ata Ser Ile ttg gaa aca Leu Giu Thr 20 ttt ccc ctc Phe Pro Leu agc Ser cag tct ttt Gin Ser Phe C tg Leu 25 t tg Leu caa ggt cag ttg Gin Gl.y Gin Leu acc Thr Val1 tgt tgt aca Cys Cys Thr tt~t tgt gtt Phe Cys Val gta aca gtg Val Thr Val gg t Gly gga Gly ggg agg gtg Gly Arg Vai ggg Gly aca ttt gtt Thr Phe Val tgagtcgatg ggtcagaact tagtatacg tcgaaaataa gcaccctggt aactaaaccc attgattaag ttactgtaaa agcttgggtt agaacatagc aagggggctc ctctgttgga gcaaaccttt aaaaaaaaaa aa 179 <211> 427 <212> DNA <213> Homo sapiens <220> <221> sig..peptide <222> 128. .268 <223> Von Heijne matrix score seq SALLFFARPCVFC/FK catgcgtcct ctctaatagc tatttttgta gtaa tgtaaa ctgagtgaca ta taaaggc t ggact taa tg tgtaarttat gggcattttg tagctgt gc taagaat t aaataaacat 117 165 213 261 309 351 411 471 531 591 613 120 169 217 265 <221> polyA-signai <222> 410. .415 <221> polyA-site <222> 424. .427 <300> <400> 179 agcttggatt tacactgggc aacgtggttg gaatgtatct caaacctggc taaaaaactt gaagaaatta aaaaggactt ctagtgc atg aga ctg cct cca gca ctg cct tca Met Arg Leu Pro Pro Ala Leu Pro Ser ggctcagaac tatgatatac ggatgccaag aagaaacccc gga tat act gat tct Gly Tyr Thr Asp Ser caa aag ctt ttg ttt Gin Lys Leu Leu Phe act gct ctt Thr Ala Leu gag ggc ctc gtt tac tat ctg aac Giu Gly Leu Val Tyr Tyr Leu Asn -25 tcg tct cca gcc tca gca ctt CtC ttc ttt gct aga ccc tgt gtt ttt Ser Ser Pro Ala Ser Ala Leu Leu Phe Phe Ala Arg Pro Cys Vai Phe WO 99/25825 WO 9925825PCTIIB98/01862 156 -10 tgc ttt aaa gca agc aaa atg ggg ccc caa ttt gag aac Cys Phe Lys Ala Ser Lys Met Gly Pro Gin Phe Glu Asn 1 5 10 ttt cca aca tac tca cct ctt ccc ata atc cct ttc caa Phe Pro Thr Tyr Ser Pro Leu Pro Ile Ile Pro Phe Gin 25 agg ttc taagactgga attatggtgc tagattagta aacatgactt Arg Phe aaaaacaaaa <210> 180 <211> 905 <212> DNA <213> Homo sapiens <220> <221> sig...peptide <222> 149. .457 <223> Von Heiine matrix tac cca aca Tyr Pro Thr ctg cat ggg Leu His Gly ttaatgaaaa ta taaaagcttc aa gagaatttcg tt ttc cct al Phe Pro 427 score 4.90000009536743 seq FLLAQTTLRNVLG/TQ poiyA_site 893. .912 <221> <222> <300> <400> 180 gccgcctgtt Ccaagaatga tgggtgtcaa cttcacactt agctccaaac ccatgaaaaa ttgccaag gatggattct agggtgtctt cacctgagaa gcaagata 4 caataaacgg cttggtgt atg tgg ctg gat cct gi Met Trp Leu Asp Pro V 4 -100 gtt ggt gat cat tac ctt ccc cat ctc cat Val Gly Asp His Tyr Leu Pro His Leu His ctc Leu ctt Leu ttt cct Phe Pro atg gat M1et Asp 9tg Val1 gaa ggt ttg atc Glu Gly Leu Ile ctg gtc ctg cca.

Leu Val Leu Pro gtt gac ctc Val Asp Leu acc aga gac Thr Arg Asp atc tat agt Ile Tyr Ser aca Thr tgc Cys -70 att Ile -85 ata Ile gat gtg ttt Asp Val Phe gtc aaa Vai Lys gtt act tgc Val Thr Cys gta act act Val Thr Thr aac Asn gta Val1 gc t Ala cct cca caa Pro Pro Gin gat gga gtt gtc Asp Gly Val Val aat gtc aac gat Asn Val Asn Asp gag atc ctc Giu Ile Leu tat tac aga Tyr Tyr Arg gtc cat caa Val His Gin gca Ala cag Gin atc Ile aca Thr ttt Phe gct gtc tca Ala Val Ser ctg ctg gct Leu Leu Ala acc act ctg Thr Thr Leu aga Arg -5 aa t Asn 120 172 220 268 316 364 412 460 508 556 604 652 700 748 gtc tta Val Leu acc ttg tcc Thr Leu Ser cag act tta Gin Thr Leu -10 cag atc Gin Ile ctt gat Leu Asp ggg aca Gly Thr 1 tta gct gga LeU Ala Gly i0 gat gcc acc Asp Ala Thr cga gaa gag atc Arg Giu Giu Ile gaa ctg tgg Giu Leu Trp gcc cat agc Ala His Ser atc cgg gtg Ile Arg Val ttg cag aga Leu Gin Arg gcc cga Ala Arg tcc atg Ser Met gtg Val1 gaa atc aaa Giu Ile Lys gat Asp 40 gag Glu gt.

Val1 cgg att ccc Arg Ile Pro gtg Val1 gca gcc gag Ala Ala Glu gcc acc cgg Ala Thr Arg ctt Leu tcc gaa Giu 60 aaa Lys gcg aga gcc aag Ala Arg Ala Lys gtc Val gca gct gaa gga Ala Ala Giu Giy atg gtg ctg gct gaa atg aat gct tcc Giu Met Asn Ala Ser tcc ctg Ser Leu aag tca gcc LYS Ser Ala cgc tac ctg tct ccc ata gct ctc cag ctg wo 99/25825 WO 9925825PCTIIB98/O1 862 Ser Met Val Leu 'Ala cag acc ttg agc acg Gin Thr Leu Ser Thr 100 cct ctg ccc atg aat Pro Leu Pro Met Asn 115 aac cac aag aag ctt Asn His Lys Lys Leu 130 caaaaaaaaa aaaa <210> 181 <211> 307 <212> PRT <213> Homno sapiens Giu Ser <220> <221> SIGNAL <222> -13. l <300> <400> 181 met Leu Ala V Leu Giu Ser P Leu Leu Leu G Arg Leu Phe G Gly Asp Val M4 Giu Asn Gly G.

Lys Thr Arg G Ala Gly Pro A: 100 Phe Giu Val A: Giu Thr Pro I 1: Val Thr Gin A.

150 Trp Gin Arg A 165 Gly Arg Leu L 180 Leu Asp Gin L Asp Phe Val L 2 Tyr Val Ser G 230 Met Pro Gly P 245 Trp Val Gly M 260 Asp Phe Leu S Lys Lys Lys <210> 182 <211> 59 <212> PRT al ro ly lu sn sn le 35 sp rg eu eu eu 15 ly he et Ser Ile Val1 Asn Phe Ile Asp Gly Pro 120 Giu Gly Asp Giu Arg 200 Val1 Leu Pro Ser gta Val1 ata Ile cca Pro 135 Leu Asp Leu 25 Gin Thr Giu Giu Thr 105 Trp Gly Arg Tyr Tyr 185 Phe Ala Met Asp Thr 265 gcc Ala Cta Leu 120 sat As n Thr Pro 10 His Leu Gly Thr Pro 90 Leu Lys Lys Lys Leu 170 Asp Pro Glu Lys Asn 250 Ile 157 Pro Ile Ala Leu Gin Leu Arg Tyr Leu 90 acc gag aag aat tct acg att gtg ttt Thr Giu Lys Asn Ser Thr Ile Val Phe 105 110 gag ggc att ggt ggC gtc agc tat gat Giu Gly Ile Gly Gly Val Ser Tyr Asp 125 aaa gec tgaggtcctc ctgcggtagt Lys Ala 796 844 891 905 Val1 Gin Pro VIal Thr Ile 75 Val1 Phe Arg Asn Ile 155 Leu Thr Asn Thr Gly 235 Ile Arg Pro Leu Pro Leu Asn Thr Gly Pro 45 Ala Asp 60 Ala Arg Cys Gly Val Ala Giu Val 125 Met Ser 140 Tyr Phe Leu Val Val Thr Gly Val 205 Thr Met 220 Gly Ala Arg Pro Pro Asn Leu Gly Ser Phe is Lys Leu 30 Glu Ser Gly Arg Phe Gly Arg Pro Asp Ala 110 Lys Leu Phe Vai Thr Asp Met Giu 175 Arg Giu 190 Gin Leu Ala Arg Asp Leu Ser Ser 255 Pro Gly 270 Arg Met Ala Met Lys Glu Arg Gin Ile Ala Val Val Ser Gly Leu Gly Tyr Lys Leu Leu Asn Asp 145 Ser Ser 160 Gly Thr Val Lys Ser Pro Ile Arg 225 Phe Val 240 Ser Gly Phe Ser Ile Phe Met Leu Pro Pro Ala Giu His Ile Lys Leu Pro Cys Ile Arg Gly Leu 115 Ser Ser 130 Leu Thr Ser Lys Asp Asp Val Leu 195 Ala Giu 210 Arg Val Glu Asn Gly Tyr Met Leu 275 Lys Val 290 er Giu Arg Pro Trp Ile Lys WO 99/25825 PCT/IB98/01862 1 <213> Homo sapiens <220> <300> <400> 182 Met Met Tyr Val Ser Ile Giu Met Ser Gly Pro Thr Ile Ser His Leu 1 5 10 Phe Asp Tyr Val Val Cys Tyr Ile Tyr Giy Leu Lys Ser Phe Ser Leu 25 Lys Gin Leu Lys Lys Lys Ser Trp Ser Lys Tyr Leu Phe Clu Ser Cys 40 Cys Tyr Arg Sor Lou Tyr Val Cys Val Phe Ile <210> 183 <211> 97 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -28. l <300> <400> 183 Met Ser Pro Ala Phe Arg Ala Met Asp Val Glu Pro Arg Ala Lys Gly -20 Val Leu Leu Glu Pro Phe Val His Gin Val Gly Gly His Ser Cys Val -5 1 Leu Arg Phe Asn Glu Thr Thr Leu Cys Lys Pro Leu Val Pro Arg Glu 10 15 His Gin Phe Tyr Glu Thr Leu Pro Ala Glu Met Arg Lys Phe Ser Pro 30 Gin Tyr Lys Gly Gin Ser Gin Arg Pro Leu Val Ser Trp Pro Ser Leu 45 Pro His Phe Phe Pro Trp Ser Phe Pro Leu Trp Pro Gin Gly Ser Val 60 Ala <210> 184 <211> 52 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -32. l <300> <400> 184 Met Leu, Gly Thr Thr Gly Leu Gly Thr Gin Gly Pro Ser Gin Gin Ala -25 Lou Gly Phe Phe Ser Phe Met Leu Leu Gly Met Gly Gly Cys Leu Pro -10 Gly Phe Leu Leu Gin Pro Pro Asn Arg Ser Pro Thr Lou Pro Ala Ser 1 5 10 Thr Phe Ala His <210> 185 <211> 124 <212> PR'r <213> Homo sapiens <220> <221> SIGNAL <222> -97. i <300> <400> 185 Met Ala Asp Asp Lou Lys Arg Phe Leu Tyr Lys Lys Leu Pro Ser Val -90 Glu Gly Leu His Ala Ile Val Val Ser Asp Arg Asp Gly Val Pro Val WO 99/25825 PCT/IB98/01862 Val Lys Val Ala As Phe Leu Ser Thr Ph -4 Leu Set Lys Asn Ly Val Gin Phe Asn Ar Ser Ala Asn Thr Gl 1 Leu Phe Glu Glu Le <210> 186 <211> 230 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -24..-1 <300> <400> 186 Met Ala Ser Leu Gl -2( Gly Leu Leu Gly Th] Ser Set Tyr Val Gl Gly Leu Trp Met Gil Asp Ile Tyr Ser Th] Gin Ala Met Met Va Ile Ser Val Val Gl Ala Lys Asp Arg Va Gly Leu Leu Gly Ph< 105 Arg Asp Phe Tyr Set 12! Gly Glu Ala Leu Ty: 140 Ala Gly Ile Ile Lei 155 Asn Tyr Tyr Asp Al 170 Pro Arg Pro Gly G1l 185 Ser Leu Thr Gly Ty <210> 187 <211> 72 n e 5 s g y u Asp -60 Ala Ser Leu Leu 5 Arg y Leu 0 r Leu y Ala u Cys 30 r Leu L Thr y Met L Ala e Ile 110 r Pro 5 r Leu j Cys a Tyr n Pro 190 r Val -75 Asn Leu Ile Pro Ile Gin Gin Val Ser 15 Ala Leu Ser Arg Val 95 Pro Leu Gly Phe Gin 175 Pro Ala Ala Ile Leu -10 Val Val Leu Ala Ile Thr Gly Ser Cys 80 Ala Val Val Ile Set 160 Ala Pro Thr Cys -25 Val Ser Val Val Met 1 Val His Leu Ala 65 Thr Gly Ala Pro Ile 145 Cys Gin Glu Asp -40 Tyr Val Leu Glu Gly -15 Leu Thr Ser Pro 50 Ile Val Gly Trp Asp 130 Ser Ser Pro -7 His -55 Gin Tyr Ser Glu 10 Val Tyr Leu Ala Thr 35 Ala Ser Phe Val Asn 115 Ser Ser Ser Leu Ala Gly Asn Phe Lys Ser Ile Pro Val Gly Asp Ser Cys Phe 100 Leu Met Leu Gln Ala 180 Leu Arg Pro Ser Lys Leu Thr Tyr Gin Ile Ala Ser Glu Leu Ala Leu Ser Gly Ile Ile Leu Gin Phe His Lys Phe Arg 165 Thr Gly Trp Phe Thr Gin Ala Glu Ile Gly Phe Ser 150 Asn Arg Leu Lys Ser Gin Ala Cys Ser Leu Ile Glu 135 Leu Arg Ser Gly Gly Val Set Pro Leu Thr Lys Cys Ala Ile Arg Gly Leu 120 Ile Ile Ser Ser Lys Val Lys Ser Glu Phe Asn Ser Tyr 195 200 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -32..-1 <300> <400> 187 Met Phe Ala Leu Ala Val Met Arg Ala Phe Arg Lys Asn Lys Thr Leu -25 WO 99/25825 PCT/IB98/01862 Gly Tyr Gly Val Pro Met Leu Leu Leu Ile Ala Gly Gly Ser Ph -10 Leu Arg Glu Phe Ser Gin Ile Arg Tyr Asp Ala Val Lys Ser Lys Met 1 5 10 15 Asp Pro Glu Leu Glu Lys Lys Pro Lys Glu Asn Lys Ile Ser Leu Glu 25 Ser Glu Tyr Glu Gly Ser Ile Cys <210> 188 <211> 88 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -33..-1 <300> <400> 188 Met Ser Gin Thr Ala Trp Leu Ser Leu Leu Ser Ser Ser Pro Phe Gly -25 Pro Phe Ser Ala Leu Thr Phe Leu Phe Leu His Leu Pro Pro Ser Thr -10 Ser Leu Phe Ile Asn Leu Ala Arg Gly Gin Ile Lys Gly Pro Leu Gly 1 5 10 Leu Ile Leu Leu Leu Ser Phe Cys Gly Gly Tyr Thr Lys Cys Asp Phe 25 Ala Leu Ser Tyr Leu Glu Ile Pro Asn Arg Ile Glu Phe Ser Ile Met 40 Asp Pro Lys Arg Lys Thr Lys Cys <210> 189 <211> 106 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -32..-1 <300> <400> 189 Met Phe Ala Pro Ala Val Thr Arg Ala Phe Arg Lys Asn Lys Thr Leu -25 Gly Tyr Gly Val Pro Met Leu Leu Leu Ile Val Gly Gly Ser Phe Gly -10 Leu Arg Glu Phe Ser Gin Ile Arg Tyr Asp Ala Val Lys Ser Lys Met 1 5 10 Asp Pro Glu Leu Glu Lys Lys Leu Lys Glu Asn Lys Ile Ser Leu Glu 25 Ser Glu Tyr Glu Lys Ile Lys Asp Ser Lys Phe Asp Asp Trp Lys Asn 40 Ile Arg Gly Pro Arg Pro Trp Glu Asp Pro Asp Leu Leu Gin Gly Arg 55 Asn Pro Glu Ser Leu Lys Thr Lys Thr Thr <21-0> 190 <211> 267 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -21..-1 <300> <400> 190 Met Trp Trp Phe Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu Val -15 e Gly t WO 99/25825 PCT/IB98/O1862 Ile Leu Trp Thr Ser Ala Ala Phe Ile Phe 1 His His Ile Asp Pro Val Ala Pro Glu L Val Leu Cys Ile A] Leu Ser Pro Giu Gl Val Leu Giy Ile Le Gin Lys Thr Thr Le Phe Giy Met Giy Se 110 Gin Met Gin Pro Ly 125 Leu Leu Val Ile Tr 140 Ser Ser Val Leu Hi 16 Leu His Trp Asn Pr 175 Thr Ala Ala Giu Tr 190 Thr Tyr Ile Arg As 205 Leu His Gly Leu Th 220 Glu Arg Thr Arg Le' 24 <210> 191 <211> 108 <212> PRT <213> Homo sapiens <220> <300> <400> 191 Met Giy Cys Vai Ph 1 5 Asp Trp Thr Leu Se Tyr Tyr Tyr Ser Asi Val His Leu Met Gi1 Gin Asp Val Gin Gl Leu Lys Giy Giu Se Leu Pro Giu Giu Pr 100 <210> 192 <211> 69

(S

La uu uu rr 's p s 0 0 p p r 0 Cys Thr Asn 65 Ser Phe Leu Ile Cys 145 Ser Clu Ser Phe Leu 225 Ala Leu Ile 50 Val Cys Ala Tyr His 130 Gly Gly Asp Met Gin 210 Tyr Leu Phe 35 Tyr Ile Lau Ala Met 115 Gly Val Asn Lys Ser 195 Lys Asp Pro Gly Val Ile Gly His 100 Phe Lys Ser Phe Giy 180 Phe Ile Thr Tyr Ala Arg Lys Leu 85 Val Val Gln Ala Gly 165 Tyr Ser Ser Ala Ile Met Tyr Leu 70 Ser Ser Gin Val Leu 150 Thr Ala Phe Leu Pro 230 Ser Leu Lys Asn Ile Gly Thr Phe 135 Ser Asp Leu Phe Arg 215 Cys Cys Asp A.rg Gly Ile Val Thr Asp Asn Gln Lys Val Ala Ile 120 Trp Met Leu His Gly 200 Val Pro Ile Glu Phe Ser Cys Val Ser Tyr Ile Thr Ala Val Thr Thr Ile Val Ala Ala Val 105 Leu Ile Leu Glu Met 185 Phe Glu lie Phe Iyr ;ln Leu Glu Leu G1 Ala His Gly Asn Lou Ser Arg Thr Gin 170 Ile Phe Ala Asn Lys Va1 Asn Leu Ile His Thr Ala Ala Leu Phe Thr Tyr Leu Cys 155 Lys Thr Leu Asn Asn 235 Ile Leu Arg Leu Arg Val Leu Ser Arg Asp Ile Arg 245 1 r n y u r 0 Gin Pro Leu Asp Ala 70 Gin Lys Ser Gly Ser Ile 55 Asp Val Gly Glu His 25 Pro Cys Gly Lys Gin 105 Asp 10 Ala Ile Asn Thr Lys 90 Met Lys Lys Gly Asp Tyr 75 Ala Leu <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -46..-l <300> <400> i92 Met Ser Val Phe Trp Gly Phe Val Gly Phe Leu Val Pro Trp Phe Ile WO 99/25825 PCT/IB98101862 162 -40 Pro Lys Gly Pro Asn Arg Gly Val Ile Ile Thr Met Leu Val Thr Cys -25 -20 Ser Val Cys Cys Tyr Leu Phe Trp Leu Ile Ala Ile Leu Ala Gin Leu -51 Asn Pro Leu Phe Giy Pro Gin Leu Lys Asn Giu Thr Ile Trp, Tyr Leu 10 Lys Tyr His Trp Pro <210> 193 <211> 251 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -28. 1 <300> <400> 193 met Trp Arg Leu Leu Ser Asp Ser Leu Leu Pro Val Lys Leu Arg Phe Glu Pro Lys Asn Giu Phe Thr Giu Leu His Trp Gly Met Asp Pro Lys Lys Pro Ile Thr Giy Ala Ile Asp 120 Val Giu Met Gly 135 Asp Gin Val Ala 150 Giy Thr Leu. Giu 165 Asn Gin Asn Pro Gly Ile Arg Lys 200 Tyr Trp, Gly Lys 215 <210> 194 <211> 99 <212> PRT Leu Ala Arg Ala Ser Ala Pro Leu Leu Trp Pro Ile Leu Gly His Asn Arg 105 His Gly His Lys Trp 185 Val1 Phe Ala Ser 10 Glu Ser Asn Phe Met 90 Lys Tyr.

Arg Lys Met 170 Thr Leu Tyr Leu Phe Arg Asp Phe Giu Phe Ser Val1 Cys Leu 155 Arg Phe Ser Met Leu Glu Ala Ile Ala 60 Met Ala Val1 Thr Giu 140 Pro Lys Giu Pro Pro 220 Pro Asp Pro Arg Ile Met Ile Gly Pro 125 Phe Phe Asp Arg Tyr 205 Lys Arg Val Pro Ala Val1 Leu 30 Gly Leu Arg Trp His 110 Val1 Giu Ala Gin Ile 190 Asp Arg Ser Ser i5 Val1 Pro Aia Leu Arg 95 Arg Lys Giu Ala Glu 175 Ala Leu Val1 Gly Pro Thr Gly Ile Pro Gly Gly Gin 145 Ala Arg Ala His Lys Lys Arg Ala Gly Arg Pro Gly 115 Leu Phe Ser Arg Met 195 Gly Thr Pro Arg Thr Tyr Ser Phe 100 Lys Val1 Leu Arg Asn 180 Leu Lys <213> Homo sapiens <220> <221> SIGNAL <222> -48. i <300> <400> 194 Met Asp Asn Val Gin Pro Lys Ile Lys His Arg Pro Phe Cys Phe Ser -40 Val Lys Gly His Val Lys Met Leu Arg Leu Asp Ile Ile Asn Ser Leu -25 WO 99/25825 PCT/IB98/01862 163 Val Thr Thr Val Phe Met Leu Ile Val Ser Val Leu Ala Leu -10 -5 Giu Thr Thr Thr Leu Thr Val Gly Gly Gly Val Phe Ala Leu Val 1 5 10 15 Ala Val Cys Cys Leu Ala Asp Gly Ala Leu Ile Tyr Arg Lys Leu 25 30 Phe Asn Pro Ser Gly Pro Tyr Gin Lys Lys Pro Val His Giu Lys 40 45 Giu Val Leu <210> 195 <211> 81 <212> PR? <213> Homo sapiens <220> <221> SIGNAL <222> <300> <400> 195 Met Ser Asn Thr His Thr Val Leu Val Ser Leu Pro His Pro His -25 -20 Aia Leu Thr Cys Cys His Leu Giy Leu Pro His Pro Val Arg Ala -10 -5 Arg Pro Leu Pro Arg Val Giu Pro Trp Asp Pro Arg Trp Gin Asp 10 15 Giu Leu Arg Tyr Pro Gin Ala Met Asn Ser Phe Leu Asn Giu Arg 25 30 Ser Pro Cys Arg Thr Leu Arg Gin Giu Ala Ser Ala Asp Arg Cys 40 45 Leu <210> 196 <211> 150 <212> PRT <213> Homo sapiens Ile Pro Thr Leu Lys Pro Pro 3er er ~sp <220> <300> <400> 196 Met Lys Val 1 Phe Ile Phe Pro Giu Ala Ser Lys Phe Giu Lys Leu Gly Leu Glu Val Giu Ile Gly Ile Leu 115 Gin His Ser 130 Val Ser Thr 145 <210> 197 <211> 273 <212> PRT Met 5 His His Lys Giu Leu His Val1 Asn Lys His Cys Arg Gin Arg 70 Leu Giu Gin His Lys 150 Thr Asn Gin Ala 55 Tyr Thr Asp Giu Leu 135 Lys His His 40 Ala Gly As n Leu Gly 120 As n Phe Cys 25 Arg Giu Giu Leu Gly 105 Lys Ser Leu Glu Met Giu Gly 75 .Leu Asp Phe Asn Ile Cys Leu LeU Thr Giu His Asp His Giy Thr GiU Leu Glu Pro Lys LYS Tyr Tyr Ile Arg Leu Ser Phe Phe Gly Giu Arg Lys Val His Vai Ser His Leu 110 His Ser His Asn His 125 Gin Thr Val Thr Ser 140 <213> Homno sapiens <220> WO 99/25825 PCT/IB98/01862 <221> SIGNAL <222> -45..-1 <300> <400> 197 Met Asn Trp Ser I] Ser Thr Ala Phe Gl -2 Val Leu Val Tyr Le Lys Asp Phe Asp Cy Phe Asp Glu Phe Ph Leu Ile Leu Val Th Tyr Arg Glu Val G1 Ser Gly Arg Leu Ty Trp Thr Tyr Val Cy Phe Leu Tyr Val Ph 100 Val Val Lys Cys Hi 12 Ile Ser Lys Pro Se 135 Thr Ala Ala Ile Cy 150 Val Ser Lys Arg Cy; 165 Met Cys Thr Gly Hi: 180 Asp Asp Leu Leu Se: Pro Pro Leu Leu Pr 215 Leu <210> 198 <211> 413 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -37..-1 <300> <400> 198 Met Ala Ser Lys Il Ile Cys Leu Glu Le Ser Leu Cys Arg Al Ser Met Gly Gly Ly Phe Glu His Leu G1 Leu Lys Glu Val Ly Cys Asp His His G1 Lys Val Ile Cys Tr le Phe Glu Gly Leu Leu Ser Gly Val Asn Lys =-3 y '5 u *s e r n r s e s 0 r s s s r 0 o Arg Val Asn Pro 25 Cys Glu Leu Ser His 105 Ala Glu Ile His His 185 Gly Asp Leu Leu Cys 1 Ser Ala Leu Glu 65 Leu Ile Thr Thr 10 Val Pro Lys Asn Leu 90 Ser Asp Lys Leu Glu 170 Pro Asp Arg Leu Thr -15 Ile Ser Asn Ser 50 Lys Cys Trp Ala Arg Ser Ser Arg Pro 75 Val Phe Pro Asn Leu 155 Cys His Leu Pro Asn -30 Glu Thr Cys Gin 35 Pro Leu Glu Leu Clu -5 Gin His Leu His 60 Gly Phe Tyr.

Cys Ile 140 Asn Leu Asp Ile Arg 220 Val Pro Val Pro 20 His Asp Leu Arg Ser -20 Arg Pro Val Leu 45 Arg Lys Lys Pro Pro 125 Phe Leu Ala Thr Phe 205 Asp Gin Leu Ser 5 Val Leu Asn Leu Ser -35 Leu Val Gly Arg 30 Val Glu Lys Ala Lys 110 Asn Thr Val Ala Thr 190 Leu His Glu Ser Asn Cys Ala Gly Phe 70 Gln Val Trp Cys Leu Val Ala Arg Ser Tyr Ile Leu Glu Arg 175 Ser Gly Val Glu Leu Lys Gly Asn Lys Cys Glu Phe Ser Ser Trp Met His Gly Val Ile Val Phe Leu 160 Lys Ser Ser Lys Val Asp Glu Ile Ile Lys Lys His Ile Asp 1 Asn Ala His Gly Gly Asp Leu Asp Met 145 Ile Ala Cys Asp Lys 225 Thr Cys Ala Ser Val Arg Glu Arg Phe Asp Val Leu Val Glu Leu Ile Pro Cys 130 Val Tyr Gin Lys Ser 210 Thr Cys Gly Val Tyr Glu Asp Asp Gly Tyr Arg His Cys Gin Ala Asn Trp Ala Pro 115 Phe Ala Leu Ala Gln 195 His Ile Pro .His Thr Ser Arg Leu Arg His e u a s n *s y

P

WO 99/25825 PCT/IB98/01862 165 85 His Thr Val Leu Thr Glu Glu Val Phe Lys Glu Cys Gin Glu Lys Leu 100 105 Gin Ala Val Leu Lys Arg Leu Lys Lys Glu Glu Glu Glu Ala Glu Lys 110 115 120 Leu Glu Ala Asp Ile Arg Glu Glu Lys Thr Ser Trp Lys Tyr Gin Val 125 130 135 Gin Thr Clu Arg Gin Arg Ile Gln Thr Glu Phe Asp Gin Leu Arg Ser 140 145 150 155 Ile Leu Asn Asn Glu Glu Gin Arg Glu Leu Gln Arg Leu Glu Clu Glu 160 165 170 Glu Lys Lys Thr Leu Asp Lys Phe Ala Glu Ala Clu Asp Glu Leu Val 175 180 185 Gin Gin Lys Gin Leu Val Arg Glu Leu Ile Ser Asp Val Glu Cys Arg 190 195 200 Ser Gin Trp Ser Thr Met Glu Leu Leu Gin Asp Met Ser Gly Ile Met 205 210 215 Lys Trp Ser Glu Ile Trp Arg Leu Lys Lys Pro Lys Met Val Ser Lys 220 225 230 235 Lys Leu Lys Thr Val Phe His Ala Pro Asp Leu Ser Arg Met Leu Gln 240 245 250 Met Phe Arg Glu Leu Thr Ala Val Arg Cys Tyr Trp Val Asp Val Thr 255 260 265 Leu Asn Ser Val Asn Leu Asn Leu Asn Leu Val Leu Ser Glu Asp Gin 270 275 280 Arg Gin Val Ile Ser Val Pro Ile Trp Pro Phe Gin Cys Tyr Asn Tyr 285 290 295 Gly Val Leu Gly Ser Gin Tyr Phe Ser Ser Gly Lys His Tyr Trp Glu 300 305 310 315 Val Asp Val Ser Lys Lys Thr Ala Trp Ile Leu Gly Val Tyr Cys Arg 320 325 330 Thr Tyr Ser Arg His Met Lys Tyr Val Val Arg Arg Cys Ala Asn Arg 335 340 345 Gin Asn Leu Tyr Thr Lys Tyr Arg Pro Leu Phe Gly Tyr Trp Val Ile 350 355 360 Gly Leu Gin Asn Lys Cys Lys Tyr Gly Ala Lys Lys Lys 365 370 375 <210> 199 <211> 393 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -19..-1 <300> <400> 199 Met Arg Thr Leu Phe Asn Leu Leu Trp Leu Ala Leu Ala Cys Ser Pro -10 Val His Thr Thr Leu Ser Lys Ser Asp Ala Lys Lys Ala Ala Ser Lys 1 5 Thr Leu Leu Glu Lys Ser Gin Phe Ser Asp Lys Pro Val Gin Asp Arg 20 Gly Leu Val Val Thr Asp Leu Lys Ala Glu Ser Val Val Leu Glu His 35 40 Arg Ser Tyr Cys Ser Ala Lys Ala Arg Asp Arg His Phe Ala Gly Asp 55 Val Leu Gly Tyr Val Thr Pro Trp Asn Ser His Gly Tyr Asp Val Thr 70 Lys Val Phe Gly Ser Lys Phe Thr Gin Ile Ser Pro Val Trp Leu Gin 85 Leu Lys Arg Arg Gly Arg Glu Met Phe Glu Val Thr Gly Leu His Asp 100 105 Val Asp Gin Gly Trp Met Arg Ala Val Arg Lys His Ala Lys Gly Leu WO 99/25825 WO 9925825PCTIIB98/01862 Ile Val Pro Arc 130 Arg Asn Val Leu Asp 145 Val Val Gin Val Ala 160 Val Trp Asn Gin Lou 175 Lau Thr His Lau Ala 190 Lou Val Ile Pro Pro 210 Phe Thr His Lys Giu 225 Ser Leu Met Thr Tyr 240 Ala Pro Leu Ser Trp 255 Ser Lys Trp Arg Ser 270 ASP Tyr Ala Thr Ser 290 Tyr Ile Gin Thr Leu 305 Gin Ala Ser Giu His 320 His Val Val Phe Tyr 335 Leu Ala Arg Giu. Leu 350 Gly Leu Asp Tyr Phe 370 <210> 200 <211> 381 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -13. i <300> <400> 200 Met Leu, Leu Ser Ile Thr Val Leu Thr Val Glu Ala Asp Ile Leu Asp ASP Leu Pro Ala Lys Gly Phe Leu Ile Val Pro Pro Pro Val Ile Arg Arg Lou Asp Arg Ala Gly Tyr Lys 100 Lou Ile Ser Met Gly 120 Ile Pro Ser Val Phe 115 Lou Ser Lys Lou Giu Ala Phe Asp Val1 Lys 275 Lys Lys Phe Pro Gly 355 Leu Phe Giu ASP Giu Asn Se r 180 Ala Ile Giu Tyr Arg 260 Ile Asp Asp Phe Thr 340 Val1 Asp Gin 165 Gin Lau Thr Gin Ser 245 Al a Leu Ala His Giu 325 Leu Gly Glu 150 His Lys His Pro Leu 230 Thr Cys Leu Arg Arg 310 Tyr Lys 135 Ile Phe Arg Gin Gly 215 Ala Ala Val1 Gly Giu 295 Pro Lys Ser 120 Trp Thr Giu. Giu Asp Gly Val Gly 185 Ala Arg 200 Thr Asp Pro Val His Gin Gin Val 265 Leu Asn 280 Pro Val Arg Met Lys Ser Leu Gin 140 Tyr Asp Asp 125 Phe Lou.

Phe 170 Lau Lou Gin Leu Pro 250 Leu Phe V/al Val1 A.rg 330 la i Ser- 155 Val Ile Lau Lau Asp 235 Gly Asp Tyr Gly Trp 315 Ser Arg Lyz Vala His Ala Gly 220 Gly Pro Pro Gly Al a 300 Asp Gly Leu lai Ser Ile Trp Glu Leu Gly Tyr Asp Leu Leu Gly Gln Ala 25 Arg 1Asn Lys Cys Ala 105 Ser Ile Met Leu 10 Tyr Phe Ser Asp As n 90 Aila Asn Gly Leu Phe Asn G ly Lys Asn 75 Phe Ile Asp Glu Met -5 Ala Phe Tyr Pro 60 Ser Asp Val1 Ile Ser 140 Leu.

Phe Giu Arg 45 Glu Ser Ile His Giu 125 Ser 360 Se r Leu Asn 30 Lou As n Gly Lys Asn 110 Vali Ala Thr *Giu Met Leu 205 Met Phe Asn Lys Met 285 Arg Ser Arg Giu Gin 365 Tyr Val1 Phe Leu Ile Leu Gin Asp 115 Asp Asp Phe Ala Asn Ala Pro Ala Thr Val1 Val1 Lou Ser Thr Pro Ser Ala Cys Phe Lou Asp Lys Ser Gin Lou Gin Giu Giu Ile Asn Ser Lys Leu 145 Pro Val Pro Thr Gly Pro Val1 Ala Asp Ile 130 Lys Glu Glu Phe Thr Tyr Giu Lys Gly GlY His Leu Ile Leu. Val WO 99/25825 PCT/IB98/01862 167 150 155 160 Ser Leu Pro Leu Glu Tyr Tyr Leu Ile Pro Phe Leu Ile Ile Val Gly 165 170 175 Ile Cys Leu Ile Leu Ile Val Ile Phe Met Ile Thr Lys Phe Val Gin 180 185 190 195 Asp Arg His Arg Ala Arg Arg Asn Arg Leu Arg Lys Asp Gin Leu Lys 200 205 210 Lys Leu Pro Val His Lys Phe Lys Lys Gly Asp Glu Tyr Asp Val Cys 215 220 225 Ala Ile Cys Leu Asp Glu Tyr Glu Asp Gly Asp Lys Leu Arg Ile Leu 230 235 240 Pro Cys Ser His Ala Tyr His Cys Lys Cys Val Asp Pro Trp Leu Thr 245 250 255 Lys Thr Lys Lys Thr Cys Pro Val Cys Arg Gin Lys Val Val Pro Ser 260 265 270 275 Gin Gly Asp Ser Asp Ser Asp Thr Asp Ser Ser Gin Glu Glu Asn Glu 280 285 290 Val Thr Glu His Thr Pro Leu Leu Arg Pro Leu Ala Ser Val Ser Ala 295 300 305 Gin Ser Phe Gly Ala Leu Ser Glu Ser Arg Ser His Gin Asn Met Thr 310 315 320 Glu Ser Ser Asp Tyr Glu Glu Asp Asp Asn Glu Asp Thr Asp Ser Ser 325 330 335 Asp Ala Glu Asn Glu Ile Asn Glu His Asp Val Val Val Gin Leu Gin 340 345 350 355 Pro Asn Gly Glu Arg Asp Tyr Asn Ile Ala Asn Thr Val 360 365 <210> 201 <211> 291 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -42..-1 <300> <400> 201 Met Asp Ser Arg Val Ser Ser Pro Glu Lys Gin Asp Lys Glu Asn Phe -35 Val Gly Val Asn Asn Lys Arg Leu Gly Val Cys Gly Trp Ile Leu Phe -20 Ser Leu Ser Phe Leu Leu Val Ile Ile Thr Phe Pro Ile Ser Ile Trp -5 1 Met Cys Leu Lys Ile Ile Arg Glu Tyr Glu Arg Ala Val Val Phe Arg 15 Leu Gly Arg Ile Gin Ala Asp Lys Ala Lys Gly Pro Gly Leu Ile Leu 30 Val Leu Pro Cys Ile Asp Val Phe Val Lys Val Asp Leu Arg Thr Val 45 Thr Cys Asn Ile Pro Pro Gin Glu Ile Leu Thr Arg Asp Ser Val Thr 60 65 Thr Gin Val Asp Gly Val Val Tyr Tyr Arg Ile Tyr Ser Ala Val Ser 80 Ala-Val Ala Asn Val Asn Asp Val His Gin Ala Thr Phe Leu Leu Ala 95 100 Gin Thr Thr Leu Arg Asn Val Leu Gly Thr Gin Thr Leu Ser Gin Ile 105 110 115 Leu Ala Gly Arg Glu Glu Ile Ala His Ser Ile Gin Thr Leu Leu Asp 120 125 130 Asp Ala Thr Glu Leu Trp Gly Ile Arg Val Ala Arg Val Glu Ile Lys 135 140 145 150 Asp Val Arg Ile Pro Val Gin Leu Gin Arg Ser Met Ala Ala Glu Ala 155 160 165 Glu Ala Thr Arg Glu Ala Arg Ala Lys Val Leu Ala Ala Glu Gly Glu WO 99/25825 WO 9925825PCTIIB98/01862 170 Met Ser Ala Ser Lys 185 Ser Pro Ilie Ala Leu 200 Ala Thr Glu Lys Asn 215 Leu Glu Gly Ile Gly 235 Asn Lys Ala <210> 202 <211> 92 <212> PRT <213> Homo sapiens <220> <300> <400> 202 Met Pro Pro Arg Asn 1 5 Thr Tyr Leu Pro Gin Asp Arg Ile Giu Asn Cys H-is Asp Lys Giu Gly Ile Gin Lys Arg Giu Asn Lys Phe Ala <210> 203 <211> 127 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -63. i <300> <400> 203 Met Ser Ala Ala Gly Pro Asp Lys Val Giu Asn His Pro Ala Gly Lys Trp Gly Leu Vai Giu Lys Leu Ser Thr Ile Trp Ser Arg Tyr Phe Ala Val Asn Phe Arg Ile Trp Arg Tyr <210> 204 <211> 84 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 204 168 175 180 Ser Leu Lys Ser Ala Ser Met Val Leu Ala 190 195 Gin Leu Arg Tyr Leu Gin Thr Leu Ser Thr 205 210 Ser Thr Ile Val Phe Pro Leu Pro Met Asn 220 225 Gly Vai Ser Tyr Asp Asn His Lys Lys Leu 240 245 Leu Ser Ile Thr Glu 70 Val1 Ala Leu Pro Cys -10 Ala Ser Phe Asn 55 Leu Tyr Asp Tyr 55 Ala Glu Arg Met Arg -25 Ala Gin Leu Val1 40 Gin Giu Leu His 40 Lys Ser Thr Gly Leu -40 Thr Gly Ser Val1 25 Gly Glu Leu Ile 25 Leu Leu Asn Leu Leu -55 Pro Val Leu Ala 10 Ile Ala Leu Leu 10 His Gly Gin Cys Ile Arg Giu Phe Ala Val1 Ile Ala Lys Ile Giu Phe Arg Phe Cys Ala Lys Phe Asp Leu Pro Gly Ala Asn Ile Lys Ala His Met Vai Ile Phe Ile Tyr Arg Arg Glu Thr Ile Ala Ile Arg His Ser Glu Val1 Ile 230 Pro Gly Thr Leu Lys Phe Leu Tyr Met Ala Phe Leu Phe Thr Leu Trp Met Met Lys Ala Lys Tyr Arg Ala Ala Ala Asn Scr Ala His Pro Pro Arg Thr Trp Gin His Arg Leu Ile Pro Gly Ser Leu Lys WO 99/25825 WO 9925825PCTJIB98/O1 862 Met Lys Gly Trp Gly Thr Ala Trp Ala Arg

I

Ala Leu Val Asp Giu Lys Thr Ilie Gin Met Ser Val Val Glu Val Ser Giy Phe Gly <210> 205 <211> 182 <212> PRT <213> Homo sapiens <220> 169 Trp, Leu Ala Leu Leu Leu Gly Ala Leu Leu Gly -15 -10 Arg Ser Gin Asp Leu His Cys Gly Ala Cys Arg 5 Leu Giu Trp Giu Ile Ala Gin Val Asp Pro Lys 20 Gly Ser Phe Arg Ile Asn Pro Asp Gly Ser Gin 35 Thr Val Thr Val Pro Pro Asn Lys Val Ala His 50 55 <221> SIGNAL <222> -1 <300> <400> 205 Met Lys Giy Trp Thr Ala Trp Ala Ala Leu Val Asp Lys Thr Ile Gin Ser Val Val Glu Leu Leu Giu Giu Asp Pro Ser Thr Gly Giu Ser Ser Ile Ser Gly Thr 110 Glu Asp Giu Leu 125 Asp Lys Leu Cys Ile Ser His Asp 160 <210> 206 <211> 71 <212> PRT Gly Trp Leu Ala Leu Leu Leu Gly Ala Leu Leu Gly Arg Glu Met Val1 Ile His Glu Leu Ile Ser 145 Clu Arg Leu Gly Pro 50 Cys Arg Leu Lys Giu 130 Lys Leu -10 Gin Trp 20 Phe Al a Arg As n Leu 100 Ala Phe Thr Asp Giu Arg Arg Met Tyr 85 Gin Cys Ser Asp Leu Ile Ile Ser Lys 70 Val1 Gly Gly Arg Leu 150 His Ala Asn Glu 55 Giu Arg Ile Ser Giu 135 Cys Cys Gin Pro Ala Tyr Val1 Arg Ile 120 Ala Asp Gly Val1 Asp His Giy Val1 Ile 105 Vali Asp His Ala Asp Gly Leu Glu Gly Asp Giu Asn Ala Cys Pro Ser Thr Gin Arg Ser Giu Val1 Leu 155 Arg Lys Gin Giu Ile As n Asp Tyr Lys 140 His <213> Homo sapiens <220> <221> SIGNAL <222> -1 <300> <400> 206 Met Pro Ala Gly Val Ser Leu Leu Ala Met Arg Pro Asp Leu Thr Lys Thr Giu Leu Leu Ser Gin Gin Giu Glu Pro Met Ser Thr Tyr Leu LYS Met Phe Ala Ala -20 -15 Cys Ala Gly Ala Glu Val Val His Arg Tyr Tyr 1 Ile Pro Giu Ile Pro Pro Lys Arg Gly Glu Leu 15 Gly Leu Lys Giu Arg Lys His Lys Pro Gin Val 30 Leu Lys WO 99/25825 WO 9925825PCTIIB98/01862 <c210> 207 <211> 73 <212> PRT <213> Homo sapiens <220> <300> <400> 207 Met Arg Ile Arg Met 1 5 Cys Thr Asp Arg Asp Leu Lys Pro Ser Asp Leu Ala Met Val Pro Glu Ser Leu Thr Giy <210> 208 <211> 169 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -150. l <300> <400> 208 170 Thr Asp Gly Arg Thr Leu Val Giy Cys Phe Leu 10 Cys Asn Val Ile Leu Gly Ser Ala Gin Glu Phe 25 Ser Phe Ser Ala Giy Glu Pro Arg Val Leu Giy 40 Gly His His Ile Val Ser 1ie Giu Val Gin Arg 55 Pro Pro Tyr Leu Met Ala Giu Thr Lys Asp Thr Ala Gin Met Leu Val Thr -145 -140 Val Ala Gin Arg Val His Leu Ser Ser Ala Phe Lys Arg Pro Gly Leu Ser Pro His Arg <210> 2C <211> 76 <212> PF Val Thr Phe Thr -130 Thr Leu Tyr Arg -115 Leu Leu Glu His -100 His Ala Thr Cys Val Xaa Arg His -65 Gly Phe Ser Cys Pro Pro Cys Pro His His Val Gly Pro Ala Ser Ala Ala Arg Gin Arg Arg Glu Gly Ala -80 Leu Leu Ala Gin Ser Lys Giu Gly Gin -95 Giu Ser Ser G ly Ala -15 Gin Thr Giu Trp Arg -125 Ile Gly Phe -110 Giu Leu Trp Phe His Ser Ser Leu Gin -60 Leu Pro Ser -45 Phe Phe Val -30 Gly Leu Giu Ser Ala Ala 5 Ala Gin Pro Ile Cys Leu Ser Phe Leu Ile Leu Lys Val1 Cys Leu Trp Leu Leu Thr Phe Lys Asp -135 Asp Leu Ala -120 Pro Glu Leu -105 Lys Arg Gly Pro Gly Trp Pro Pro Giu Asp Tyr Arg Val Giu Thr Thr Ser Cys Gly Val Ser <213> Homo sapiens <220> <221> SIGNAL <222> -22. i <300> <400> 209 Met Glu Leu Ile Se Leu Phe Leu Leu Le Lys Gly Trp Ile Pr Pro Thr Val Ile Ile Ile Leu Gly Cys Leu Ala -15 Gin Arg Lys Asn Leu Arg Arg Pro Pro Cys Ile 1 5 Trp Ile Giy Val Gly Phe Glu Phe Gly Lys Ala WO 99/25825 PCT/IB98/01862 171 Pro Leu Giu Phe Ile Giu Lys Ala Arg Ile Lys Val Cys Gly Arg Gly 35 Arg Arg Gly Leu Gin Arg Arg Gin Cys Phe Leu Phe <210> 210 <211> <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 210 Met Ala Giu Thr Lys Asp Ala Ala Gin Met Leu Val Thr Phe Lys Asp -45 Val Ala Val Thr Phe Thr Arg Glu Giu Trp Arg Gin Leu Asp Leu Ala -30 Gin Arg Thr Leu Tyr Arg Glu Val Met Leu Giu Thr Cys Gly Leu Leu -15 Val Ser Leu Val Giu Ser Ile Trp Leu His Ile Thr Glu Asn Gin Ile 1 5 Lys Leu Ala Ser Pro Gly Arg Lys Phe Thr Asn Ser Pro Asp Giu Lys' 20 Pro Giu Val Trp Leu Ala Pro Gly Leu Phe Gly Ala Ala Ala Gin 35 <210> 211 <211> 92 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -22. l <300> <400> 211 Met Glu Leu Ile Ser Pro Thr Val Ile Ile Ile Leu Gly Cys Leu Ala -15 Leu Phe Leu Leu Leu Gin Arg Lys Asn Leu Arg Arg Pro Pro Cys Ile 1 5 Lys Gly Trp Ile Pro Trp Ilie Gly Val Gly Phe Glu Phe Gly Lys Ala 20 Pro Leu Giu Phe Ile Giu Lys Ala Arg Ile Lys Tyr Gly Pro Ile Phe 35 Thr Vai Phe Ala Met Gly Asn Arg Met Thr Phe Val Thr Giu Giu Giu 50 Gly Ile Asn Val Phe Leu Lys Ser Lys Lys Lys Lys 65 <210> 212 <211> 89 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -1 <300> <400> 212 Met Ile Ile Ser Leu Phe Ile Tyr Ile Phe Leu Thr Cys Ser Asn Thr -10 Ser Pro Ser Tyr Gin Gly Thr Gin Leu Gly Leu Gly Leu Pro Ser Ala 1 5 10 Gin Trp Trp Pro Leu Thr Giy Arg Arg Met Gin Cys Cys Arg Leu Phe 25 Cys Phe Leu Leu Gin Asn Cys Leu Phe Pro Phe Pro Leu His Leu Ile 40 WO 99/25825 WO 9925825PCTIIB98/O1 862 172 Gin His Asp Pro Cys Glu Leu Val Leu Thr Ile Ser Trp Asp Trp, Ala 55 Glu Ala Gly Ala Ser Leu Tyr Ser Pro <210>-213 <211> 109 <212> PRT <213> Homo sapiens <220> <300> <400> 213 Met Lys Val Asp Lys Asp Arg Gin Met Val Val Leu Giu Glu Giu Phe 1 5 10 Arg Asn Ile Ser Pro Glu Giu Leu Lys Met Giu Leu Pro Giu Arg Gin 25 Pro Arg Phe Val Val Tyr Ser Tyr Lys Tyr Val Arg Asp Asp Gly Arg 40 Val Ser Tyr Pro Leu Cys Phe Ile Phe Ser Ser Pro Val Gly Cys Lys 55 Pro Glu Gin Gin Met Met Tyr Ala Gly Ser Lys ASn Arg Leu Val Gin 70 75 Thr Ala Giu Leu Thr Lys Val Phe Glu Ile Arg Thr Thr Asp Asp Leu 90 Thr Giu Ala Trp Leu Gin Glu Lys Leu Ser Phe Phe Arg 100 105 <210> 214 <211> 114 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -103. l <300> <400> 214 Met Val Ile Arg Val Tyr Ile Ala Ser Ser Ser Gly Ser Thr Ala Ile -100 -95 Lys Lys Lys Gin Gin Asp Val Leu Gly Phe Leu Glu Ala Asn Lys Ile -80 Gly Phe Glu Giu Lys Asp Ile Ala Ala Asn Giu Giu Asn Kr-g Lys Trp -65 Met Arg Giu Asn Val Pro Giu Asn Ser Arg Pro Ala Thr Gly Asn Pro -50 -45 Leu Pro Pro Gin Ile Phe Asn Glu Ser Gin Tyr Arg Gly Asp Tyr Asp -30 Ala Phe Phe Giu Ala Arg Giu Asn Asn Ala Val Tyr Ala Phe Leu Gly -15 Leu Thr Ala Pro Ser Gly Ser Lys Glu Aia Giu Val Gin Ala Lys Gin 1 Gin Ala <210> 215 <211> 124 <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -97. l <300> <400> 215 Met Ala Asp Asp Leu Lys Arg Phe Leu Tyr Lys Lys Leu Pro Ser Val -90 Giu Gly Leu His Ala Ile Val Val Ser Asp Arg Asp Gly Val Pro Val -75 -7 0 WO 99/25825 WO 9925825PCT/IB98/01862 Ile Lys Val Ala Asn Phe Leu Ser Thr Phe Leu Ser Lys Asn Lys Val Gin Phe Asn Arg Ser Ala Asn Thr Gly 1 Leu Phe Giu Giu Leu <210> 216 <211> 93 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -22. l <300> <400> 216 Met Lys Pro Val Leu Leu Gin Leu Val Pro Leu Glu Pro Pro Pro Cys Thr Met Gin Giu Phe Cys Gly Ile Val Ile LYS His Lys Giy <210> 217 <211> 207 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -42. 1 Asp -60 Ala Ser Leu Leu Arg Pro Gly Cys Asp Cys Ser Leu Asp Giu Thr Gin Ile Lys 60 Asp Cys Ala Asn Leu Ile Pro Ile Gin Leu Ser Ile Cys Ser Giu 65 Leu Thr Phe Val1 Thr Asp 45 Pro Leu Giu Gin Ala Ala Ile Leu -10 Val1 Val1 Gin -15 Pro Ser Giu Ser 50 Val1 Thr -35 Gly Cys Leu Giu 30 Se r Glu Thr Phe Gly 110 Pro Thr Cys -25 Val1 Se r Val1 Phe Lys Ala Lys 35 Giu Ile Thr Lys Gin Ala 15 Gin Leu Asn Gin Leu 95 Val1 Asp -40 Tyr Val1 Leu Clu Leu Gin Pro 20 Gly Thr Met Val1 Asp Ser Se r Giu Ile Se r Asp 80 Se r Lys Giu His Ala Leu Arg Pro Gly -55 Gin Gly Ser Lys Leu Giy Tyr Asn Thr Tyr Gin Val Ser Phe Ile Ala Ser Ser Giu Lys Giu Leu Ala Pro 10 Val Ser <300> <400> 217 Met His I Ile Val H Asp Leu V Leu Gin G Ala Ile T 2 Val ASP L Giu Gin C Giu Thr L le is al iu 'yr 5 eu .ys 'ys ~sp ,iu .05 Leu Cys Cys Gln Ala Pro Gin Arg Ile 90 Thr Gin Pro His Lys Ser Leu Lys Thr Leu Val Val1 Arg Giu Phe Phe Pro Asp Ile Asp Val1 Tyr Arg Ala 65 Asp Asn Glu Val1 Val1 Asn Gin Gin Ala Asp Trp Asp Phe Leu Val Giu Phe Ile Gly Phe Cys Leu Ala Leu Lys Tyr Ile Cys Thr His Leu Cys Cys Ser Ser Lys Arg Asn Arg Asn G ly Asn Pro Ser Lys Leu Ser His Phe Gin Ile Leu Pro Val1 Ile Gin Asn Leu Gin 100 Leu Gin Leu Ile Leu Giu Asn Thr Lys Ala Ser Ala Phe Ile Lys Met Giu Ile Leu Lys Leu Thr Lys Lys ,b c Gin Lys Cys Ser 5cr Ala Phe Gin Asn Leu Leu Pro Phe Tyr Ser Pro WO 99/25825 PCT/IB98/01862 174 120 125 130 Val Val Glu Asp Phe Ile Lys Ile Leu Arg Glu Val Asp Lys Ala Leu 135 140 145 150 Ala Asp Asp Leu Glu Lys Asn Phe Pro Ser Leu Lys Val Gin Thr 155 160 165 <210> 218 <211> 59 <212> PRT <213> Homo sapiens <220> <300> <400> 218 Met Pro His Ser Lys Pro Leu Asp Trp Gly Leu Ser Ser Val Ala Glu 1 5 10 Cys Pro Ala Glu Leu Phe Pro Ser Thr Gly Gly Leu Ala Gly Lys Gly 25 Pro Gly Leu Asp Ile Leu Arg Cys Val Leu Ser Pro Trp Ala Ser His 40 Phe Pro Ser Leu Ser Leu Gly Val Phe Asn Leu <210> 219 <211> 56 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -27..-1 <300> <400> 219 Met Asn Arg Val Pro Ala Asp Ser Pro Asn Met Cys Leu Ile Cys Leu -20 Leu Ser Tyr Ile Ala Leu Gly Ala Ile His Ala Lys Ile Cys Arg Arg -5 1 Ala Phe Gin Glu Glu Gly Arg Ala Asn Ala Lys Thr Gly Val Arg Ala 15 Trp Cys Ile Gin Pro Trp Ala Lys <210> 220 <211> 162 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -94..-1 <300> <400> 220 Met Leu Gin Thr Ser Asn Tyr Ser Leu Val Leu Ser Leu Gin Phe Leu -85 Leu Leu Ser Tyr Asp Leu Phe Val Asn Ser Phe Ser Glu Leu Leu Gin -70 Lys Thr Pro Val Ile Gin Leu Val Leu Phe Ile Ile Gin Asp Ile Ala -55 Val Leu Phe Asn Ile Ile Ile Ile Phe Leu Met Phe Phe Asn Thr Phe -40 Val Phe Gin Ala Gly Leu Val Asn Leu Leu Phe His Lys Phe Lys Gly -25 -20 Thr Ile Ile Leu Thr Ala Val Tyr Phe Ala Leu Ser Ile Ser Leu His -5 1 Val Trp Val Met Asn Leu Arg Trp Lys Asn Ser Asn Ser Phe Ile Trp 10 Thr Asp Gly Leu Gin Met Leu Phe Val Phe Gin Arg Leu Ala Ala Val 25 Leu Tyr Cys Tyr Phe Tyr Lys Arg Thr Ala Val Arg Leu Gly Asp Pro WO 99/25825 WO 9925825PCTIIB98/01862 175 40 45 His Phe Tyr Gin Asp Ser Leu Trp, Leu Arg Lys Clu Phe Met Gin Val 60 Arg Arg <210> 221 <211> 154 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -l <300> <400> 221 met Ala Ser Ala Ser Ala Arg Gly Asn Gin Asp Lys Asp Ala His Phe -60 Pro Pro Pro Ser Lys Gin Ser Leu Leu Phe Cys Pro Lys Ser Lys Leu -45 His Ile His Arg Ala Giu Ile Ser Lys Ile Met Arg Glu Cys Gin Giu -30 Giu Ser Phe Trp Lys Arg Ala Leu Pro Phe Ser Leu Val Ser Met Leu -15 -10 Val Thr Gin Gly Leu Val Tyr Gin Gly Tyr Leu Ala Ala Asn Ser Arg 1 5 Phe Gly Ser Leu Pro Lys Val Ala Leu Ala Gly Leu Leu Gly Phe Gly is 20 Leu Gly Lys Val Ser Tyr Ile Gly Val Cys Gin Ser Lys Phe His Phe 35 Phe Giu Asp Gin Leu Arg Gly Ala Gly Phe Gly Pro Gin His Asn Arg 50 55 His Cys Leu Leu Thr Cys Giu Giu Cys Lys Ile Lys His Gly Leu Ser 70 Giu Lys Gly Asp Ser Gin Pro Ser Ala Ser <210> 222 <211> 99 <212> PRT <213> Homo sapiens <220> <300> <400> 222 Met Lys Val Glu Glu Glu His Thr Asn Ala Ile Gly Thr Leu His Gly 1 5 10 Gly Leu Thr Ala Thr Leu Val Asp Asn Ile Ser Thr Met Ala Leu Leu 25 Cys Thr Giu Arg Gly Ala Pro Gly Val Ser Val Asp Met Asn Ile Thr 40 Tyr Met Ser Pro Ala Lys Leu Gly Giu Asp Ile Val Ile Thr Ala His 55 Val Leu Lys Gin Gly Lys Thr Leu Ala Phe Thr Ser Val Gly Leu Thr 70 75 Asn Lys Ala Thr Gly Lys Leu Ile Ala Gin Gly Arg His Thr Lys His 90 Leu Gly Asn <210> 223 <211> 43 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -24. l <300> <400> 223 Met Gln Cys Phe Ser Phe Ile Lys Thr Met Met Ile Leu Phe Asn Leu WO 99/25825 WO 9925825PCT/1B98/01862 176 20 15 Leu Ile Phe Leu Cys Giy Phe Thr Asn Tyr Thr Asp Phe Giu Asp Ser 1 Pro Tyr Phe Lys Met His Lys Pro Val Thr Met <210> 224 <211> 69 <212> PRT <213> Homo sapiens <220> <221> SIGNAL 4222> -21. l <300> <400> 224 Met Trp Trp Phe Gin Gin Gly Leu Ser Phe Leu Pro Ser Ala Leu Val -15 Ile Trp Thr Ser Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala Val Thr 1 5 Leu His His Ile Asp Pro Ala Leu Pro Tyr Ile Ser Asp Thr Gly Thr 20 Val Ala Pro Glu Lys Cys Leu Phe Gly Ala Met Leu Asn Ile Ala Ala 35 Val Leu Cys Gin Lys <210> 225 <211> 78 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> -18. l <300> <400> 225 Met Ser Pro Giy Ser Ala Leu Ala Leu Leu Trp Ser Leu Pro Ala Ser -10 Asp Leu Gly Arg Ser Val Ile Ala Giy Leu Trp Pro His Thr Gly Val 1 5 Leu Ile His Leu Giu Thr Ser Gin Ser Phe Leu Gin Gly Gin Leu Thr 20 25 Lys Ser Ile Phe Pro Leu Cys Cys Thr Ser Leu Phe Cys Val Cys Val 40 Val Thr Val Gly Gly Gly Arg Val Gly Ser Thr Phe Val Ala 55 <210> 226 <211> <212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -47. l <300> <400> 226 Met Arg Leu Pro Pro Ala Leu Pro Ser Giy Tyr Thr Asp Ser Thr Ala -40 Leu Giu Gly Leu Val TIyr Tyr LeU Asn Gin Lys Leu Leu Phe Ser Ser -25 Pro Ala Ser Ala Leu Leu Phe Phe Ala Arg Pro Cys Val Phe Cys Phe -10 -5 1 Lys Ala Ser Lys Met Gly Pro Gin Phe Giu Asn Tyr Pro Thr Phe Pro 10 Thr Tyr Ser Pro Leu Pro Ile Ile Pro Phe Gin Leu His Gly Arg Phe 25 <210> 227 WO 99/25825 PCT/1B98/01862 177 <211> 242.

<212> PRT <213> Homno sapiens <220> <221> SIGNAL <222> -103. l <300> <400> 227 Met Trp Leu Asp Pro Val. Phe Pro Leu Phe Pro Val Gly Asp His Tyr -100 -95 Lou Pro His Leu His Met Asp Val. Lou Giu Gly Leu Ile Leu Val Lou -80 Pro Cys Ile Asp Val Phe Val Lys Val Asp Leu Arg Thr Val Thr Cys -65 Asn Ile Pro Pro Gin Glu Ile Leu Thr Arg Asp Ser Val Thr Thr Gin -50 -45 Val Asp Gly Val Val Tyr Tyr Arg Ile Tyr Ser Ala Val Ser Ala Val -30 Ala Asn Val Asn Asp Vai His Gin Ala Thr Phe Leu Leu Ala Gin Thr -15 Thr Leu Arg Asn Val Leu Gly Thr Gin Thr Leu Ser Gin Ile Leu Ala 1 Gly Arg Glu Giu Ile Ala His Ser Ile Gin Thr Leu Leu Asp Asp Ala 15 20 Thr Giu Leu Trp Gly Ile Arg Val Ala Arg Val Giu Ile Lys Asp Val 35 Arg Ile Pro Val Gin Leu Gin Arg Ser Met Ala Ala Giu Ala Glu Ala 50 Thr Arg Giu Ala Arg Ala Lys Val Leu Ala Ala Giu Gly Glu Met Asn 65 Ala Ser Lys Ser Leq, Lys Ser Ala Ser Met Val Leu Ala Glu Ser Pro 80 Ile Ala Lou Gin Leu Arg Tyr Leu Gin Thr Lou Ser Thr Val Ala Thr 95 100 105 Glu Lys Asn Ser Thr Ile Val Phe Pro Leu Pro Met Asn Ile Leu Glu 110 115 120 Gly Ile Gly Gly Val Ser Tyr Asp Asn His Lys Lys Leu Pro Asn Lys 125 130 135

Claims (11)

1. A purified or isolated polypeptide comprising a sequence of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

2. A purified or isolated polypeptide comprising a mature protein of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

3. A purified or isolated polypeptide comprising a signal peptide of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

4. A purified or isolated polypeptide comprising a sequence which is at least 95% identical to any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227, wherein the polypeptide has at least one biological activity of the polypeptide according to any one of claims 1 to 3. The purified or isolated polypeptide of claim 4, wherein the biological activity is cell adhesion.

6. A purified or isolated nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 5, or a sequence complementary thereto.

7. The purified or isolated nucleic acid sequence of claim 6, wherein the nucleic acid comprises a sequence selected from SEQ ID NOs: 139, 154, 25 150, 167, 176 or 180.

8. A method of making a polypeptide comprising one of the sequences of SEQ ID NO: 186, 197, 201, 214, 223 or 227, the method comprising the steps of: 30 obtaining a nucleic acid comprising one of the sequences of SEQ ID NO: 139, 154, 150, 167, 176 or 180; inserting said nucleic acid in an expression vector such that said nucleic acid is operably linked to a promoter; and introducing said expression vector into a host cell whereby said host cell produces the polypeptide encoded by said nucleic acid. 112

9. The method of claim 8, further comprising the step of isolating said polypeptide. A polypeptide obtainable by the method of claim 9.

11. A host cell containing a recombinant nucleic acid according to claim 6 or claim 7.

12. A purified or isolated antibody capable of specifically binding to a polypeptide having a sequence of any one of SEQ ID NOs: 186, 197, 201, 214, 223 or 227.

13. An array of polynucleotides comprising at least one polynucleotide selected from the group consisting of: a) a polynucleotide sequence of SEQ ID NOs: 139, 150, 154, 167, 176 or 180; b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: o oo 186, 197, 201, 214, 223 or 227, wherein the polypeptide fragment has at least one biological activity of the polypeptide provided as SEQ ID NO: 186, 197, 201, 214, 223 or 227; c) a polynucleotide encoding a signal sequence of SEQ ID NO: 186, 197, 201, 214, 223 or 227, d) a polynucleotide encoding a polypeptide which is at least identical to any of one of SEQ ID NOs: 186, 197, 201, 214, 223 or 25 227, wherein the polypeptide has at least one biological activity of the polypeptide provided as SEQ ID NO: 186, 197, 201, 214, 223 or 227; and e) a polynucleotide sequence complementary to any one of a) to d). Dated this twenty-ninth day of July 2002 Genset Patent Attorneys for the Applicant: F B RICE CO