JP2002539767A - Complementary DNA-encoding protein containing signal peptide - Google Patents

Abstract

  (57) [Summary] Disclosed is the sequence of a cDNA encoding a secreted protein. Using this cDNA, it is possible to express a secreted protein or a fragment thereof, and to obtain an antibody capable of specifically binding to the secreted protein. Such cDNAs may also be used in diagnostics, forensic methods, gene therapy and chromosome mapping. Furthermore, expression vectors and secretion vectors can be designed using this cDNA.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION It is very likely that an estimated 50,000-100,000 genes scattered on the human chromosome will be used to understand, diagnose and treat various diseases that occur in humans. . In addition, probes that can specifically hybridize to loci dispersed throughout the human genome have applications in constructing high-resolution chromosome maps and identifying individuals.

In the past, the process of characterizing only one human gene was laborious, requiring years of work. The recent integration of cloning vector, DNA sequencing and computer technology developments has greatly increased the rate at which human genes can be isolated, sequenced, mapped, and characterized. ing.

[0003] Currently, two approaches are being pursued to identify and characterize genes that are dispersed along the human genome. One involves isolating, cloning, and sequencing large fragments of genomic DNA. That is, the potential reading frames contained in these genomic sequences are identified using bioinformatics software. However, in order to find protein coding sequences scattered throughout the genome by this method, the sequence of a large non-protein-coding fragment of human DNA must be determined. Extensive sequencing is required, and bioinformatics software can mischaracterize the resulting genomic sequence (i.e., label noncoding DNA as coding DNA and vice versa) There is also.

[0004] Another method is to identify and characterize human genes by more direct means. In this method, an isolated messenger RN encoding a human protein is used.
From A (mRNA), complementary DNA (cDNA) is synthesized. If this method is used, sequencing need only be performed on DNA derived from protein-coding fragments of the genome. Often, only short fragments of the cDNA are sequenced, resulting in a sequence called an expressed sequence tag (EST). Using this EST, cDNA containing a sequence adjacent to the EST sequence can be isolated or purified. In the cDNA, the ES used to obtain these cDNAs
In some cases, the entire sequence of T is included, in other cases, the EST used to obtain the above cDNA
In some cases, only fragments of the sequence Also, these cDNAs
In some cases, the complete coding sequence of the gene from which the EST was derived may be included, and in other cases, fragments of the coding sequence of the gene from which the EST is derived may be included in the cDNA. CDNA as a result of alternative splicing or due to the activity of an alternative promoter
It can be understood that the EST may contain an EST sequence.

Heretofore, most of the above short EST sequences have been obtained from cDNA libraries primed with oligo dT. Therefore, EST mainly corresponded to the 3 'untranslated region of mRNA. For one thing, EST sequences from the 3 'end of the mRNA are dominant, because common techniques for obtaining cDNA are not suitable for isolating the cDNA sequence from the 5' end of the mRNA. (Adams et al., Nature 377: 3-174, 1996; Hillier et al., Genome Re
s. 6: 807-828, 1996). Also, in reported cases where a longer cDNA sequence was obtained, the reported sequence generally corresponds to the coding sequence, and does not include the complete 5 'untranslated region (5' UTR) of the mRNA from which the cDNA is derived. Absent. In particular, it has become clear that the 5′UTR affects mRNA stability or translation. For this reason, it has been described in translation of tissue inhibitors of metalloprotease mRNA contained in mitogenically activated cells (Waterhouse et al., J Biol Chem. 265: 5585-9.1990).
In some cases, selective 5 'UTRs can be used to regulate gene expression. further,
Modification of the 5'UTR by events such as mutations, insertions, or translocations may even be manifested as disease development. For example, the fragile X syndrome, the most common cause of genetic mental retardation, has multiple 5'UTRs in fragile X mRNA.
It is somewhat implicated that CGG trinucleotide insertion results in inhibition of protein synthesis due to ribosome stall (Feng et al., Science 268: 73).
1-4, 1995). Aberrant mutations in the 5'UTR region, which is known to inhibit the translation of the c-myc proto-oncogene, up-regulate c-myc protein levels in cells from multiple myeloma patients. (Willis et al
., Curr Top Microbiol Immunol 224: 269-76, 1997). In addition, even when using a cDNA library primed with oligo dT, the incomplete sequence obtained by this method may not include the first exon of mRNA, especially when the first exon is short, The complete 5'UTR cannot be isolated. In addition, some exons (often short exons) located upstream of the splice site may not be included. For this reason, a sequence derived from the 5 'end of the mRNA must be obtained.

In addition, it has been obtained in a large sequencing project (Adams et al., Nature 37
7: 174, 1996, Hillier et al., Genome Res. 6: 807-828, 1996) Despite the large amount of EST data, information on the biological function of mRNA corresponding to the cDNA obtained therefrom It is clear that there is a limit. In particular, although studying the biological function of mRNA requires absolute knowledge of the complete coding sequence, only partial coding sequences can be obtained using ESTs. To date, methods for constructing full-length cDNA libraries have been very inefficient, with only limited success in large-scale full-length cDNA cloning. In particular, such methods require large amounts of mRNA (Ederly et al., 1995).
Therefore, if only a small amount of tissue is available, the full-length library will not be representative, or PCR amplification will be necessary to obtain a considerable number of clones (Maruyama
et al., 1994, CLONTECHniques, 1996), resulting in a highly biased cDNA library with long and rare cDNAs lost. Therefore,
It is necessary to obtain a full-length cDNA, ie, a cDNA containing the complete coding sequence of its corresponding mRNA.

While many sequences from the human chromosome have practical uses, methods that utilize the identification and characterization of chromosomal sequences encoding protein products are particularly relevant for diagnostic and therapeutic uses. Of 50,000-100,000 protein-encoding genes,
Genes encoding proteins secreted from cells that are the site of protein synthesis, as well as secreted proteins themselves, are particularly useful as potential therapeutics. Such proteins are often involved in intercellular communication and may produce clinically relevant responses in their own target cells. In fact, at present, including tissue plasminogen activator, G-CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon-α, interferon-β, interferon-γ and interleukin-2 Several secreted proteins are in clinical use. These proteins are used to treat a variety of conditions, including acute myocardial infarction, acute cerebral infarction, anemia, diabetes, growth hormone deficiency, hepatitis, kidney cancer, neutropenia and multiple sclerosis due to chemotherapy It is used for From these facts, the cDNA encoding the secreted protein or a fragment thereof,
It is a particularly valuable source for potential therapeutics. Therefore, there is a need to identify and characterize secreted proteins and nucleic acids encoding the same.

In addition to being therapeutically useful in itself, secreted proteins include a short peptide called a signal peptide at the amino terminus that directs secretion. These signal peptides are encoded by a signal sequence located at the 5 'end of the coding sequence of the gene encoding the secreted protein. These signal peptides direct the extracellular secretion of a protein to which they are operably linked, and thus utilize the signal sequence by operably linking the signal sequence to a gene encoding the protein desired to be secreted. It may be possible to direct the efficient secretion of proteins. In addition, a fragment of a signal peptide called a transmembrane sequence may be used to instruct intracellular transfer of a target peptide or protein. This method may be successful in gene therapy strategies where it is desirable to deliver a particular gene product to cells other than the cell from which the gene product is produced. A signal sequence encoding a signal peptide can also be applied to simplify protein purification methods.
In such applications, extracellular secretion of the desired protein reduces the number of unwanted proteins that must be distinguished from the desired protein, making purification much easier. Thus, there is a need to identify and characterize a 5 'fragment of a gene for a secretory protein encoding a signal peptide.

[0009] Sequences encoding secreted proteins may also find use as therapeutic or diagnostic agents. In particular, when such a sequence is used, when a mutation occurs in a coding sequence for a secretory protein, it may be possible to determine whether or not there is a possibility that a detectable phenotype such as a disease is expressed in an individual. If an individual is at risk of contracting a disease or other undesired phenotype due to a mutation in such a coding sequence, the undesired phenotype can be achieved by introducing a normal coding sequence using gene therapy. Could be corrected. Alternatively, if the undesired phenotype is due to overexpression of the protein encoded by the coding sequence, antisense or triple helix-based strategies may be used to reduce protein expression. .

[0010] By direct administration of a human secreted polypeptide encoded by the coding sequence to an individual in a condition such as a disease caused by a mutation in the sequence encoding the polypeptide,
These polypeptides may also be available as therapeutics. In such cases, the condition can be cured or reduced by administering the polypeptide to the individual.

It is also possible that human secreted polypeptides or fragments thereof may be used to generate antibodies useful in determining the source species or tissue type of a biological sample. These antibodies may be used to determine the cellular location of the polypeptide fused to the human polypeptide or the cellular location of the human secreted polypeptide. Also,
Antibodies may be utilized in immunoaffinity chromatography to isolate, purify, or concentrate a human polypeptide or a target polypeptide fused to a human polypeptide.

[0012] Very limited information is published on the number of human genes for which the promoter and upstream regulatory region have already been identified and characterized. This may be due in part to the difficulty in isolating such control sequences. Upstream regulatory sequences, such as transcription factor binding sites, are generally too short for probes to isolate promoters from human genomic libraries. In recent years, several methods for isolating human promoters have been developed. One of these
One consists in creating a library of CpG islands (Cross et
al., Nature Genetics 6: 236-244, 1994). The second method consists in isolating a human genomic DNA sequence containing a SpeI binding site using a SpeI binding protein (Mortlock et al., Genome Res. 6: 327-335, 1996). These methods are also limited because of lack of specificity and comprehensiveness. Therefore, the gene
There is a need to identify and systematically characterize 5 'fragments.

[0013] Using the cDNA containing the 5 'end of its corresponding mRNA, the 5' UTR and upstream regulatory regions that control the location, developmental stage, ratio and amount of protein synthesis and mRNA stability are efficiently used. Potential for identification and isolation (Theil et al., BioFactors
4: 87-93, (1993). Once identified and characterized, these regulatory regions can be used in gene therapy or protein purification schemes to synthesize proteins in desired amounts and locations, or to inhibit, reduce or reduce the synthesis of unwanted gene products. Can interfere.

The cDNA containing the 5 ′ end of the secretory protein gene may contain a sequence useful as a probe for chromosome mapping and individual identification. Therefore, there is a need to identify and characterize sequences upstream of the 5 'coding sequence of a gene encoding a secretory protein.

DISCLOSURE OF THE INVENTION [0015] The present invention provides a purified protein encoding a secreted protein or a fragment thereof.
Related to cDNA, isolated cDNA or recombinant cDNA. Preferably, the purified cDNA
The isolated or recombinant cDNA includes the entire reading frame, including the start and stop codons of the corresponding mRNA. For example, the cDNA may include a signal peptide as well as a nucleic acid encoding a mature protein. Such cDNAs are referred to herein as "full-length" cDNAs. Alternatively, the cDNA may include a reading frame fragment. Such a cDNA is referred to herein as "EST" or "5'EST". In some embodiments, the above fragments may encode only the sequence of the mature protein. Alternatively, the fragment may encode only a fragment of the mature protein.
Yet another aspect of the present invention is a nucleic acid encoding a signal peptide of a secreted protein.

The term “corresponding mRNA” means the mRNA that was the template for cDNA synthesis that produced the cDNA of the invention. As used herein, the expression "purified" does not require absolute purity, but is intended to be relatively defined. cDNA
Each cDNA clone isolated from the library was conventionally purified until electrophoretic homogeneity was obtained. Sequences obtained from these clones could not be obtained directly from either the library or total human DNA. This cDNA clone does not occur in itself, but is obtained by manipulating a partially purified substance (messenger RNA) of a substance produced in nature. mRN
When A is converted to a cDNA library, a synthetic substance (cDNA) is generated. By selecting a clone, a pure cDNA clone can be isolated from the synthetic library. Thus, by creating a cDNA library from messenger RNA and subsequently isolating each clone from that library, the purification will be about 10 ⁴ to 10 ⁶ times that of the original message. It is specifically contemplated that the starting material or natural material be purified to at least one order, preferably two or three orders, more preferably up to four or five orders.

As used herein, a substance designated by the term “isolated” must be outside its intended environment (eg, the natural environment if naturally occurring). For example, the naturally occurring polynucleotide of a living animal is not said to be isolated, but if the same polynucleotide is separated from some or all of the coexisting substances in the natural system, it will be isolated. It will be.

The term “recombinant,” as used herein, means that the cDNA is adjacent to a “backbone” nucleic acid that is not adjacent in its natural environment. Further, “enriched” means that cDNA becomes 5% or more of the number of nucleic acid inserts inserted into the 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, integrated nucleic acids and other vectors, or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. Preferably, the enriched cDNA is at least 15% of the number of nucleic acid inserts inserted into the population of recombinant backbone molecules. More preferably, it is at least 50% of the number of nucleic acid inserts inserted into the population of recombinant backbone molecules. In a highly preferred embodiment, it is 90% or more of the number of nucleic acid inserts inserted into the population of recombinant backbone molecules.

Accordingly, one or more cDNAs encoding a secreted polypeptide or a fragment thereof may be present in a cDNA library comprising at least 5% of the number of nucleic acid inserts inserted into the backbone molecule. The encoding cDNA is "enriched recombinant cDNA" as defined herein. Similarly, one or more of the cDNAs of the present invention encode secreted polypeptides or fragments thereof contained in a population of plasmids into which such cDNAs have been inserted so that they are at least 5% of the inserts contained in the plasmid backbone. cDNA is also "enriched recombinant cDNA" as defined herein. However, cDNAs encoding secreted polypeptides or fragments thereof, such as libraries in which backbone molecules containing cDNA inserts encoding secreted polypeptides are extremely rare, are less than 5% of the number of nucleic acid inserts contained in the population of backbone molecules. The cDNA encoding the secreted polypeptide or a fragment thereof contained in the cDNA library is not “enriched recombinant cDNA”.

The term “polypeptide” refers to a polymer of amino acids independent of the length of the polymer. Thus, the definition of polypeptide includes peptides, oligopeptides and proteins. The term also does not specify or exclude post-expression modifications of the polypeptide, for example, polypeptides to which a glycosyl, acetyl, phosphate, lipid group, or the like, has been covalently attached. Explicitly encompassed by the term polypeptide. Further, the definition includes one or more amino acid analogs (e.g., includes non-naturally occurring amino acids, amino acids that occur only biologically independently and naturally, modified amino acids from mammalian systems, and the like).
, Polypeptides having substitution bonds, as well as other modifications of natural and non-natural origin well known in the art.

As used interchangeably herein, the terms “nucleic acid”, “oligonucleotide” and “polynucleotide” are composed of either single or double stranded two or more nucleotides Includes RNA, DNA or RNA / DNA hybrid sequences. In the present specification, the term `` nucleotide '' as an adjective refers to a single-stranded or double-stranded RNA sequence, DNA sequence or
1 shows a molecule containing an RNA / DNA hybrid sequence. As used herein, the term `` nucleotide '' refers to individual nucleotides or a wide variety of nucleotides or 1
Individual molecules contained in a large nucleic acid molecule containing a purine or pyrimidine, ribose or deoxyribose sugar moiety and a phosphate group or a phosphodiester linkage if it is a nucleotide contained in an oligonucleotide or polynucleotide. Also used as a noun to indicate units. As used herein, the term `` nucleotide '' refers to a modification of at least one of (a) a selective linking group, (b) a form similar to a purine, (c) a form similar to a pyrimidine, and (d) a similar saccharide. Used to include `` modified nucleotides '', including those containing at least one of similar linking groups, purines, pyrimidines, sugars and the like (e.g., International Patent Application Publication No. See WO 95/04064). The polynucleotide sequence of the present invention may be prepared by any method known in the art, such as synthesis, recombination, ex vivo production or a combination thereof, as well as using purification methods known in the art.

The expressions “base-paired” and “Watson & Crick base-paired” are used interchangeably herein, and as in double-stranded DNA, where an adenine residue has two hydrogen atoms. It binds to a thymine residue or a uracil residue by bonding, and since the sequences are identical in that a cytosine residue and a guanine residue are bonded by three hydrogen bonds, nucleotides capable of hydrogen bonding to each other are shown (Stryer, L., Biochemistry, 4 ^th e
dition, 1995).

As used herein, the term “complementary” or “complement thereof” refers to a sequence of a polynucleotide whose entire complementary region can form a Watson & Crick base pair with another specific polynucleotide. Show. For the purposes of the present invention, a first polynucleotide is considered to be complementary to a second polynucleotide if each base of the first polynucleotide is paired with its complementary base. The complementary base is generally A
And T (or A and U) or C and G. As used herein, the term "complementary" is used as a synonym for "complementary polynucleotide,""complementary nucleic acid," and "complementary nucleotide sequence." These terms apply to a pair of polynucleotides based solely on their sequence, and not to a particular set of conditions under which the two polynucleotides actually bind. Preferably, a `` complementary '' sequence is one in which A is located where T is on the opposite strand, T is where A is on the opposite strand, and G is where C is on the opposite strand. , Where C is located where G is on the opposite strand.

“Stringent hybridization conditions”, “moderate” hybridization conditions and “low” hybridization conditions are defined below.

In particular, the present invention relates to cDNAs derived from genes encoding secreted proteins. As used herein, a `` secreted '' protein is one which, when expressed in a suitable host cell, is translocated across or across a membrane (transport resulting from the inclusion of a signal peptide in its amino acid sequence). (Including) proteins. A “secreted” protein includes, but is not limited to, a protein that is completely secreted from the cell in which it is expressed (such as a soluble protein) or a protein that is partially secreted (such as a receptor) . "Secreted" proteins also include, but are not limited to, proteins that are transported across the endoplasmic reticulum membrane.

The cDNA encoding a secreted protein may include a nucleic acid sequence called a signal sequence, encoding a signal peptide that directs extracellular secretion of the protein encoded by the cDNA. Generally, the signal peptide is located at the amino terminus of the secreted protein.

[0027] Secreted proteins are translated by ribosomes associated with "rough" ERs. In general, secreted proteins co-translate to the endoplasmic reticulum membrane. The association of ribosomes with the endoplasmic reticulum during translation of secreted proteins is mediated by signal peptides. This signal peptide is generally cleaved after co-translational incorporation into the endoplasmic reticulum. After reaching the endoplasmic reticulum, secreted proteins may travel through the Golgi. In the Golgi apparatus, proteins may undergo post-translational modifications before entering secretory vesicles that release the protein out of the cell membrane.

[0028] The cDNAs of the present invention have several important uses. For example, these cDNAs can be used to express the entire secreted protein encoded by these cDNAs. Alternatively, these cDNAs can be used to express fragments of secreted proteins. This fragment may include a signal peptide encoded by the above-described cDNA, or a mature protein encoded by the cDNA (that is, a protein generated upon cleavage of the signal peptide). These fragments also contain at least 5,10,15,2 contiguous sequences encoded by the above cDNAs.
It may comprise a polypeptide having 0, 25, 30, 35, 40, 50, 75, 100 or 150 amino acids.

An antibody specifically recognizing the whole secreted protein encoded by the cDNA, or the above cDNA containing at least 10 consecutive amino acids, at least 15 consecutive amino acids, at least 25 consecutive amino acids, or at least 40 consecutive amino acids An antibody that specifically recognizes the fragment can be obtained as described below.
An antibody that specifically recognizes a mature protein generated upon cleavage of a signal peptide can also be obtained as described below. Similarly, an antibody that specifically recognizes a signal peptide encoded by cDNA can be obtained.

In some embodiments, the cDNA includes a signal sequence. In another embodiment, the complete coding sequence for the mature protein (ie, the protein produced upon cleavage of the signal polypeptide) may be included in the cDNA. Also this c
DNA may include regulatory regions upstream of the translation start site or downstream of the stop codon, which control the amount, location or stage of development of the gene. As mentioned above,
Secreted proteins are of therapeutic importance. Thus, proteins expressed from the above cDNAs can be useful in treating or controlling various human conditions. The corresponding genomic DNA can also be obtained using cDNA.
The term "corresponding genomic DNA" refers to genomic DNA encoding mRNA comprising the sequence of one of the strands of the cDNA, in which a thymidine residue in the sequence of the cDNA has been replaced by a uracil residue of the mRNA.

An individual who is identified by using the cDNA or genomic DNA obtained therefrom in a forensic method or used in a diagnostic method, and suffers from a genetic disease caused by abnormal expression of a gene corresponding to the cDNA. May be identified. Further, the present invention is useful for constructing a high-resolution map of human chromosomes.

[0032] The present invention also relates to a secretion vector capable of instructing secretion of a target protein. Such vectors may be used in gene therapy strategies where it is desirable to produce the gene product in one cell that is to be delivered to another location in the body. In some cases, the use of a secretion vector facilitates purification of a desired protein.

[0033] The present invention also relates to an expression vector capable of directing the expression of the inserted gene in a desired spatial or temporal manner or at a desired level. Such vectors include cDNAs such as promoters or upstream regulatory sequences.
It may include a sequence upstream of the sequence.

In addition, the present invention may be used in gene therapy to control or treat genetic diseases. Alternatively, the signal peptide may be fused with a heterologous protein to direct its extracellular secretion.

One embodiment of the invention is a purified or isolated nucleic acid comprising one of SEQ ID NOs: 24-73 or a sequence complementary thereto. In one aspect of this embodiment, the nucleic acid is a recombinant nucleic acid.

Another embodiment of the present invention is a purified or isolated comprising at least 8 contiguous bases of one of SEQ ID NOs: 24-73 or one of its complementary sequences. It is a nucleic acid. In one aspect of this embodiment, the nucleic acid is at least 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75 contiguous at least one of the sequences of SEQ ID NOs: 24-73.
, 100, 150, 200, 300, 400, 500, 1000 or 2000 bases or one of its complementary sequences. The nucleic acid may be a recombinant nucleic acid.

Another embodiment of the present invention is capable of hybridizing under stringent conditions with a sequence that is complementary to one of SEQ ID NOs: 24-73 or one of SEQ ID NOs: 24-73. Purified or isolated nucleic acid consisting of at least 15 bases. In one aspect of this embodiment, the nucleic acid is a recombinant nucleic acid.

Another embodiment of the present invention comprises a complete coding sequence for one of SEQ ID NOs: 24-73, wherein said complete coding sequence optionally encodes a sequence encoding a signal peptide as well as a mature protein. Purified or isolated nucleic acid containing the sequence In one aspect of this embodiment, the nucleic acid is a recombinant nucleic acid.

[0039] Yet another embodiment of the present invention relates to SEQ ID NOs: 24-
A purified or isolated nucleic acid comprising one nucleotide of 73. In one aspect of this embodiment, the nucleic acid is a recombinant nucleic acid.

[0040] Yet another embodiment of the present invention relates to a SEQ ID NO: 24 encoding a signal peptide.
Purified or isolated nucleic acid comprising one nucleotide of -73. In one aspect of this embodiment, the nucleic acid is a recombinant nucleic acid.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of one of SEQ ID NOs: 74-123.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a mature protein comprised in one of the sequences SEQ ID NOs: 74-123.

Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a signal peptide comprised in one of the sequences SEQ ID NOs: 74-123.

Yet another embodiment of the present invention is a purified or isolated protein having the sequence of one of SEQ ID NOs: 74-123.

Another embodiment of the invention is a purified or isolated polypeptide comprising at least 5 or 8 contiguous amino acids of one of the sequences of SEQ ID NOs: 74-123. In one aspect of this embodiment, the purified or isolated polypeptide is SEQ ID NO: 74-12
Consecutive at least 10, 12, 15, 20, 25, 30, 35, 40, 50 of one of the three sequences
, 60, 75, 100, 150 or 200 amino acids.

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: 74-123.

[0047] 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: 74-123.

[0048] Yet another embodiment of the present invention is a method of producing a protein comprising one of the sequences of SEQ ID NOs: 74-123, comprising a cDNA comprising one of the sequences of SEQ ID NOs: 24-73. And inserting the cDNA into an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell that produces a protein encoded by the cDNA by introducing the expression vector. And producing a protein. In one aspect of this embodiment, the method further comprises isolating the protein.

Another embodiment of the present invention is a protein obtained by the method described in the above paragraph.

Another embodiment of the present invention is a method for producing a protein comprising the amino acid sequence of a mature protein contained in one of the sequences SEQ ID NOs: 74-123, comprising the sequence of SEQ ID NO: 24 encoding the mature protein. A cDN comprising one of the nucleotide sequences of ~ 73 sequences
Obtaining A, inserting the cDNA into an expression vector such that the cDNA is operably linked to a promoter, and transferring the expression vector to a host cell that produces a mature protein encoded by the cDNA by introducing such an expression vector. And a method for producing a protein. In one aspect of this embodiment, the method further comprises isolating the protein.

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

Another embodiment of the present invention relates to a host cell comprising a purified or isolated nucleic acid comprising one of SEQ ID NOs: 24-73 as described herein or a sequence complementary thereto. It is.

Another embodiment of the present invention is a host cell comprising a purified or isolated nucleic acid comprising the complete coding sequence of one of SEQ ID NOs: 24-73, wherein the complete coding sequence is , A host cell comprising a sequence encoding a signal peptide described herein, as well as a sequence encoding a mature protein.

[0054] Another embodiment of the present invention provides a method for encoding a mature protein as described herein.
A host cell comprising a purified or isolated nucleic acid comprising one nucleotide of SEQ ID NOs: 24-73.

Another embodiment of the present invention is directed to a host cell comprising a purified or isolated nucleic acid comprising a nucleotide of one of SEQ ID NOs: 24-73 encoding a signal peptide described herein. is there.

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: 74-123. In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 10 contiguous amino acids of one of SEQ ID NOs: 74-123.

Another embodiment of the invention is an array of cDNAs or fragments thereof of at least 15 nucleotides in length, wherein at least one of the sequences of SEQ ID NOs: 24-73 or the sequence of SEQ ID NOs: 24-73. And an array comprising one of the sequences complementary to, or a contiguous fragment of at least 15 nucleotides. In one aspect of this embodiment, the array comprises at least two of the sequences of SEQ ID NOs: 24-73,
It includes a sequence complementary to the sequence of SEQ ID NOs: 24 to 73, or a contiguous fragment of at least 15 nucleotides. In another embodiment of the invention, the array comprises:
It includes at least five of the sequences of SEQ ID NOs: 24-73, a sequence complementary to the sequence of SEQ ID NOs: 24-73, or a contiguous fragment of at least 15 nucleotides.

[0058] Yet another embodiment of the present invention provides an accession number 9906735 and the name SignalTag 150619.
99, comprising the sequence of SEQ ID NOS: 25-40 and 42-46, or a clone deposited at the depository ECACC, or accession number 98121805, named SignalTag 166-191, comprising SEQ ID NOs: 47-73, A purified polynucleotide comprising an insert obtained from a clone deposited with the depository ECACC, or at least 8 of said insert
, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 40
Include fragments of these nucleic acids, including a continuous span of 0, 500, 1000 or 2000 nucleotides. Another embodiment of the present invention relates to accession number 99061735 and the name Sign
alTag 15061999, comprising the sequences SEQ ID NOS: 25-40 and 42-46, the depository EC
A clone deposited with the ACC or accession number 98121805 and named SignalTag 166
-191 and comprising an amino acid sequence encoded by an insert obtained from a clone deposited with the depository ECACC, comprising SEQ ID NOs: 47-73, or consisting essentially of an amino acid sequence as described above, or Consisting of the amino acid sequence as described above,
Purified polypeptide and signal peptide, mature protein, or at least 5, 8, 10, 12, 15, 20, 25, 30, 35, encoded by the insert
Includes polypeptides comprising fragments of the above amino acid sequences consisting of a continuous span of 40, 50, 60, 75, 100, 150 or 200 amino acids.

Another embodiment of the present invention is directed to a subject of at least 5, 8, 10, 12, 15, 2 of SEQ ID NOs: 74-123.
A purified polypeptide comprising a continuous span of 0, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 amino acids, wherein said continuous span is any of FIGS. 10-13. And polypeptides comprising at least one of the amino acid positions that have not been confirmed to be identical to the published sequence shown in Table 1. Further, a purified polynucleotide encoding the polypeptide is also included in the present invention.

Another embodiment of the present invention provides a cDNA encoding SEQ ID NOS: 24-73 and SEQ ID NOs: 74-12.
A computer-readable medium storing a sequence selected from the group consisting of 3 polypeptide codes.

Another embodiment of the present invention is a computer system comprising a processor and a data storage device, wherein the data storage device is derived from the cDNA code of SEQ ID NOS: 24-73 and the polypeptide code of SEQ ID NOS: 74-123. A computer system storing an array selected from the group consisting of: In some embodiments, the computer system further comprises an array comparer, wherein the reference array is stored on the data storage device. For example, the sequence comparer may include a computer program that indicates a polymorphism. In another aspect of the computer system, the system further comprises an identifier for identifying a feature of the array.

Another embodiment of the present invention provides a cDNA encoding SEQ ID NOS: 24-73 and SEQ ID NOs: 74-12.
A method for comparing a first sequence selected from the group consisting of the polypeptide codes of 3 and a reference sequence, using a computer program for comparing the sequences, reading the first sequence and the reference sequence. , Using a computer program
Determining the difference between the one sequence and the reference sequence. In some aspects of this embodiment, the step of determining a difference between the first sequence and the reference sequence comprises identifying a polymorphism.

Another embodiment of the present invention provides a cDNA encoding SEQ ID NOS: 24-73 and SEQ ID NOs: 74-12.
A method for identifying a sequence characteristic selected from the group consisting of the polypeptide codes of 3, wherein the sequence is read using a computer program for identifying the sequence characteristic, and the sequence characteristic is identified using the computer program. Performing the steps.

[0064]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (I. Creation of cDNA Library Containing the 5 ′ End of Each Corresponding mRNA) The cDNA of the present invention comprises a true translation initiation site, a signal sequence, It may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the sequence encoding the mature protein remaining after cleavage of the signal peptide. Such a cDNA is referred to herein as "full-length cDNA". Alternatively, the cDNA may include only the sequence encoding the mature protein remaining after cleavage of the signal peptide, or may include only the sequence encoding the signal peptide.

Further, by using the method described here, it is possible to obtain a cDNA encoding a portion smaller than the entire coding sequence of the secretory protein encoded by the gene corresponding to the cDNA. In some embodiments, the cDNA isolated using these methods
Has at least 5 of one of the proteins encoded by the sequences of SEQ ID NOs: 24-73.
Encodes amino acids. In yet other embodiments, the cDNA comprises at least 10, 12, 15, 20, 25, 30, 35, contiguous of the protein encoded by the sequences of SEQ ID NOs: 24-73.
, 40, 50, 60, 75, 100, 150 or 200 amino acids. In a preferred embodiment, the cDNA encodes a full length protein sequence, including the protein coding sequences of SEQ ID NOs: 24-73.

From a cDNA library derived from mRNA containing an intact 5 ′ end, the cD of the present invention can be prepared using either chemical or enzymatic methods as described in Examples 1-5.
I got NA.

Example 1 (Preparation of mRNA) Total human RNA or polyA + RNA from different tissues were separated by LABIMO and CLONT, respectively.
It was purchased from ECH and used to create a cDNA library as described below. RNA purchased
Is extracted with acid guanidine thiocyanate-phenol-chloroform (Chomczyniski
and Sacchi, Analytical Biochemistry 162: 156-159, 1987). Aviv to remove ribosomal RNA
Natl.Acad.Sci. USA 69: 1408-1412, 1972) and isolate polyA + RNA from total RNA (LABIMO) by two rounds of oligo dT chromatography as described in did.

The quality and integrity of the poly A + RNA was checked. Using a Northern blot hybridized with a probe corresponding to ubiquitous mRNA such as elongation factor 1 or elongation factor 2, it was confirmed that mRNA was not degraded. Northern blot and 28S rR
Using a probe derived from the NA sequence, it was checked whether the ribosomal sequence was mixed in the poly A ⁺ mRNA. For library construction, mRNA preparations with less than 5% rRNA were used. To avoid the library being constructed with RNA mixed with exogenous sequences (prokaryotic or fungal), PCR was used to detect the presence or absence of bacterial 16S ribosomal sequences or two highly expressed fungal sources. The presence or absence of mRNA was examined.

Example 2 (Method for Obtaining mRNA with Intact 5 ′ End) After preparing mRNA from various tissues as described above, using a chemical method or an enzymatic method, Select an mRNA that has an intact 5 'end and the oligonucleotide tag specifically binds to the 5' end of the mRNA. Both techniques, "
This cap structure, which takes advantage of the existence of a `` cap '' structure,
Characterized at the intact 5 'end of NA, generally methylated guanosine
Included in the rank.

In the method by chemical modification, the 2 ′, 3′-cis diol of the 3 ′ terminal ribose is selectively removed, and the 2 ′, 3 ′, − of the ribose bound to the 5 ′ terminal cap of the mRNA is removed. The cis diol is oxidized to a dialdehyde, which is coupled to a derivatized oligonucleotide tag. Details of a chemical method for obtaining mRNA having an intact 5 'end are disclosed in International Patent Application Publication No. WO 96/34981 published November 7, 1996.

The enzymatic method for ligating an oligonucleotide tag to the 5 ′ end of an mRNA having an intact 5 ′ end removes the phosphate group present at the 5 ′ end of the incomplete, uncapped mRNA, Subsequently, the cap of the mRNA having the intact 5 'end is removed, and the phosphate at the 5' end of the mRNA after the removal of the cap is ligated to the oligonucleotide tag. For more information on enzymatic methods for obtaining mRNAs with intact 5 'ends, see Dumas Milne Edwards J. B. (Doctoral Thesis of Pa
ris VI University, Le clonage des ADNc complets: difficultes et perspect
ives nouvelles.Apports pour l'etude de la regulation de l'expression de
la tryptophane hydroxylase de rat, 20 Dec. 1993), EP 625572 and Kato et al., Gene 150: 243-250 (1994).

[0072] In both the chemical method and the enzymatic method, the oligonucleotide tag for facilitating subsequent cloning has a restriction enzyme site (such as an EcoRI site). After binding the oligonucleotide tag to the mRNA, Northern blot was performed using a probe complementary to the oligonucleotide tag to confirm the integrity of the mRNA.

Example 3 (Synthesis of cDNA Using mRNA Template Having Intact 5 ′ End) For mRNA to which an oligonucleotide tag was bound using either a chemical method or an enzymatic method, First strand cDNA synthesis with reverse transcriptase was performed using dT primers or random nonamer. In some cases, this oligo dT
Primers contained an internal tag of at least 4 nucleotides that differed from tissue to tissue. To avoid digestion of the internal EcoRI site of the cDNA in subsequent steps of this method, methylated dCTP was used for first strand synthesis. After removing the RNA by alkaline hydrolysis, the first strand of the cDNA was precipitated using isopropanol to remove the remaining primer.

Next, using a primer corresponding to the 5 ′ end of the ligated oligonucleotide, the second strand of cDNA was synthesized using the Klenow fragment. Preferably, the primer is between 20 and 25 bases in length. To ensure that the internal EcoRI site of the cDNA was not digested during the cloning process, the second strand synthesis also utilized methylated dCTP.

Example 4 Cloning of cDNA from mRNA with an Intact 5 ′ End into BlueScript Following synthesis of the second strand, the cDNA was cloned into the phagemid vector pBlueScript II SK-vector (Stratagene). did. Insert both ends of the cDNA with T4 DNA polymerase (Bio
labs) and the cDNA was digested with EcoRI. Methylated dCTP for cDNA synthesis
Was used, the only EcoRI site contained in the tag was the hemimethylated site,
Only sites susceptible to digestion by coRI. In some cases, a Hind III adapter was added to the 3 'end of the cDNA to facilitate subcloning.

Next, size exclusion chromatography (AcA,
CDNA was size fractionated using either Biosepra) or electrophoretic separation. In addition, these cDNAs were prepared using EcoRI and SmaI restriction sites or using HindIII
If an adapter was included in the cDNA, it was directionally cloned into pBlueScript using EcoRI and HindIII restriction sites. The ligation mixture was electroporated into bacteria and grown under selection of appropriate antibiotics.

(Example 5) (Selection of clone to which oligonucleotide tag was bound) Next, a clone containing an oligonucleotide tag bound to cDNA was selected as follows.

The plasmid DNA containing the cDNA library created as described above was purified (
Qiagen). Tagged clones were positively selected as follows. Briefly, this selection method uses the gene II endonuclease of phage F1
Plasmid DNA was converted to single-stranded DNA in combination with an exonuclease such as exonuclease III or T7 gene 6 exonuclease (Chang et al., Gene 127: 95-8, 1993). The obtained single-stranded DNA was subjected to Fry et al., Biotechniques, 13: 124.
Purified using paramagnetic beads as described in -131, 1992. In this method, single-stranded DNA was hybridized with a biotinylated oligonucleotide having a sequence corresponding to the 3 ′ end of the oligonucleotide tag described in Example 2.
Preferably, the primer is between 20 and 25 bases in length. Incubation with streptavidin magnetic beads was followed by magnetic selection to capture clones containing sequences complementary to the biotinylated oligonucleotide. After capture of the positive clones, the plasmid DNA was released from the magnetic beads and
Double-stranded D using a DNA polymerase such as ThermoSequenase available from otech.
Converted to NA. Alternatively, a protocol such as the Gene Trapper kit (Gibco BRL) may be used. Subsequently, the double-stranded DNA was electroporated into bacteria. Dot blot analysis was used to estimate the percentage of positive clones with the 5 'tag oligonucleotide, ranking primarily between 90-98%.

Following electroporation, a 384-well microtiter plate (MT
Libraries are arranged in P). A copy of the MTP was kept for future use. These libraries were then transferred to 96-well MTP.

II. Characterization of the 5 'end of the clone In order to sequence only the cDNA containing the 5' end of the corresponding mRNA, the 5 'end of the clone was sequenced as described in Example 6. The first round of decision was made. In some cases, only partial sequences of clones referred to herein as "5'EST" were obtained. On the other hand, the complete sequence of the clone, referred to herein as "cDNA", may be obtained. Evaluate the quality of the cDNA library and at the same time
In order to select a clone containing the target sequence from the cDNA sequences containing the 5 'end of NA, computer analysis was performed on the 5' EST or cDNA as described in Examples 7 and 8.

(Example 6) (Sequence determination of 5 'end of cDNA clone) The sequence of the 5' end of the cloned cDNA was determined as follows. First, standard SETA-A and SETA-B primers (Genset SA), AmpliTaqGold (Pe
r9-Elmer), dNTPs (Boehringer), buffers and cycling conditions recommended by Perkin-Elmer Corporation using a PE 9600 thermocycler (Perkin-Elmer, Applie
d The plasmid insert was amplified by PCR on Biosystems Division, Foster City, CA).

The PCR products were then sequenced using the ABI Prism 377 automated sequencer (Perkin-Elmer). Standard dye-primer chemistry and ThermoSequenase (Amersham
The sequencing reaction was performed on a Pharmacia Biotech) using a PE 9600 thermocycler. As primers, T7 or 21M13 (available from Genset SA), whichever was convenient, was used. Primers were labeled with JOE, FAM, ROX and TAMRA dyes. DNTPs and ddNTPs used in the sequencing reaction were purchased from Boehringer. Sequencing buffers, reagent concentrations and cycling conditions were those recommended by Amersham.

Following the sequencing reaction, samples were sedimented with ethanol, resuspended in formamide loading buffer, and loaded onto a standard 4% acrylamide gel. Perform electrophoresis at 3000 V for 2.5 hours using an ABI 377 sequencer, and perform ABI Prism DNA Sequenci
Sequence data was collected and analyzed with ng Analysis Software version 2.1.2.

The sequence data obtained by sequencing the 5 ′ end of all the cDNA libraries created as described above was transferred to a dedicated database where quality control and validation steps were performed. Suspicious peaks were automatically flagged by a dedicated dedicated base reader that operates using a Unix system, taking into account peak shape, peak-to-peak resolution and noise levels. Automatic trimming was also performed with a dedicated base reader. Fragments of 25 bases or less containing 5 or more suspicious peaks were excluded because they were unreliable. Sequences corresponding to cloning vectors or ligation oligonucleotides were automatically removed from the sequence. However, the resulting sequence may contain at the 5 'end 1-5 nucleotides belonging to the sequences described above. If necessary, these sequences can be easily removed from case to case.

Following sequencing as described above, the sequence of the cDNA clone was entered into a database for storage and manipulation as described below. Prior to exploring the sequence of interest from the cDNA clones in the database, cDNA from non-target mRNAs, i.e., endogenous contaminants (ribosomal RNA, transfer tRNA, mitochondrial RNA), using the parameters and software shown in FIG. ) As well as foreign contaminants (prokaryotic RNA and fungal RNA) were identified and eliminated. In addition, cDNA sequences exhibiting homology to the repetitive sequences (Alu, L1, THE and MER repetitive sequences, SSTR sequences or satellite, microsatellite or telomere repetitive sequences) were identified and masked in subsequent processing.

(Example 7) (Determination of 5 'end selection efficiency) In order to determine the efficiency when the cDNA containing the 5' end of the corresponding mRNA was isolated by the above-described selection method, the FASTA algorithm was used. , 5 'EST or cDNA sequence and EMBL
The complete mRNA / cDNA extracted from release 57 was aligned with a reference pool. Obtain a reference mRNA / cDNA starting from the most 5 'transcription start site, then 5'EST or
It was compared with the position of the 5 'transcription start site of cDNA. Greater than 75% of the 5 'EST or cDNA contained a 5' end near the 5 'end of the known sequence. Although some mRNA sequences obtained from the EMBL database are inferred from genomic sequences, the 5 'end that matches these sequences is counted as an internal match. Therefore, the method used here will underestimate the yield of 5 ′ EST or cDNA containing the true 5 ′ end of the corresponding mRNA.

(Example 8) (Identification of Reading Frame Encoding Potential Signal Peptide) Next, the obtained nucleic acid sequence was screened, and using dedicated software,
Sequences with an open reading frame (ORF) without interruption and good coding probability were identified. Total length c
After obtaining the DNA, only the complete ORF, ie, nucleic acid sequences longer than 150 nucleotides, starting at the start codon and ending at the stop codon, were considered. If only the 5 'EST sequence was obtained, complete ORF longer than 150 nucleotides and incomplete ORF
That is, nucleic acid sequences longer than 60 nucleotides starting at the start codon and ending at the 5 ′ EST were considered.

Next, using a slightly modified version of the method disclosed in Von Heijne, Nucleic Acids Res. 14: 4683-4690, 1986, the searched ORF was probed to identify potential signal motifs. Identified. At least the 5 'EST or cDNA sequence encoding the polypeptide has a score on the Von Heijne signal peptide identification matrix.
The sequence of 3.5 was considered to be the sequence containing the signal sequence. Matches with a known human mRNA sequence or EST sequence, and is 5 'at least 31 nucleotides downstream from the known 5' end.
The 5 'EST or cDNA having a terminal was excluded from the subsequent analysis.

(Example 9) (Confirmation of Identification Accuracy of Potential Signal Sequence in 5 ′ EST) The accuracy of the above-described identification method for a signal sequence encoding a signal peptide was determined by comparing this method with the N-terminal of the whole human SwissProt protein. Was evaluated by applying to 43 amino acids located at Von Heij calculated by computer for each protein
The ne scores were compared with characteristics of known proteins as secreted or non-secreted proteins. In this way, the number of non-secreted proteins with a score greater than 3.5 (false positive) and the number of secreted proteins with a score less than 3.5 (false negative) could be calculated.

Using the above analysis results, the peptide encoded by the 5 ′ region of the mRNA
The probability of being a real signal peptide, as judged from the n Heijne score, was calculated based on either the assumption that 10% of the human protein was secreted or the assumption that 20% of the human protein was secreted. FIG. 2 shows the results of this analysis.

Using the secretory protein identification method described above, human glucagon, γ-interferon-derived monokine precursor, secreted cyclophilin-like protein, human pleiotrophin and human biotinidase precursor (all secreted Which is a polypeptide known to be 5) EST. Thus, the 5'EST encoding the signal peptide could be identified by the above method.

To confirm that the signal peptide encoded by 5 ′ EST or cDNA actually functions as a signal peptide, a signal sequence obtained from 5 ′ EST or cDNA is designed for signal peptide identification. What is necessary is just to clone into the vector made. Such vectors are designed to confer the ability to grow on selective media only for host cells that contain the vector with an operably linked signal sequence. For example, to confirm that the 5 'EST or cDNA encodes a genuine signal peptide, the 5' EST or cDNA signal sequence may be in-frame with the non-secreted form of the yeast invertase gene and upstream thereof. US Patent 5,536,63
It may be inserted into a signal peptide selection vector as described in No. 7. Five
If the host cell containing the signal sequence selection vector into which the 'EST or cDNA signal sequence was correctly inserted grows, the 5' EST or cDNA encodes a real signal peptide.

Alternatively, 5′EST or cDNA is cloned into an expression vector such as pXT1 as described below, or a promoter-signal sequence-reporter gene vector encoding a fusion protein between a signal peptide and an assayable reporter protein. May be used to confirm the presence of the signal peptide.
After introducing these vectors into suitable host cells such as COS cells or NIH 3T3 cells, the growth medium can be collected and analyzed for the presence or absence of secreted proteins. The medium obtained with these cells is compared with the medium obtained with control cells containing a vector without the signal sequence or cDNA insert, to identify a vector encoding a functional signal peptide or a true secreted protein.

(Example 10) (Evaluation of mRNA expression level and pattern corresponding to 5 'EST or cDNA) Spatial and temporal expression pattern of mRNA corresponding to 5' EST or cDNA, and its expression level Can be determined. As will be described in more detail below, characterizing the spatial and temporal expression patterns as well as the expression levels of these mRNAs can be performed in a desired spatial or temporal manner as described in more detail below. Is useful in constructing an expression vector capable of producing E. coli.

[0095] cDNAs or 5'ESTs whose corresponding mRNA is associated with a disease state can also be identified. For example, a state in which mRNA corresponding to cDNA or 5 ′ EST is not expressed,
Certain diseases may arise due to their overexpression or underexpression. By comparing the expression pattern and amount of mRNA in a sample obtained from a healthy individual with a sample obtained from an individual suffering from a specific disease, cDNA and 5'ES involved in the disease are examined.
T may be identified in some cases.

Performing solution hybridization using a long probe to analyze the expression level and pattern of mRNA corresponding to 5′EST or cDNA as described in International Patent Application Publication No. WO 97/05277. Can be. Briefly, a 5 'EST, cDNA or fragment thereof corresponding to the gene encoding the mRNA to be characterized was inserted into a cloning site immediately downstream of the bacteriophage (T3, T7 or SP6) RNA polymerase promoter. Produce antisense RNA. Preferably, the 5 'EST or cDNA is at least 100 nucleotides in length. The plasmid is linearized and transcription is performed in the presence of ribonucleotides containing modified ribonucleotides (ie, biotin-UTP and DIG-UTP). Excess double-labeled RNA is hybridized in solution with mRNA collected from the cell or tissue of interest. This hybridization was carried out under stringent standard conditions (80% formamide, 0.
In 4M NaCl buffer, pH 7-8 at 40-50 ° C. for 16 hours). The unhybridized probe was replaced with a ribonuclease specific for single-stranded RNA (i.e., RNAse CL3
, T1, Phy M, U2 or A). The presence of the biotin-UTP modification allows capture of the hybrid on streptavidin-coated microtiter plates. The presence of the DIG modification allows the hybrids to be detected and quantified by ELISA using an anti-DIG antibody conjugated to alkaline phosphatase.

As disclosed in GB-A-2 305 241 A, the 5 ′ EST, cDNA or a fragment thereof can also be tagged with a nucleotide sequence for continuous gene expression analysis (SAGE). Good. In this method, cDNA is prepared from a cell, tissue, organism or other nucleic acid source for which it is desired to determine a gene expression pattern. CDNA obtained
Into two pools. Cleavage each pool of cDNAs with a first restriction endonuclease called an "anchoring enzyme" that contains a recognition site likely to occur at least once in most cDNAs. The fragment containing the most 5 'or 3' region of the cleaved cDNA is isolated by binding to a capture medium such as streptavidin beads. A first oligonucleotide linker comprising a first sequence for hybridization of an amplification primer and an internal restriction site for a "tagging endonuclease" is ligated to the digested cDNA contained in the first pool. Digestion with a second endonuclease generates a short "tag" fragment from the cDNA.

A second oligonucleotide comprising a second sequence for hybridization of an amplification primer and an internal restriction site is ligated to the digested cDNA contained in the second pool. The cDNA fragments of the second pool are also digested with "tagging endonucleases" to generate short "tag" fragments from the cDNAs of the second pool. The “tags” obtained by digesting the first and second pools with an anchoring enzyme and a tagging endonuclease are ligated to each other to create a “ditag”. In some embodiments, the dual tags are concatamerized to produce a ligation product comprising between 2-200 double tags. The tag sequence is then determined and compared to the sequence of the 5 'EST or cDNA to determine which 5' EST or cDNA is expressed in the cell, tissue, organism or other nucleic acid source from which the tag was derived. judge. In this way, the expression pattern of the 5 ′ EST or cDNA in a cell, tissue, organism or other nucleic acid source is obtained.

[0099] Quantitative analysis of gene expression may be performed using an array. In the present specification,
The term array refers to full-length cDNA (i.e., the coding sequence for the signal peptide, the coding sequence for the mature protein and the cDNA containing the stop codon), cDNA,
One-, two-, or multi-dimensional fragments of full-length cDNA, cDNA, or 5 'EST, or fragments of the 5' EST that are long enough to allow specific detection of gene expression Means an aggregate. Preferably, the fragment is at least 15 nucleotides in length. More preferably, the fragment is at least 100 nucleotides in length. More preferably, the fragment is longer than 100 nucleotides. In some embodiments, the fragment is 500
It may be longer than the nucleotide length.

For example, see Schena et al. (Science 270: 467-470, 1995, Proc. Natl. Acad. S.
ci. USA 93: 10614-10619, 1996).
Using cDNA, 5 ′ EST or a fragment thereof, gene expression may be quantitatively analyzed on a complementary DNA microarray. The full-length cDNA, cDNA, 5'EST or a fragment thereof is PCR amplified and arrayed from a 96-well microtiter plate on a silylation-treated microscope slide using a high-speed apparatus. Incubate the printed array in a wet chamber, rehydrate the array elements, and add 0.2% SD
One 1 minute rinse with S, two 1 minute rinses with water, and one 5 minute rinse with sodium borohydride solution. Submerge this array at 95 ° C for 2 minutes and add 0.2% SDS
Transfer 1 minute, wash twice with water, air dry and store at 25 ° C in the dark.

Isolating mRNA of a cell or tissue, or obtaining one commercially available,
One cycle of reverse transcription is performed to prepare a probe. The probe is hybridized with a 1 cm ² microarray under a 14 x 14 mm cover glass at 60 ° C for 6-12 hours. Wash the array with low stringency wash buffer (1 × SSC / 0.2% SDS)
After washing at 25 ° C. for 5 minutes in a high stringency washing buffer (0.1 × S
Wash for 10 minutes at room temperature in SC / 0.2% SDS). The array is scanned in 0.1 × SSC with a fluorescent laser scanner using a uniquely prepared filter set. By taking the average of the ratios of the two independent hybridizations, an accurate measure of the differential expression is obtained.

As described by Pietu et al. (Genome Research 6: 492-503, 1996), quantification of gene expression using full-length cDNA, cDNA, 5′EST or a fragment thereof in a complementary DNA array An analysis may be performed. The full-length cDNA, cDNA, 5'EST or a fragment thereof is PCR amplified and spotted on a membrane. Next, mRNA from various tissues or cells is labeled with a radionucleotide. After hybridization and washing under controlled conditions, the hybridized mRNA is detected by phosphoimaging or autoradiography. After performing the experiment twice in duplicate, quantitative analysis of mRNA differentially expressed is performed.

Alternatively, as described by Lockhart et al. (Nature Biotechnology 14: 1675-1680, 1996) and Sosnowsky et al. (Proc. Natl. Acad. Sci. 94: 1119-1123, 1997) 5 'EST or cDNA using high density nucleotide sequences
Can also be analyzed for expression. An oligonucleotide consisting of 15 to 50 nucleotides corresponding to the sequence of the 5 ′ EST or cDNA is directly synthesized on the chip (Lockhar
t et al., supra) or address the chip after synthesis (Sosnowski et al.
al., supra). Preferably, the oligonucleotide is about 20 nucleotides in length.

CDNA labeled with a suitable compound such as biotin, digoxigenin or a fluorescent dye
After the probes are synthesized from the appropriate mRNA population, they are randomly fragmented to an average size of 50-100 nucleotides. Next, the probe is hybridized with the chip. Wash as described by Lockhart et al., Supra, and apply different electric fields (Sosnowsky et al., Proc. Natl. Acad. Sci. 94: 1119-1123.
), The dye or labeled compound is detected and quantified. Hybridization is performed twice in duplicate. Differential expression of the mRNA corresponding to the 5'EST or cDNA from which the oligonucleotide sequence was designed by comparing and analyzing the signal intensity from the cDNA probe for the same target oligonucleotide in different cDNA samples I understand.

III. Characterization of cDNA Containing the 5 'End of the Corresponding mRNA Example 11 Characterization of the Complete Sequence of the cDNA Clone Including the 5' End of the Corresponding mRNA Clones encoding a novel protein containing a signal peptide as determined by the method were completely sequenced as follows.

First, in order to confirm the identity of clones, cloned cD was confirmed by the dye terminator method using the AmpliTaq DNA polymerase FS kit available from Perkin Elmer.
Both the 5 'and 3' ends of the NA were sequenced twice. Next, if the complete coding region is not yet available, software such as OSP for selecting primers and automated computer software such as ASMG (Sutton et al., Genome Science Technol. 1: 9-19, 1995) Perform primer walking using and the first 5 '
A contig of the walking sequence containing the tag was constructed. Next, a contig map was created using the 5 ′ sequence and the 3 ′ sequence, and finally using primer walking. If the resulting contig contained a complete coding region and overlapping sequences with vector DNA at both ends, this sequence was considered complete.
Clones were also completely sequenced so that at least two sequences were obtained per clone. Preferably, these sequences are derived from both the sense and antisense strands. In this way, a consensus sequence was obtained using all contig sequences for each clone, and this was used as a target for computer analysis described later.

Alternatively, a clone encoding the novel protein containing the 5 ′ end of the corresponding mRNA and containing a signal peptide as determined by the above-described method is called pED6dpc2 (Dis
The whole sequence may be determined after subcloning into an appropriate vector such as coverEase, Genetics Institute, Cambridge, Mass.

Example 12 Determination of Structural and Functional Characteristics Following identification of contaminants and masking of repetitive sequences, the sequence of the cDNA was determined using the algorithms, parameters and criteria defined in FIG. Structural features such as polyA tail and polyadenylation signal were determined. Briefly, at least 11
The poly-A tail was defined as a homopolymer fragment composed of A in which only one base was different at most. Since sequencing reactions often cannot be read after such polyA, the search for the polyA tail was narrowed down to the last 100 nt of the sequence and limited to fragments consisting of 11 consecutive A's. To probe for the polyadenylation signal, a polyA tail was excised from the full length sequence. The normal polyadenylation signal AAUAAAA was replaced with polyA so that one mismatch could reveal possible sequencing errors in the normal sequence of the polyadenylation signal as well as known changes.
Searched 50bp before the tail.

Next, functional characteristics such as the ORF and signal sequence of the cDNA sequence were determined as follows. The ORF, defined as the largest fragment that starts at the translation start codon and ends at the stop codon, is placed in the frame of the three upstream strands of the cDNA (upper strand).
frame). ORFs encoding at least 80 amino acids were preferred. Next, each ORF found was scanned, and the presence or absence of a signal peptide was examined using the matrix method described in Example 10.

The published sequence and the sequence of the cDNA, which were available at the time of filing, were compared on a nucleotide or protein basis.

(Example 13) (Selection of full-length sequence) In order to preselect a cDNA containing a target sequence, a cDNA already characterized by the above-mentioned computer analysis was subjected to automatic processing.

A) Preliminary Sequence Selection Performed Automatically All cDNAs excised for each vector at both ends were examined. First, to eliminate sequences generated by either contaminants or artifacts,
A negative selection was performed as follows. Sequences consistent with the contaminating sequence as well as those encoding ORF sequences that were extensively homologous to the repetitive sequence were discarded. Sequences without poly A tail were also discarded. A cDNA that matches the known human mRNA or EST sequence and contains a 5 'end 31 nucleotides or more downstream from the known 5' end was also excluded from the analysis. Only those ORFs that terminated before the poly A tail were left.

Next, for each of the remaining cDNAs containing some ORFs, apply the following criteria to
A preliminary selection of RF was performed. The longest ORF was preferred. If the ORF sizes were equivalent, the selected ORF had the highest signal peptide score according to the Von Heijne method defined in Example 10.

Subsequently, after the repetitive sequences were masked, the sequences of the cDNA clones were compared with BLAST in pairs. Sequences that were at least 90% homologous over more than 30 nucleotides were clustered into the same class. Next, cluster analysis was performed for each cluster to detect a sequence obtained by internal priming or alternative splicing, an identical sequence, or a sequence having some frame shifts. This automated analysis was the basis for manual sequence selection.

B) Manual Sequence Selection Manual selection was performed using an automatically generated report for each sequenced cDNA clone. At the time of manual selection, selection was performed from clones belonging to the same class as follows. OR coded by clones belonging to the same class
The F sequences were aligned and compared. If the homology of the nucleotide sequences of the clones belonging to the same class exceeds 90% in the fragment having more than 30 nucleotides, or if the homology between the amino acid sequences of the clones belonging to the same class exceeds 20 amino acids,
If above 80%, these clones were considered identical. The selected ORF is
It either matched the known amino acid sequence, or met the best criteria described in the section on automatic sequence pre-selection. If the nucleotide homology was less than 90% and the amino acid homology was less than 80%, these clones were all said to encode alternative proteins that could be selected if they contained the desired sequence.

A full-length cDNA clone encoding the sequence of interest was selected using the following criteria.
First check structural parameters (initiation tag, polyadenylation site and signal, ultimately those that match the published EST at 5 'or 3' of the sequence) and confirm that the 5 'and 3' cDNAs are complete. It was confirmed whether it was a thing. Next, homology with known nucleic acids and proteins is examined to determine whether the clone sequence matches the known nucleic acid or protein sequence, and in the latter case, the coverage and the date on which the sequence was published. Was also determined. The clone sequence contains substantially new information, such as a lack of extensive matches with sequences other than ESTs or genomic DNA, or encoding a protein obtained by alternative splicing of an mRNA encoding a known protein. If so, this sequence was retained. An example of a full-length cloned cDNA containing the sequence of interest as described above is described in Example 14. In this method, sequences resulting from double inserts or chimeras evaluated based on homology to other sequences were discarded.

(Example 14) (Characterization of full-length cDNA) Using the above-described method, full-length cDNAs derived from various tissues were obtained. An example of the cDNA thus obtained is listed below.

Using this method, the full-length cDN of SEQ ID NO: 1 (in-house identification number 108-005-5-0-F9-FLC)
A got. Such a cDNA encodes a potential secreted protein (SEQ ID NO: 2) with a von Heijne score of 4.1 for the signal peptide.

Using this method, the full-length cD of SEQ ID NO: 3 (in-house identification number 108-004-5-0-G10-FLC)
I got NA. Such a cDNA encodes a potential secreted protein (SEQ ID NO: 4) with a von Heijne score of 5.3 for the signal peptide.

Using this method, the full-length cD of SEQ ID NO: 5 (in-house identification number 108-004-5-0-B12-FLC)
I got NA. Such a cDNA encodes a potential secreted protein (SEQ ID NO: 6) with a von Heijne score of 7.0 for the signal peptide.

Using this method, the full-length cDN of SEQ ID NO: 7 (in-house identification number 108-013-5-0-G5-FLC)
A got. Such a cDNA encodes a potential secreted protein (SEQ ID NO: 8) with a von Heijne score of 9.4 for the signal peptide.

In addition, polypeptides encoded by the extended or full-length cDNAs may be tested for the presence or absence of known structural or functional motifs, or for the presence or absence of signatures that are amino acid sequences that are well conserved among members of the protein family. Screening may be performed. Some of the results obtained for polypeptides encoded by full-length cDNAs screened for known protein signatures and motifs using the Proscan software and Prosite database included in the GCG package are described below.

The protein of SEQ ID NO: 10 encoded by the full-length cDNA of SEQ ID NO: 9 (in-house accession number 108-013-5-O-H9-FLC) is a eukaryote (yeast, rabbit, rodent and human) Shows homology with the family of lysophospholipases conserved in. Also, some members of this family exhibit calcium-independent phospholipase A2 activity (Portilla et al., J. Am. Soc. Nephro., 9: 1178-1186 (1998)). All members of this family are also found in the protein of SEQ ID NO: 10 (positions 54-58)
It exhibits the active site consensus GXSXG motif for carboxylesterase. Also, TopPred II software (Claros and von Heijne, CABIOS applic. Not
es, 10: 685-686 (1994)), this protein has a transmembrane domain.
It may be a membrane protein with one. Taken together, these data suggest that the protein of SEQ ID NO: 10 probably plays a role in fatty acid metabolism as a phospholipase. Thus, this protein or a portion thereof may be useful in the diagnosis and / or treatment of several dysfunctions, including but not limited to cancer and diabetes, as well as neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. May be useful. Infectious agents
) May help modulate the inflammatory response to and / or control graft rejection.

The protein of SEQ ID NO: 12 encoded by the full-length cDNA of SEQ ID NO: 11 (in-house identification number 108-004-5-0-D10-FLC) is widely used in animals (human, rodent, cow, chicken). Low homology to the conserved β4-galactosyltransferase subfamily.
Such enzymes are typically type II membrane proteins, present in the endoplasmic reticulum or Golgi apparatus, and catalyze the biosynthesis of glycoproteins, glycolipid glycans, and lactose. Br
eton et al., J. BioChem., 123: 1000-1009 (1998), which was defined as a feature of subfamily A, which is involved in the protein of SEQ ID NO: 12, in particular, UDP binding or the catalytic process itself. Region I containing the DVD motif (163-16
5th place) is well preserved. Further, the protein of SEQ ID NO: 12 has a structure typical of a type II protein. In particular, a software called TopPred II (Claro
s and von Heijne, CABIOS applic.Notes, 10: 685-686 (1994)), a short N-terminal tail 28 amino acids in length, a transmembrane segment from positions 29 to 49, 278
There is a long C-terminal tail of amino acids. Taken together, these data suggest that the protein of SEQ ID NO: 12 may play some role in polysaccharide biosynthesis, carbohydrate biosynthesis of glycoproteins and glycolipids, and / or intercellular recognition. Can be Thus, this protein has several types of functions, including but not limited to cancer, atherosclerosis, cardiovascular disorders, autoimmune disorders, rheumatic diseases such as rheumatoid arthritis Diagnosis of faults and / or
Or may be useful for treatment.

[0125] The protein of SEQ ID NO: 14 encoded by the extended cDNA of SEQ ID NO: 13 (in-house identification number 108-004-5-0-E8-FLC) is an integral membrane protein involved in the transport of amino acids to cells. One amino acid permease (positions 5 to 66) has a typical PROSITE signature. In addition, a software called TopPred II (Claros and von Heijne, CABIOS app
lic. Notes, 10: 685-686 (1994)), the protein of SEQ ID NO: 14 has a transmembrane segment from position 9 to position 29. Taken together, from these data,
It is likely that the protein of SEQ ID NO: 14 is involved in amino acid transport. Thus, this protein includes, but is not limited to, cancer, amino aciduria, neurodegenerative disorders, eating disorders, chronic fatigue, arterial tract disease, diphtheria, hypoglycemia, male infertility, muscular disorders and myopathy Rather, it may be useful in diagnosing and / or treating some types of dysfunction.

At present, bacterial clones containing a plasmid containing the above-described full-length cDNA are stored in the research laboratory of the present inventor with the above-mentioned in-house identification number. The insert may be recovered from the storage location by growing an aliquot of the appropriate bacterial clone in the appropriate medium. In this way, these plasmid DNAs can be isolated using a plasmid isolation method familiar to those skilled in the art, such as a miniprep by the alkaline method or a plasmid isolation method by the large-scale alkaline method. If necessary, the plasmid DNA may be further concentrated by centrifugation on a cesium chloride gradient, size exclusion chromatography or anion exchange chromatography. Plasmid DNA obtained by these methods can be manipulated using standard cloning techniques familiar to those skilled in the art. Alternatively, PCR can be performed using primers designed at both ends of the cDNA insertion. This allows the PCR product corresponding to the cDNA to be manipulated using standard cloning techniques familiar to those skilled in the art.

Using the above method, the cDNA of the present invention containing the sequences of SEQ ID NOS: 24 to 73 can also be obtained. Table I shows the sequence identification number of the cDNA of the invention, SEQ ID NO: 24 of the complete coding sequence.
Positions of the first and last nucleotides at ~ 73 (i.e., nucleotides encoding both the signal peptide and the mature protein; listed in the FCS position column of Table I), SEQ ID NOs for the first and last nucleotides encoding the signal peptide twenty four
-73 (listed in the position column of SigPep in Table I), the positions in SEQ ID NOs: 24-73 of the first and last nucleotides encoding the mature protein produced by cleavage of the signal peptide (of the mature polypeptide of Table I). The stop codon positions in SEQ ID NOs: 24-73 (listed in the stop codon position column in Table I), the first and last nucleotides of the poly A signal in SEQ ID NOs: 24-73 (listed in Table I). The positions of the first and last nucleotides of the poly A site (listed in the poly A signal position column)
Listed in the position column of the site).

Table II includes the sequence identification numbers of the polypeptides of SEQ ID NOs: 74-123, SEQ ID NOs: 74-123.
Positions of the first and last amino acid residues in the full length polypeptide (second column), positions of the first and last amino acid residues of SEQ ID NOs: 74 to 123 in the signal peptide (third column)
The positions (column 4) of the first and last amino acid residues of SEQ ID NOs: 74-123 in the mature polypeptide (generated by cleavage of the signal peptide from the full length polypeptide) are listed.

The nucleotide sequences of SEQ ID NOS: 24-73 and the amino acid sequences encoded by SEQ ID NOs: 24-73 (ie, amino acid sequences of SEQ ID NOs: 74-123) are shown in the attached Sequence Listing. Some of these are preliminary sequences that may contain some incorrect or ambiguous sequences or amino acids. In each case where the nucleic acid sequence includes the symbol "n", it means that the nucleotide can be adenine, guanine, cytosine or thymine. For each amino acid sequence, the present applicant identified an open reading frame that was considered to have the highest identification rate using sequence information available at the time of filing. Some polypeptides in the sequence listing include the symbol "Xaa".
This `` Xaa '' symbol is (1) a residue whose nucleotide sequence is ambiguous and cannot be identified, or (2) a stop codon contained in the determined sequence. Indicates any stop codons that may not be present. Thus, "Xaa" indicates that the residue may be any of the 20 amino acids. Also, the genetic code may indicate some possible identity of the unknown amino acid.

The sequences of SEQ ID NOs: 24-73 allow for easy screening of errors in the sequence, so resequencing fragments containing such errors or ambiguity in both strands will Can be resolved. Nucleic acid fragments for resolving sequencing errors or ambiguity may be obtained from the deposited clones or isolated using the techniques described herein. Utilization of a primer that hybridizes to a sequence located near the ambiguous or erroneous sequence can facilitate the elimination of such ambiguity or error. For example, the primer may hybridize to a sequence within 50 to 75 bases from the ambiguity or error. Upon resolution of an error or ambiguity, a corresponding correction sequence can be created in the protein sequence encoded by the DNA containing the error or ambiguity. By expressing the clone in a suitable host cell, collecting this protein and determining its sequence,
It is also possible to determine the amino acid sequence of the protein encoded by a particular clone.

Example 15 Categorization of cDNA of the Present Invention Nucleic acid sequences of the present invention (SEQ ID NOS: 24-73) were grouped as follows according to homology to known sequences. All sequences were compared to EMBL release 58 and daily update data available at the time of filing using BLASTN.

Some of the cDNAs did not match any of the known vertebrate sequences or the publicly available EST sequences and were entirely new.

All sequences with greater than 90% homology to known sequences over a range of at least 30 nucleotides were searched and further analyzed. Table III shows the sequence identification numbers of these cDNAs (column 1) and the location of the preferred fragments in these sequences (column 2 titled "Preferred fragment location"). Each fragment is shown in the form of xy, where x is the start position and y is the end position of certain preferred fragments. Preferred fragments are separated by commas. In the present specification, "
The expression "polynucleotide shown in Table III" means all preferred polynucleotide fragments as defined above in Table III. The invention consists of a continuous span of one of the sequences of SEQ ID NOs: 24-73 or a sequence complementary thereto.
An isolated nucleic acid, purified or recombinant nucleic acid substantially consisting of or comprising such a continuous span, wherein said continuous span comprises SEQ ID NO: 24-
At least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 1 of 73 sequences
Contains sequences of 00, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides or complementary thereto, provided that consecutive spans of these lengths do not conflict with the length of the particular sequence, and The continuous span is at least 1, 2, 3, 5, 10, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 10 of the polynucleotides shown in Table III.
Includes isolated, purified or recombinant nucleic acids comprising 0, 150, 200, 300, 400 or 500 or a sequence complementary thereto. The present invention also relates to at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75 of the polynucleotide shown in Table III.
, 100, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides or a sequence complementary thereto (provided that the continuous span of these lengths is
(Consistent with the length of the particular sequence shown in II), consisting essentially of such a continuous span or a sequence complementary thereto, or consisting of such a continuous span or a sequence complementary thereto. Nucleic acids, purified or recombinant nucleic acids.
The invention comprises a polynucleotide set forth in Table III or a sequence complementary thereto, consists of such a polynucleotide or a sequence complementary thereto, or consists essentially of such a polynucleotide or a sequence complementary thereto , An isolated nucleic acid,
Also encompasses purified or recombinant nucleic acids. The invention further includes all combinations of the nucleic acids listed above.

Cells containing the cDNA of the present invention (SEQ ID NOS: 24-73) in the vector pBluescriptII SK- (Stratagene) were placed in our permanent storage at Genset SA, 24 Rue Royale, 75008 Paris France. Hold.

A cDNA capable of obtaining a cell containing a specific polynucleotide (SEQ ID NOS: 24 to 73)
A pool of cells, including Portsdown, Salisbury, Wiltshire, UK
Vaccine Research and P of the European Animal Cell Culture Collection (ECACC) at SP4 OJG
Deposited on June 17, 1999 with roduction Laboratory, Public Health Laboratory Service, Center for Applied Microbial Research. In addition, a pool of cells containing extended cDNAs (SEQ ID NOs: 47-73) from which cells containing specific polynucleotides can be obtained is collected from the European Animal Cell Culture Collection (ECACC), SP4 OJG, Portsdowne, Salisbury, Wiltshire, UK. ) Vaccine Research and Production Laboratory, Publi
c Deposited with the Health Laboratory Service, Applied Microbial Research Center on December 18, 1998. For these composite deposits, each cDNA clone was transfected into separate bacterial cells (E-coli). In particular, cells containing the sequences of SEQ ID NOs: 25-40 and 42-46 were deposited on June 17, 1999 in the pool with the ECACC accession number 98121805 and the name SignalTag 166-191. Further, a cell containing the sequence of SEQ ID NOs: 47 to 73 is
December 1998 in pool with CACC accession number 99061735 and name SignalTag 15061999
Deposited on the 18th. Table IV shows the in-house reference numbers assigned to each sequence number, and specifies whether the sequence is a nucleic acid sequence or a protein sequence.

If the cDNA clone sequence did not contain BsH II, each cDNA was attached by double digestion with BsH II to produce the appropriate fragment for each clone.
It is possible to remove cDNA from Bluescript vectors. Alternatively, the desired insert may be recovered using other restriction enzymes at the multicloning site of the vector according to the manufacturer's instructions.

[0137] Bacterial cells containing a particular clone can be obtained from the composite deposit as follows.

[0138] One or more oligonucleotide probes need to be designed for the sequence known for that particular clone. This sequence can be derived from the sequences described herein, or it can be derived from a combination of these sequences. The design of the oligonucleotide probe is preferably according to the following parameters:

(A) When an ambiguous base (“N”) is present, it is necessary to design an area having a sequence having the least number of ambiguous bases. (b) Preferably, the probe has a Tm of about 80 ° C (2 ° C for each A or T, each G
Or 4 ° C for C). However, a probe having a melting point between 40 ° C. and 80 ° C. may be used as long as the specificity is not impaired.

Using techniques commonly used for labeling oligonucleotides, (-[ ³² P] A
Preferably, the oligonucleotide is labeled with TP (specific activity 6000 Ci / mmol) and T4 polynucleotide kinase. Other labeling techniques can be used.
Preferably, labels that have not been incorporated are removed by gel filtration chromatography or other established methods. It is necessary to measure and quantify the amount of radioactivity incorporated into the probe with a scintillation counter. Preferably, the resulting probe has a specific activity of about 4 × 10 ⁶ dpm / pmol.

Preferably, the bacterial culture containing the pool of full-length clones is thawed and inoculated with 100 μl of the stock solution into a sterile culture flask containing 25 ml of sterile L-broth containing 100 μg / ml ampicillin. Preferably, the culture is grown to saturation at 37 ° C., and the saturated culture is diluted with fresh L-broth. Aliquots of these dilutions were plated on culture dishes, cultured overnight at 37 ° C., and placed in a 150 mm Petri dish on solid bacteriological medium containing L-broth containing 100 μg / ml ampicillin and 1.5% agar. hand,
It is preferable to determine the dilution and volume that will yield about 5,000 well distinguishable distinct colonies. Other known methods for obtaining well-distinguished distinct colonies can also be used.

Subsequently, colonies were transferred to nitrocellulose filters, lysed, denatured and baked using standard colony hybridization techniques.

The filter was washed with 6 × SSC (175.3 g NaCl in 1 × 20 × stock solution, 88.2 g citric acid containing 0.5% SDS, 100 pg / ml yeast RNA and 10 mM EDTA (about 10 ml per 150 mm filter). (Na / liter, adjusted to pH 7.0 with NaOH) at 65 ° C. for 1 hour with gentle stirring. Preferably, the probe is added to the hybridization mixture at a concentration of 1 × 10 ⁶ dpm / ml or higher. The filter is then preferably incubated overnight at 65 ° C. with gentle agitation. Preferably, filters are washed in 500 ml of 2 × SSC / 0.1% SDS for 15 minutes at room temperature with gentle shaking. It is optional whether or not to perform the third wash with 0.1 × SSC / 0.5% SDS at 65 ° C. for 30 minutes to 1 hour. The filter is preferably dried and subjected to autoradiography for a time sufficient to visualize the positive with X-ray film. Other known hybridization methods can be used.

Positive colonies were picked using standard methods, grown in culture, and plasmid DNA was isolated. In this way, clones can be identified by restriction analysis, hybridization analysis or DNA sequencing.

The plasmid DNA obtained by these methods may be manipulated by standard cloning techniques familiar to those skilled in the art. Alternatively, PCR can be performed using primers designed at both ends of the cDNA insertion. In this way, it is possible to manipulate the PCR product corresponding to the cDNA using standard cloning techniques familiar to those skilled in the art.

Alternatively, the cDNA clone obtained by the method described in Examples 1 to 13 does not contain a sequence derived from the 5 ′ end of the corresponding mRNA, but the corresponding mRNA
May not include the entire coding sequence of the protein encoded by By using such a 5 ′ EST, an extended cDNA containing a sequence adjacent to the 5 ′ EST can be isolated. The extended cDNA thus obtained may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the true translation initiation site. Examples 16 and 17 below describe a method for obtaining an extended cDNA using 5'EST. Example 17 also describes a method for obtaining genomic DNA homologous to cDNA, mRNA, or cDNA, 5 ′ EST or a fragment thereof.

Further, using the methods of Examples 16 and 17, it is possible to obtain a cDNA encoding a range shorter than the entire coding sequence of the protein encoded by the gene corresponding to 5 ′ EST. In some embodiments, the cDNA isolated by these methods comprises at least 5 consecutive ones of the proteins encoded by the sequences of SEQ ID NOs: 24-73
It encodes 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 amino acids.

Example 16 Generic Method Using 5 ′ EST for Cloning and Sequencing a cDNA Containing the Entire Coding Region and the Standard 5 ′ End of the Corresponding mRNA) Generic Method Following Can be used to rapidly and efficiently isolate a cDNA containing a sequence adjacent to the sequence of the 5 ′ EST used to obtain the cDNA. Figure 3 illustrates this method.
As shown in the figure, cDNA can be obtained for any 5 'EST using this.

This method makes use of the known 5 ′ sequence of mRNA. Using a poly dT primer containing a nucleotide sequence at the 5 'end, perform a reverse transcription reaction on the purified mRNA so that a known sequence can be added to the end of the cDNA corresponding to the 3' end of this mRNA. . Such primers and a commercially available reverse transcriptase are added to the buffered mRNA sample,
A reverse transcript anchored to the 3 'poly A site of the RNA is obtained. Next, nucleotide monomers are added to terminate the synthesis of the first strand. MRNA hybridized to first strand cDNA
Can be removed by alkaline hydrolysis and the alkaline hydrolysis product and residual poly dT primer can be removed on an exclusion column.

Subsequently, based on the known 5 ′ sequence from the 5 ′ EST and the known 3 ′ end added by the poly dT primer used in the synthesis of the first strand, a pair of nests at each end Design mold primers. The software used for primer design is OSP (Illier a
nd Green, PCR Meth.Appl.
c.il/software/PC-Rare/doc/manuel.html)
rity) method (Griffais et al., Nucleic Acids Res. 19: 3887-3891, 1991). Preferably, the nested primer at the 5 'end and the nested primer at the 3' end are separated by 4 to 9 bases. With respect to these primer sequences, those having a melting point and specificity suitable for use in PCR may be selected.

A first round of PCR is performed using the outer primers of each of the nested pairs. next,
A second PCR is performed on a small aliquot of the first PCR product using primers inside each of the nested pairs. Thereafter, the primer and residual nucleotide monomers are removed.

There are no positional constraints in the design of 5 'nested primers suitable for use in PCR using OSP software, resulting in two types of amplicons. 2nd 5 '
Nested PC with primer located upstream of translation initiation codon and containing entire coding sequence
Preferably, the R product has been generated. Such a cDNA may be used in a direct cloning method as described in Example 4.

However, in some cases, since the second 5 ′ primer is located downstream of the translation initiation codon, a PCR product containing only a part of the ORF is generated. For amplicons that do not contain the complete coding sequence, an intermediate step is necessary to obtain both the complete coding sequence and a PCR product that contains the complete coding sequence. Different PCR
The complete coding sequence can be assembled from several subsequences determined directly from the product. After the complete coding sequence has been completely determined, new primers are designed that are suitable for use in PCR to obtain an amplicon containing the entire coding region.
However, in this case, the 3 'primer suitable for use in PCR is the 3'U of the corresponding mRNA.
Because it is located inside the TR, it becomes an amplicon lacking part of this region, ie, the polyA tract and, in some cases, the polyadenylation signal, as shown in FIG.
The cDN obtained in a suitable vector in substantially the same manner as described in Example 4.
Clone A.

The full-length PCR product is sequenced in a manner similar to that described in Example 11. Whether or not the sequencing of a specific cDNA fragment has been completed may be determined by comparing the sequence length with the size of the corresponding nested PCR product. If Northern blot data is available, the size of the mRNA detected for a particular PCR product may also be used to ultimately determine whether the sequence is complete. Sequences that do not meet these criteria are discarded and a new isolation method is attempted.

Next, the full-length PCR product is cloned into an appropriate vector. For example, in Example 4
The cDNA can be cloned into a vector in a manner similar to that described above. Such a full-length cDNA clone was subjected to double-sequence, and Example 1 was used.
Computer analysis is performed using a method substantially the same as the method described in 1 to 13. However, it will be understood that a full-length cDNA clone obtained from an amplicon in which a part of the 3′UTR has been deleted may lack a polyadenylation site and a polyadenylation signal.

(Example 17) (Method for Obtaining Nucleic Acid Homologous to cDNA or cDNA or a Fragment of cDNA) Not only a method by PCR for obtaining cDNA but also a method by conventional hybridization. Good. In addition, these methods may be used for obtaining mRNA from which cDNA is derived, mRNA corresponding to cDNA, or genomic DNA encoding a nucleic acid homologous to cDNA or a fragment thereof. In particular, a cDNA library or genomic DNA using the cDNA of the present invention or a fragment thereof containing 5 ′ EST
From the library, cDNA or a nucleic acid homologous to cDNA can be isolated as follows. Such a cDNA library or a genomic DNA library may be obtained from commercial sources, or may be produced by techniques familiar to those skilled in the art, such as those described in Examples 1 to 5. An example of such a hybridization method is described below.

Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold S
The Spring Harbor Laboratory Press, 1989 discloses a technique for identifying cDNA clones in a cDNA library that hybridizes to a particular probe sequence. Genomic DNA can be isolated using this same technique.

Briefly, a cDNA or genomic DNA clone that hybridizes to a detectable probe is identified and isolated for further manipulation. Probes containing at least 10 contiguous nucleotides from the cDNA or fragment thereof are labeled with a detectable label, such as a radioisotope or a fluorescent molecule. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the cDNA or a fragment thereof. More preferably, the probe comprises at least 20-30 contiguous nucleotides from the cDNA or a fragment thereof. In some embodiments, the probe comprises 31 or more nucleotides from the cDNA or a fragment thereof.

Techniques for labeling probes are well known and include phosphorylation with polynucleotide kinase, nick translation, in vitro transcription and non-radioactive techniques. Transfer the library cDNA or genomic DNA to a nitrocellulose or nylon filter and denature. After blocking the non-specific sites, the filter is incubated with the labeled probe for a time sufficient to allow the probe to bind to the cDNA or genomic DNA containing the sequence hybridizable to the probe.

As described below, by changing the stringency of the hybridization conditions used to identify a cDNA or genomic DNA that hybridizes to a detectable probe, cDNAs or genomic DNAs with different levels of homology to the probe can be identified. And can be isolated.

(1. Isolation of cDNA sequence or genomic DNA sequence having high homology to labeled probe) To identify cDNA or genomic DNA having high homology to the probe sequence,
The melting point of the probe can be calculated using the following equation.

For a 14-70 nucleotide long probe, the formula: Tm = 81.5 + 16.6 (log (Na +)) + 0.
The melting point (Tm) is calculated using 41 (fraction G + C)-(600 / N), where N is the length of the probe.

When hybridization is performed in a solution containing formamide, the melting point may be calculated using the following equation. Tm = 81.5 + 16.6 (log (Na +)) + 0.41 (fraction G + C)-
(0.63% formamide)-(600 / N) (where N is the length of the probe)

6 × SSC, 5 × Denhardt's reagent, 0.5% SDS, 100 μg of denatured and fragmented salmon sperm DNA or
Prehybridization is performed in 6 × SSC, 5 × Denhardt's reagent, 0.5% SDS, 100 μg of denatured and fragmented salmon sperm DNA, and 50% formamide. The compositions of SSC and Denhardt's solution are listed in Sambrook et al., Supra.

A detectable probe is added to the prehybridization solution described above,
Perform hybridization. If the probe contains double-stranded DNA, denature it before addition to the hybridization solution. The filter is contacted with the hybridization solution for a time sufficient to allow the probe to hybridize with cDNA or genomic DNA containing a sequence complementary to the probe or a homologue to the probe. For probes longer than 200 nucleotides, hybridization may be performed at a temperature 15 to 25 ° C. below Tm. For shorter probes such as oligonucleotide probes, hybridization may be performed at a temperature 15 to 25 ° C. lower than Tm. Preferably, hybridization at 6 × SSC is performed at about 68 ° C. Preferably, hybridization in a solution containing 50% formamide is performed at about 42 ° C.

All of the above hybridizations are considered to be under “stringent” conditions.

After hybridization, filters are washed in 2 × SSC, 0.1% SDS at room temperature for 15 minutes. Next, the filter was placed in 0.1 × SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour.
Wash over time. Thereafter, the solution is washed at a hybridization temperature in 0.1 × SSC, 0.5% SDS. Perform a final wash in 0.1 × SSC at room temperature.

[0168] The cDNA or genomic DNA hybridized to the probe is identified by autoradiography or other conventional techniques.

(2. Isolation of cDNA or Genomic DNA Sequences with Low Homology to Labeled Probes) The above-described method may be partially modified to identify cDNAs or genomic DNAs with slightly lower levels of homology to probe sequences. it can. For example, to obtain cDNA or genomic DNA with low homology to a detectable probe, conditions with low stringency are used. For example, in a hybridization buffer having a sodium concentration of about 1M, the hybridization temperature may be lowered from 68 ° C to 42 ° C in 5 ° C increments. After hybridization, the filter may be washed at a hybridization temperature in 2 × SSC, 0.5% SDS. Regarding these conditions, when the temperature is higher than 50 ° C, the condition is regarded as “medium”, and when the temperature is lower than 50 ° C, the condition is regarded as “low”.

Alternatively, hybridization may be performed at a temperature of 42 ° C. in a buffer such as 6 × SSC containing formamide. In this case, the formamide concentration in the hybridization buffer is reduced from 50% to 0% in 5% steps, and clones having reduced homology levels to the probe can be identified. After hybridization, the filter may be washed at 50 ° C. in 6 × SSC, 0.5% SDS. These conditions are considered “moderate” conditions above 25% formamide and “low” conditions below 25% formamide. CD hybridized to probe
NA or genomic DNA is identified by autoradiography or other conventional techniques.

(3. Between the obtained cDNA or genomic DNA and the cDNA or a fragment thereof used as the labeling probe, or the obtained cDNA or genomic DNA and the cDNA or the cDNA used as the labeling probe Determination of the homology ratio between the polypeptide encoded by the fragment) The hybridized cDNA or genomic DNA is hybridized to determine the level of homology between the probe-derived cDNA or its fragment. The nucleotide sequence of the nucleic acid obtained was compared with the nucleotide sequence of the cDNA from which the probe was derived or a fragment thereof. The sequence of the cDNA or its fragment from which the probe was derived and the cDNA or genome hybridized to the detectable probe
The DNA sequence may be stored on a computer readable medium as described below and compared with one another using any of a variety of algorithms familiar to those skilled in the art, such as those described below.

To determine the level of homology between the polypeptide encoded by the hybridizing cDNA or genomic DNA and the polypeptide derived from the cDNA or fragment thereof from which the probe was derived, the hybridized The polypeptide sequence encoded by the nucleic acid was compared with the polypeptide sequence encoded by the cDNA from which the probe was derived or a fragment thereof. The polypeptide sequence encoded by the cDNA or fragment thereof from which the probe was derived and the polypeptide sequence encoded by the cDNA or genomic DNA hybridized to the detectable probe were converted to a computer-readable medium as described below. It may be stored and compared with one another using any of a variety of algorithms familiar to those skilled in the art, such as those described below.

The homology of protein and / or nucleic acid sequences may be evaluated using an appropriate one among various sequence comparison algorithms and programs known in the prior art. Such algorithms and programs include TBLA
STN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc.
Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990, J. Mol. Bi
ol. 215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res. 22 (2): 4673.
-4680, Higgins et al., 1996, Methods Enzymol. 266: 383-402, Altschul et a
l., 1990, J. Mol. Biol. 215 (3): 403-410, Altschul et al., 1993, Nature Ge.
netics 3: 266-272), but is not limited thereto.

In a particularly preferred embodiment, the Basic Local Alignment well known in the prior art
Search Tool (BLAST) (e.g., Karlin and Altschul, 1990, Proc. Natl. Aca
d. Sci. USA 87: 2267-2268, Altschul et al., 1990, J. Mol. Biol. 215: 403-4.
10, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1
997, Nuc. Acids Res. 25: 3389-3402) to evaluate protein and nucleic acid sequence homology. In particular, the following tasks are performed using five dedicated BLAST programs.

(1) Comparison of amino acid query sequence with BLASTP and BLAST3 to protein sequence database (2) Comparison of nucleotide query sequence with BLASTN with nucleotide sequence database (3) Comparison of nucleotide query sequence (both strands) with BLASTX Compare conceptual translation products converted in six reading frames with protein sequence database (4) Compare protein query sequence with nucleotide sequence database converted in all six reading frames (both strands) with TBLASTN (5) Use TBLASTX The nucleotide query sequence converted in six reading frames is 6
Compare with nucleotide sequence database converted in one reading frame

The BLAST program uses a “high score segment pair” as described herein between an amino acid or nucleic acid query sequence and a test sequence, preferably obtained from a protein or nucleic acid sequence database. Homologous sequences are identified by identifying similar segments to be called. The high score segment pairs are preferably identified (ie, aligned) by a scoring matrix, various of which are known in the prior art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science
256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61). Although less preferred than this matrix, PAM or PAM250 matrices can also be used (e.g., Schwartz and Dayhoff, eds., 1978, Matrices for
Detecting Distance Relationships: Atlas of Protein Sequence and Structu
re, Washington: See National Biomedical Research Foundation).

The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology. I do. Finding statistical significance
It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268).
See).

The parameters used in the above algorithms may be varied according to the length of the sequence to be studied and the degree of homology. In some embodiments, these parameters may be default parameters used by the algorithm when not instructed by the user.

[0179] In some embodiments, the hybridized nucleic acid and probe derived cD
The level of homology between NA and its fragments is determined by Brutlag et al. Comp. App.
Biosci. 6: 237-245, 1990. In these analyses, parameters may be selected as follows. Ma
trix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomi
zation Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty = 0
.05, Window Size = 500 or the length of the sequence hybridized to the probe, whichever is shorter. Since the FASTDB program does not consider the 5 'or 3' cleavage when calculating the homology level, if the sequence hybridized with the probe is cleaved compared to the sequence of the cDNA from which the probe was derived or the fragment thereof, Calculate the number of nucleotides in the cDNA or fragment thereof that do not match or align with the sequence for soy, determine the ratio of the total number of nucleotides in the hybridizing sequence that do not match or align with the nucleotides, and subtract this ratio from the homology level. And manually adjust the homology level. For example, the sequence for hybridization is 700 nucleotides in length, the cDNA sequence or its fragment sequence is 1000 nucleotides in length, and the first 300 bases at the 5 ′ end of the cDNA or its fragment are missing in the sequence for hybridization, If the overlapping 700 nucleotides are identical, adjust the level of homology as follows. Where the cDNA
Alternatively, 30% of the length of the fragment is 300 bases that do not match and are not aligned. If the overlapping 700 nucleotides are 100% identical, the adjusted homology level will be 100-30 = 70% homology. Note that the above adjustments are made only when the mismatched or unaligned nucleotides are at the 5 'or 3' end. No adjustments are made if there is a mismatched or unaligned sequence internally or under other conditions.

For example, using the methods described above, the nucleic acid homology to the cDNA or fragment thereof from which the probe was derived is at least 95%, the nucleic acid homology is at least 96%, the nucleic acid homology is at least 97%, and the nucleic acid homology is at least 98%, nucleic acid homology at least 99
% Or greater than 99% nucleic acid homology can be obtained and identified. Such nucleic acids may be allelic variants or related nucleic acids obtained from other species. Similarly, using progressively decreasing stringency hybridization conditions, one obtains and identifies nucleic acids at least 90%, at least 85%, at least 80% or at least 75% homologous to the probe-derived cDNA or fragment thereof. can do.

Using the above method and an algorithm such as FASTA, which changes parameters depending on the length and homology degree of the sequence to be studied, such as default parameters used in the algorithm when there is no instruction from the user. And derived the probe c
At least 99% of the protein encoded by the DNA or fragment thereof,
At least 98%, at least 97%, at least 96%, at least 95%, at least 90
%, At least 85%, at least 80% or at least 75% homologous nucleic acids can be obtained. In some embodiments, the "default" op
ening penalty and "default" gap penalty and PAM 250 (standard score matrix, Dayhoff et al., Atlas of Protein Sequence and Structure, V
ol. 5, Supp. 3 (1978)) can be used to determine the level of homology.

Alternatively, the level of polypeptide homology may be determined using the method of Brutlag et al. Comp. App. Bios.
ci. 6: 237-245, 1990. In these analyses, parameters may be selected as follows. Matrix = P
AM 0, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization G
roup Length = 0, Cutoff Score = 1, Window Size = Sequence Length, Gap Penalty =
5, Gap Size Penalty = 0.05, Window Size = 500 or homologous sequence length,
Whichever is shorter. If the homologous amino acid sequence is shorter than the amino acid sequence encoded by the cDNA or a fragment thereof due to N-terminal and / or C-terminal deletions, the results can be manually corrected as follows. First, the number of amino acid residues in the amino acid sequence encoded by the cDNA or its fragment that does not match or align with the homologous sequence is determined. Next, the ratio of amino acids whose lengths do not match or are not aligned in the sequence encoded by the cDNA or fragment thereof is calculated. This ratio is subtracted from the homology level. For example, the amino acid sequence encoded by the cDNA or fragment thereof is 100 amino acids long, the length of the homologous sequence is 80 amino acids, and the amino acid sequence encoded by the cDNA or fragment thereof is smaller than the homologous sequence. When truncated at the N-terminus, the homology level is calculated as follows. In the above assumptions, there are 20 unmatched and unaligned amino acids in the sequence encoded by the cDNA or fragment thereof. This accounts for 20% of the length of the amino acid sequence encoded by the cDNA or fragment thereof. Assuming that the remaining amino acids are 100% identical between the two sequences, the homology level will be 100% -20% = 80% homology. No adjustments are made if there is a mismatched or unaligned sequence internally or under other conditions.

In addition to the methods described above, as outlined in the following paragraphs, the cDNA of the invention
Alternatively, other protocols can be used to obtain homologous cDNA using the fragments.

The cDNA may be prepared by obtaining mRNA from the tissue, cell or organism of interest, using mRNA preparation methods utilizing poly A selection methods or other techniques well 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 strand of cDNA.

[0185] The first strand of this cDNA is hybridized to a second primer comprising at least 10 contiguous nucleotides of the sequence of SEQ ID NOs: 24-73. Preferably, the primers are at least 10, 12, 15, 17, 18, 20, contiguous from the sequence of SEQ ID NOs: 24-73.
Contains 23, 25 or 28 nucleotides. In some embodiments, the primer is
It contains 31 or more nucleotides from the sequence of SEQ ID NOs: 24-73. If it is desired to obtain a cDNA containing the entire protein coding sequence, including the true translation start site, the second primer used will include sequences located upstream from the translation start site. The second primer is extended to produce a second strand of cDNA that is complementary to the first strand of cDNA. Alternatively, RT-PC may be performed from both ends of the obtained cDNA using primers as described above.

The mRNA containing the sequence of SEQ ID NOs: 24-73 is hybridized with a known cDNA fragment that hybridizes the mRNA with the primer, a primer containing a sequence complementary to the genomic DNA or a fragment thereof, and hybridized. The first strand of the cDNA may be prepared from the mRNA by reverse transcription of the primers to prepare a cDNA containing the 5 ′ fragment of the mRNA. Preferably, the primers have SEQ ID NOS: 24-
At least 10, 12, 15, 17, 18, 20, 23, 25 or more contiguous sequences of 73
Contains 28 nucleotides.

Thereafter, a second strand of cDNA complementary to the first strand of cDNA is synthesized. A primer complementary to the sequence contained in the first strand of the cDNA is hybridized to the first strand of the cDNA, and the primer is extended to produce the second strand of the cDNA, thereby producing the second strand of the cDNA. You may.

The double-stranded cDNA produced using the method described above is isolated and cloned. this
The cDNA may be cloned into a vector such as a plasmid or viral vector that is replicable in a suitable host cell. For example, the host cell may be a bacterial cell, a mammalian cell, an avian cell or an insect cell.

[0189] The mRNA is isolated and the primers hybridized to the mRNA are reverse transcribed to obtain a cDNA primer.
Techniques for generating one strand, extending the primers to produce a second strand of cDNA complementary to the first strand of cDNA, isolating the double-stranded cDNA, and cloning this double-stranded cDNA include:
Well known to those skilled in the art, Current Protocols in Molecular Biology, John Wiley
& Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.

Alternatively, other methods may be used to obtain full-length or homologous cDNA. One method is to prepare cDNA from mRNA and clone it into a double-stranded phagemid as follows. After cloning, double-stranded phagemid cD
The NA library is treated with endonucleases and exonucleases such as the Gene II product of phage F1 to make it single-stranded (Chang et al., Gene 127: 95-8, 1993).
. A biotinylated oligonucleotide comprising a sequence of a known cDNA fragment, genomic DNA or a fragment thereof is hybridized to a single-stranded phagemid.
Preferably, the fragment is at least 10 contiguous of the sequence of SEQ ID NOs: 24-73,
Includes 12, 15, 17, 18, 20, 23, 25 or 28 nucleotides.

Hybrids of biotinylated oligonucleotides and phagemids are incubated with paramagnetic streptavidin beads, and the beads are collected with a magnet to isolate the hybrids (Fry et al., Biotechniques, 13: 124-131). , 1992). Thereafter, the obtained phagemid is released from the beads and converted into double-stranded DNA using a primer specific to the cDNA used for designing the biotinylated oligonucleotide or a fragment thereof. Alternatively, a protocol such as Gene Trapper kit (Gibco BRL) may be used. The resulting double-stranded DNA is transformed into bacteria. Homologous or full-length cDNAs containing the cDNA sequence or its fragment sequence are identified by colony PCR or colony hybridization.

Using any of the methods described above, a plurality of cDNAs, including full-length protein coding sequences or fragments of protein coding sequences, are provided as a cDNA library, and the encoded proteins can be evaluated or used in diagnostic assays as described below. Can be used.

The cDNA prepared by any of the methods described herein can be manipulated to obtain nucleic acids containing the desired cDNA fragments using conventional techniques such as subcloning, PCR or in vitro oligonucleotide synthesis. Is also good. For example, a nucleic acid containing only the complete coding sequence (ie, the sequence encoding the signal peptide and the mature protein remaining after cleavage of the signal peptide peptide) can be obtained using techniques well known to those skilled in the art. Alternatively, a conventional technique may be applied to obtain a nucleic acid containing only the coding sequence for the mature protein remaining after cleavage of the signal peptide, or a nucleic acid containing only the coding sequence for the signal peptide.

Similarly, nucleic acids containing other desired fragments of the coding sequence for the encoded protein can be obtained. For example, the nucleic acid may comprise at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200,
It may contain 300, 400, 500, 1000 or 2000 bases.

Once the cDNA has been obtained, it can be sequenced to determine the amino acid sequence encoded by the cDNA. Once the encoded amino acid sequence can be determined, many possible cDNAs encoding that protein can be generated and identified using only the degeneracy of the genetic code. For example, as described below, it is possible to identify allelic variants or other homologous nucleic acids. Alternatively, a nucleic acid encoding the desired amino acid sequence can be synthesized in vitro.

In a preferred embodiment, the coding sequence may be selected using known codons or codon pairs for the host organism in which the cDNA is to be expressed.

(IV. Using cDNA or Fragments thereof to Express Proteins and Using These Expressed Proteins) Using any of the methods described above, the corresponding cDNAs, such as cDNAs encoding mature proteins, Using a cDNA containing the entire protein coding sequence of mRNA or a part thereof,
The secretory protein encoded by the cDNA or a part thereof can be expressed as described below. If necessary, replace the sequence encoding the signal peptide with cD
It may be included in NA to facilitate secretion of the expressed protein. It will be appreciated that a plurality of extended cDNAs containing the entire protein coding sequence or a portion thereof can be simultaneously cloned into an expression vector to create an expression library for analysis of the encoded protein, as described below.

Example 18 Expression of Protein Encoded by cDNA or Fragment thereof To express a protein encoded by cDNA or a fragment thereof,
A nucleic acid containing the coding sequence for the protein or fragment thereof to be expressed is obtained as described above and cloned into a suitable expression vector. If necessary, the sequence encoding the signal peptide may be included in the cDNA to facilitate secretion of the expressed protein. For example, the nucleic acid may include one of SEQ ID NOs: 24-73 as shown in Table I and the attached Sequence Listing.
Alternatively, the nucleic acid is one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above.
It may comprise the nucleotides of one complete coding sequence.

Due to sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modifications of the encoded protein, enzymatic cleavage of the encoded protein or other biological factors, the complete coding sequence ( That is, if the range of the sequence encoding the signal peptide and the mature protein generated by cleavage of the signal peptide) is different from those of the sequences listed in Table I, those skilled in the art will recognize SEQ ID NOS: 24-73.
It will be appreciated that the complete coding sequence range in the sequence of can be easily identified. Accordingly, as set forth in the claims appended hereto, the numbers 24-73
It is not intended to exclude variations or equivalents of the complete coding sequence listed in Table I that are readily identifiable from the scope for nucleic acids comprising the complete coding sequence of one of the sequences. Similarly, if the range of the full-length polypeptide differs from the range shown in Table II due to any of the above-described elements, the claims set forth in the claims below refer to the range for the polypeptide comprising the amino acid sequence of the full-length polypeptide. It is not intended to exclude variants or equivalents of the coding sequences listed in Table II that are readily identifiable.

Alternatively, the nucleic acid used to express the protein or fragment thereof is a mature protein encoded by one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above (ie, (Protein produced by cleavage of the signal peptide).

Encoding a mature protein due to sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modifications of the encoded protein, enzymatic cleavage of the encoded protein or other biological factors If the sequence range differs from that of the sequences listed in Table I, it will be understood by those skilled in the art that the sequence range encoding the mature protein in the sequence of SEQ ID NOs: 24-73 can be easily identified. Accordingly, SEQ ID NOs: 24-73, as set forth in the claims appended hereto.
It is not intended to exclude variants or equivalents of the sequences listed in Table I that are readily identifiable from the scope for nucleic acids comprising the sequence encoding the mature protein encoded by one of the above. Thus, the claims relating to nucleic acids comprising the sequence encoding the mature protein arise from post-translational modifications, enzymatic cleavage or cleavage of signal peptides as well as other readily identifiable variants or equivalents of secreted proteins. Includes equivalents of the sequences listed in Table I, such as those encoding biologically active proteins. Similarly, if the range of the mature polypeptide differs from the range set forth in Table II due to any of the above-mentioned elements, the mature polypeptide is sequenced into one of SEQ ID NOs: 74-123, as set forth in the claims. It is not intended to exclude variations or equivalents of the coding sequences listed in Table II that are readily identifiable from the scope of the polypeptide comprising the sequence of the protein. Thus, the claims relating to polypeptides comprising the sequence of the mature protein are intended to cover not only post-translational modifications, enzymatic cleavage or cleavage of signal peptides, but also organisms resulting from other readily identifiable variants or equivalents of secreted proteins. Includes the equivalents of the sequences listed in Table II, such as chemically active proteins. Also, due to sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modifications of the encoded protein, enzymatic cleavage of the encoded protein or other biological factors, A biologically active form of the polypeptide contained in one of the sequences, or a nucleic acid encoding a biologically active form of the polypeptide, is identified as a mature polypeptide shown in Table II, or If different from the nucleotides that encode the mature polypeptide shown in Table I, one of skill in the art can readily ascertain amino acids in the biologically active form of the polypeptide and nucleic acids that encode the biologically active form of the polypeptide. It will be understood that it can be identified. In such cases, a claim relating to a polypeptide comprising a mature protein comprised in one of SEQ ID NOs: 74-123 or a nucleic acid comprising one nucleotide of SEQ ID NOs: 24-73 encoding the mature protein. Is not intended to exclude readily identifiable variations from the sequences listed in Tables I and II.

[0202] In some embodiments, the nucleic acid used to express the protein or fragment thereof is encoded by one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above. May comprise a nucleotide encoding a signal peptide.

Encodes a signal protein due to sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modifications of the encoded protein, enzymatic cleavage of the encoded protein or other biological factors If the sequence range differs from that of the sequences listed in Table I, it will be understood by those skilled in the art that the range of the sequence encoding the signal protein in the sequence of SEQ ID NOs: 24-73 can be easily identified. Accordingly, a table that is easily identifiable from the scope described in the claims appended hereto for nucleic acids comprising a sequence encoding a signal protein encoded by one of SEQ ID NOs: 24-73. It is not intended to exclude variants of the sequences listed in I. Similarly, if the range of the signal polypeptide differs from the range set forth in Table II due to any of the above-described elements, a signal to one of SEQ ID NOs: 74-123, as set forth in the claims. It is not intended to exclude variations of the coding sequences listed in Table II that are readily identifiable from the scope of polypeptides comprising the sequence of the protein.

Alternatively, the nucleic acid is at least one contiguous one of the sequences of SEQ ID NOs: 74-123.
It may encode a polypeptide containing 5 amino acids. In some embodiments, the nucleic acid is at least 8, 10, 12, 15, 2, 2 consecutive ones of the sequences of SEQ ID NOs: 74-123.
A polypeptide comprising 0, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 amino acids may be encoded.

The nucleic acid to be inserted into the expression vector may include a sequence upstream of the sequence encoding the signal peptide, such as a sequence that regulates the expression level or a sequence that confers tissue-specific expression.

A nucleic acid encoding a protein or polypeptide to be expressed is operably linked to a promoter with an expression vector using conventional cloning technology. The expression vector may be any mammalian, yeast, insect or bacterial expression system known in the art. Genetics Institute (C
Obtain commercially available vectors and expression systems from a variety of sources, including Ambridge, MA), Stratagene (La Jolla, California), Promega (Madison, WI) and Invitrogen (San Diego, CA). Can be. If necessary, see Hatfield, et al. U.S. Patent No. 5,082,767.
The codon context and codon pairing of the sequence may be optimized for the particular expression organism into which the expression vector is to be introduced, as described in the above section, to facilitate expression and facilitate proper protein folding.

An example of a method for expressing a protein encoded by the above-described cDNA or nucleic acid will be described below. First, the methionine start codon for the gene and the poly A signal of this gene are identified. If the nucleic acid encoding the polypeptide to be expressed does not have a methionine functioning as a starting site, the starting methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the cDNA does not have a polyA signal, the polyA signal is spliced out of pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes, which are incorporated into the mammalian expression vector pXT1 (Stratagene). The above sequence can then be added to the construct. pXT1 contains the LTR obtained from Moloney murine leukemia virus and a fragment of the gag gene. Since the LTR is at the above-mentioned position in the construct, stable transfection can be performed efficiently. The vector contains the herpes simplex virus thymidine kinase promoter and the selectable neomycin gene. A restriction endonuclease sequence for Pst I, which is complementary to the cDNA or a fragment thereof, and incorporated into the 5 ′ primer, and a corresponding restriction endonuclease sequence for BglII at the 5 ′ end of the cDNA 3 ′ primer. Using oligonucleotide primers containing the cDNA, the cDNA encoding the polypeptide to be expressed or a fragment thereof can be transferred to a bacterial vector, paying careful attention to position the cDNA in frame with respect to the polyA signal. Obtained by PCR from The purified fragment obtained by the PCR reaction thus generated is digested with PstI, blunt-ended with exonuclease, digested with BglII, purified and ligated to pXT1 (now containing the polyA signal), BglII Digest with.

The ligation product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Grand Island, NY) under the conditions outlined in the product description. Positive transfectants are selected after growth of transfected cells in G418 (Sigma, St. Louis, Mo.) at 600 ug / ml. Preferably, the expressed protein is released into the medium to facilitate purification.

Alternatively, the cDNA was cloned into pED6dpc2 (DiscoverEase, Genetics Institute, Cambridge,
MA). The pED6dpc2 construct thus obtained may be transfected into a suitable host cell, such as a COS 1 cell. Select and expand methotrexate resistant cells. Preferably, the protein expressed from the cDNA is released into the medium to facilitate purification.

The proteins in the medium are separated by gel electrophoresis. If necessary, the proteins may be precipitated with ammonium sulfate or separated according to size or charge before performing the electrophoresis.

As a control, an expression vector without a cDNA insert is introduced outside the host cell or organism, and the medium is expressed using techniques such as Coomassie or silver staining, or using antibodies to the protein encoded by the cDNA. Collect the protein. The secreted protein present in the medium is detected. The techniques of Coomassie staining and silver staining are familiar to those skilled in the art.

An antibody capable of specifically recognizing a protein of interest may be generated using a 15-mer synthetic peptide containing the sequence encoded by the appropriate 5 ′ EST, cDNA or fragment thereof. Synthetic peptides are injected into mice to generate antibodies against the polypeptide encoded by 5'EST, cDNA or a fragment thereof.

A secreted protein obtained from a host cell or organism, including an expression vector comprising a cDNA or fragment thereof, is compared to a protein obtained from a control cell or organism. The presence of a band in the medium obtained from the cells containing the expression vector, which is not present in the medium obtained from the control cells, indicates that the cDNA encodes a secreted protein. In general, the mobility of a band corresponding to a protein encoded by cDNA is close to that expected based on the number of amino acids contained in the reading frame of cDNA. However, the mobility of this band may be different from the mobility that would result from modifications such as glycosylation, ubiquitination or enzymatic cleavage.

Alternatively, when a protein expressed from the above expression vector does not contain a sequence directing its secretion, expression from a host cell containing an expression vector containing an insert encoding a secreted protein or a fragment thereof is performed. The compared protein can be compared to a protein expressed from a host cell containing an expression vector without the insert. Samples obtained from cells containing the expression vector containing the insert include:
The presence of bands not present in samples obtained from cells containing the expression vector without the insert indicates that the secreted protein or fragment thereof is being expressed. Usually, this band has the same mobility as expected for a secreted protein or fragment thereof. However, the mobility of this band may be different from the mobility that would result from modifications such as glycosylation, ubiquitination or enzymatic cleavage.

[0215] The protein encoded by the nucleic acid insert may be purified using standard immunochromatographic techniques. In such a method, such as a cell extract or a medium,
The solution containing the secreted protein is placed in a column charged with antibodies to the secreted protein, which is attached to a chromatography matrix. The secreted protein is bound to an immunochromatographic column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound secreted protein is separated from the column and recovered using standard techniques.

If antibody production is not possible, the cDNA or fragment thereof may be incorporated into an expression vector designed for use in a purification scheme using the chimeric polypeptide. In such a strategy, the coding sequence of the cDNA or fragment thereof is inserted in frame with the gene encoding the other chimera. The other chimera may be a β-globin or nickel binding polypeptide coding sequence. In this way, the chimeric protein is purified using a chromatographic matrix having antibodies to β-globin or nickel. The protease cleavage site may be engineered between the β-globin gene or nickel binding polypeptide and the cDNA or a fragment thereof. This allows the two polypeptides of the chimera to be digested with the protease and separated from each other.

One useful expression vector for producing β-globin chimeras is pSG5 (Stratagene). It encodes rabbit β-globin. The intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the level of expression is increased by the polyadenylation signal incorporated into the construct. These techniques are well known to those skilled in the field of molecular biology. Standard methods are described in Davis et al., ( Basic Methods in Molecular Biology , LG Davis, MD
Dibner, and JF Battey, ed., Elsevier Press, NY, 1986). In addition, many methods available from Stratagene, Life Technologies or Promega are available. Also, In vitro Expre
The polypeptide may be produced from the construct using an in vitro translation system such as the ss® Translation Kit (Stratagene).

Following expression and purification of the secreted protein encoded by the 5 ′ EST, cDNA or fragment thereof, the ability of the purified protein to bind to the surface of various cell types, as described below, may be tested. A plurality of proteins expressed from these cDNAs are proteins to be simultaneously evaluated for the activities specifically described below and other biological roles capable of performing an assay for determining the activities. It will be understood that it may be included in the panel.

Alternatively, the polypeptide to be expressed is a product of a transgenic animal,
That is, it may be a product as a component of milk of a transgenic cow, goat, pig or sheep, which is characterized by having a somatic cell or a germ cell containing a nucleotide sequence encoding a protein of interest.

Example 19 Analysis of Secreted Protein to Determine Whether Secreted Protein Binds to Cell Surface The protein encoded by the cDNA or fragment thereof was prepared as described in the previous example. Cloned into an expression vector such as The protein is purified by size chromatography, charge chromatography, immunochromatography or other techniques familiar to those skilled in the art. After purification, the protein is labeled using techniques well known to those skilled in the art. The labeled protein is incubated with cells or cell lines from various organs or tissues to bind the protein to receptors present on the cell surface. After incubation, cells are washed to remove non-specifically bound proteins. The labeled protein is detected by autoradiography. Alternatively, unlabeled protein may be incubated with the cells and detected using an antibody having a detectable label, such as a fluorescent molecule, attached thereto.

By performing a competition analysis in which various amounts of unlabeled protein are incubated with the labeled protein, the specificity of cell surface binding can be analyzed. As the amount of competitive unlabeled protein increases, the amount of labeled protein bound to the cell surface decreases. As a control, in some binding reactions, various amounts of unlabeled protein independent of the labeled protein are used in combination. In a binding reaction in which the amount of unrelated unlabeled protein increases, the amount of labeled protein bound to the cell surface does not decrease, indicating that the protein encoded by the cDNA is specifically bound to the cell surface.

As noted above, secreted proteins have a number of important physiological effects that have proven to be valuable therapeutic sources. A secreted protein encoded by cDNA or a fragment thereof produced by any of the methods described herein can be evaluated to determine its physiological activity, as described below.

Example 20 Assay of Proteins Expressed from cDNA or Fragments thereof for Cytokine, Cell Proliferation or Differentiation Activity As described above, secreted proteins function as cytokines, And may affect differentiation. Many protein factors discovered to date, including the well-known cytokines, exhibit activity in one or more factor-dependent cell proliferation assays, making the assay convenient for confirming cytokine assays. It is a good means. 32D, DA2, DA1G, T10
, B9, B9 / 11, BaF3, MC9 / G, M ⁺ (preB M ⁺ ), 2E8, RB5, DA1, 123, T1165, HT2, C
The activity of the proteins of the present invention is demonstrated by any of a variety of factor-dependent cell proliferation assays for cell lines, including but not limited to TLL2, TF-1, Mo7c and CMK. Assays as described above, or Current Protocols in Immunology , Ed. By JE Coligan et al., Greene Publishing Associa
tes and Wiley-Interscience, Takai et al. J. Immunol. 137: 3494-3500, 1986
, Bertagnolli et al. J. Immunol. 145: 1706-1712, 1990; Bertagnolli et al.
, Cellular Immunology 133: 327-341, 1991, Bertagnolli, et al. J. Immunol.
149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994. The ability to regulate growth can be assessed.

In addition, cytokine production and / or cytokine production by spleen cells, lymph node cells and thymocytes
Numerous assays for proliferation are well known. These assays include: Current Protocols in Immunology. J.E.Coligan et al. Eds., Vol 1 pp. 3.1
2.1-3.12.14 John Wiley and Sons, Toronto. 1994 and Schreiber, R.D.Curr ent Protocols in Immunology. , Supra Vol 1 pp. 6.8.1-6.8.8, John Wiley and
Sons, Toronto. 1994.

[0225] The ability of the protein encoded by the cDNA to regulate the growth and differentiation of hematopoietic cells or lymphopoietic cells can be assayed. Many assays for such activity are familiar to those of skill in the art and include, by way of example, Bottomly, K., Da.
vis, LS and Lipsky, PE, Measurement of Human and Murine Interleukin
2 and Interleukin 4, Current Protocols in Immunology. , JE Coligan et a
l. Eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991, deVr.
ies et al., J. Exp.Med. 173: 1205-1211, 1991, Moreau et al., Nature 36: 6.
90-692, 1988, Greenberger et al., Proc. Natl. Acad. Sci. USA 80: 2931-
2938, 1983, Nordan, R., Measurement of Mouse and Human Interleukin 6 Cur
rent Protocols in Immunology. JE Coligan et al. Eds. Vol 1 pp. 6.6.1-6
.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad.
Sci. USA 83: 1857-1861, 1986; Bennett, F., Giannotti, J., Clark, SC.
and Turner, KJ, Measurement of Human Interleukin 11 Current Protocols in Immunology. JE Coligan et al. Eds. Vol 1 pp. 6.15.1 John Wiley an
d Sons, Toronto. 1991, Ciarletta, A., Giannotti, J., Clark, SC and Tur
ner, KJ, Measurement of Mouse and Human Interleukin 9 Current Protocols in Immunology. JE Coligan et al., Eds. Vol 1 pp. 6.13.1, John Wiley
and Sons, Toronto. 1991.

[0226] The ability of a cDNA-encoded protein to modulate a T cell response to an antigen can also be assayed. Many assays for such activity are familiar to those of skill in the art, and include, for example, Chapter 3 (In vitro Assays for Mou
se Lymphocyte Function), Chapter 6 (Cytokines and Their Cellular Recepto
rs) and Chapter 7, (Immunologic Studies in Humans) in Current Protocols in Immunology , JE Coligan et al. Eds. Greene Publishing Associates and
Wiley-Interscience, Weinberger et al., Proc. Natl. Acad. Sci. USA 77:60
91-6095, 1980, Weinberger et al., Eur. J. Immun. 11: 405-411, 1981, Takai
137: 3494-3500, 1986; Takai et al., J. Immunol. 140:
508-512, 1988.

[0227] Cytokines, proteins that exhibit cell proliferation or cell differentiation activity can be formulated as pharmaceuticals and used in the treatment of clinical conditions that are beneficial for inducing cell proliferation or differentiation. Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary.

(Example 21) (Assay of activity of protein expressed from cDNA or fragment thereof as immune system regulator) [0228] The effect of a protein encoded by cDNA as an immune regulator can also be evaluated. For example, proteins may be evaluated for activity that affects thymocyte or splenocyte cytotoxicity. Various assays for such activity are familiar to those of skill in the art, and include, for example, Chapter 3 (In vitro Assa
ys for Mouse Lymphocyte Function 3.1-3.19) and Chapter 7 (Immunologic st
udies in Humans) in Current Protocols in Immunology , JE Coligan et al.
Eds, Greene Publishing Associates and Wiley-Interscience, Herrmann et a
Natl. Acad. Sci. USA 78: 2488-2492, 1981, Herrmann et al., J. I.
mmunol. 128: 1968-1974, 1982, Handa et al., J. Immunol. 135: 1564-1572, 19
85, Takai et al., J. Immunol. 137: 3494-3500, 1986, Takai et al., J. Immu
nol. 140: 508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2.
488-2492, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1982, Handa
et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3.
494-3500, 1986, Bowman et al., J. Virology 61: 1992-1998, Takai et al., J.
Immunol. 140: 508-512, 1988, Bertagnolli et al., Cellular Immunology 13
3: 327-341, 1991, Brown et al., J. Immunol. 153: 3079-3092, 1994.

The effect of the protein encoded by the cDNA on T cell-dependent immunoglobulin response and isotype switching can also be evaluated. Various assays for such activity are familiar to those of skill in the art, and by way of example, Mali
szewski, J. Immunol. 144: 3028-3033, 1990; Mond, JJ and Brunswick, M As.
says for B Cell Function: In vitro Antibody Production, Vol 1 pp. 3.8.1-
3.8.16 in Current Protocols in Immunology. JE Coligan et al Eds., John
Assays disclosed in Wiley and Sons, Toronto. 1994.

The effect of the protein encoded by the cDNA on immune effect cells, such as the effect on Th1 cells and cytotoxic lymphocytes, can also be evaluated. Various assays for such activity are familiar to those of skill in the art, and by way of example,
Chapter 3 (In vitro Assays for Mouse Lymphocyte Function 3.1-3.19) and C
hapter 7 (Immunologic Studies in Humans) in Current Protocols in Immunolgy , JE Coligan et al. Eds., Greene Publishing Associates and Wiley-In
terscience, Takai et al., J. Immunol. 137: 3494-3500, 1986, Takai et al.
J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Immunol. 149: 377.
8-3783, 1992.

The effect of the protein encoded by the cDNA on the activation of naive T cells by dendritic cells can also be evaluated. Various assays for such activity are familiar to those of skill in the art, and include, for example, Guery et al., J. Immunol. 13
4: 536-544, 1995, Inaba et al., Journal of Experimental Medicine 173: 549-
559, 1991; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995;
Porgador et al., Journal of Experimental Medicine 182: 255-260, 1995, Nai
r et al., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science
264: 961-965, 1994; Macatonia et al., Journal of Experimental Medicine 16
9: 1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94
: 797-807, 1994, Inaba et al., Journal of Experimental Medicine 172: 631-6
40, 1990.

The effect of the protein encoded by the cDNA on lymphocyte longevity can also be assessed. Various assays for such activity are familiar to those of skill in the art, and include, by way of example, Darzynkiewicz et al., Cytometry 13: 795-808, 1992,
Gorczyca et al., Leukemia 7: 659-670, 1993, Gorczyca et al., Cancer Resea
rch 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243, 1991, Zacharchuk,
Journal of Immunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14:89
1-897, 1993, Gorczyca et al., International Journal of Oncology 1: 639-64
8, 1992.

Assays for proteins that influence T cell differentiation decisions and early stages of development include 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, To
Nat. Acad Sci. USA 88: 7548-7551, 1991, including but not limited to ki.

A protein exhibiting activity as an immune system modulator activity can be formulated as a formulation and used in the treatment of a clinical condition that has advantages in modulating immune activity. For example, this protein helps regulate (up or down) the growth and proliferation of T and / or B lymphocytes, as well as acting on the cytolytic activity of NK cells and other cell populations. Immunodeficiency and dysfunction (Severe combined immunodeficiency (
SCID)). These immunodeficiencies may be genetic or may be due to viral (such as HIV) and bacterial or fungal infections, and may be due to autoimmune disorders. Specifically, using the protein of the present invention, HIV, hepatitis virus, herpes virus, actinomycetes, Leishmania parasite, other malaria parasites, as well as various fungal infections such as candidiasis, viruses, Bacterial, fungal or other infections can be treated. Of course, in this regard, the proteins of the present invention are also useful where boosting the immune system is desirable, ie, treating cancer.

Possible autoimmune disorders that can be treated using the proteins of the present invention include:
For example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune lung disease, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease And autoimmune inflammatory eye diseases. Such proteins of the invention may also be useful in treating allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory disorders. Other conditions where immunosuppression is desirable (including organ transplantation) may be treatable using the proteins of the present invention.

Using the proteins of the present invention, it may be possible to modulate the immune response in various ways. Down-regulation may be in the form of suppression or inhibition of an immune response that is already in progress, or it may be involved in preventing the induction of an immune response. The function of the activated T cell can be suppressed by suppressing the T cell response or inducing a specific resistance in the T cell, or both. Immunosuppression of a T cell response is generally an active, non-antigen-specific process that requires continuous exposure of the T cell to a suppressor. Resistance is involved in the induction of allergies or unresponsiveness in T cells, but is generally antigen-specific and distinguishes it from immunosuppression in that it persists after exposure to the tolerizing agent has ceased. it can. The absence of a T cell response upon re-exposure to a specific antigen in the absence of a tolerizing agent can operatively indicate resistance.

In the context of tissue, skin and organ transplantation and graft-versus-host disease (GVHD), one or more antigenic functions (B lymphocyte The sphere antigen function (including but not limited to B7, for example) may be down-regulated or prevented. For example, inhibiting T cell function reduces tissue destruction during tissue transplantation. Generally, in tissue grafts, T cells recognize the graft as foreign and begin rejection of the graft, followed by an immune response that destroys the graft. Prior to transplantation, a molecule that suppresses or inhibits the interaction between the B7 lymphocyte antigen and the natural ligand of the immune cell before transplantation (in the form of a soluble monomer of a peptide having only B7-2 activity, or other B lymphocyte antigen (B7 -1 and B7-3) (in connection with the monomeric form of the peptide that has activity or inhibits antibodies) binds this molecule to the natural ligand of the immune cell without transmitting the corresponding costimulatory signal be able to. When the antigen function of B lymphocytes is inhibited in this way, cytokine synthesis by immune cells such as T cells is inhibited, and thus functions as an immunosuppressant. Furthermore, there is a case where T cell immunity can be reduced by simply not causing costimulation, and resistance can be induced in a subject. Inducing long-term resistance by B lymphocyte antigen inhibitory reagents may eliminate the need for repeated administration of these inhibitory reagents. To achieve sufficient immunosuppression or tolerance in a subject, B
In some cases, the function of the lymphocyte antigen combination must be inhibited.

The efficiency of a particular inhibitory agent to prevent organ transplant rejection or GVHD can be assessed using animal models that help predict the efficiency in humans. Examples of suitable systems that can be used include allogeneic heart transplants in rats and xenogeneic Engelhans islet cell transplants in mice; see Lenschow et al., Science 257.
: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89: 11102-1.
As described in 1105 (1992), both in vivo of CTLA4Ig fusion protein
Used to study immunosuppressive effects in o. In addition, a mouse model of GVHD (Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.
846-847) can be used to determine the effect of inhibiting B lymphocyte antigen function in vivo on the development of this disease.

[0239] Inhibition of antigen function can also be therapeutically useful in treating autoimmune diseases. Many autoimmune disorders are reactive to autologous tissues and result from inappropriate activation of T cells that promote the production of cytokines as well as autoantibodies involved in the pathogenesis of the disease. Preventing activation of self-reactive T cells may reduce or eliminate disease symptoms. By administering a reagent that disrupts T cell costimulation by disrupting the receptor-ligand interaction of B lymphocyte antigens, it suppresses T cell activation and may be involved in disease processes It can interfere with the production of antibodies or T cell-derived cytokines. In addition, antigen-specific resistance of autoreactive T cells may be induced by the inhibitory reagent, and the disease may be suppressed for a long period of time. A number of well-characterized animal models of human autoimmune disease can be utilized to determine the efficacy of an inhibitory agent in preventing or reducing autoimmune disorders. Examples include experimental autoimmune encephalitis in mice, systemic lupus erythematosus in MRL / pr / pr mice or NZB hybrid mice, autoimmune collagen-induced arthritis in mice, diabetes mellitus in OD and BB rats, experimental myasthenia in mice Disease (Paul ed., Fundamental Immunology, Raven Press, New York, 1989)
, pp. 840-856).

Up-regulation of antigen function (preferably B lymphocyte antigen function) as a means to up-regulate an immune response may be useful for treatment. Up-regulation of the immune response may be in the form of augmenting an existing immune response or eliciting an early immune response. For example,
In cases of viral infection, it may be useful to boost the immune response by stimulating the antigenic function of B lymphocytes. In addition, systemic administration of influenza or B lymphocyte antigens in an activated form may reduce systemic viral diseases such as common cold and encephalitis.

Alternatively, T cells are removed from the patient and pulsed with a viral antigen that expresses a peptide of the invention or reintroduces in vitro activated T cells into the patient along with a stimulated form of a soluble peptide of the invention. By co-stimulating T cells in vitro with APC,
The antiviral immune response in infected patients may be increased.

In other applications, information control or increase in antigen function (preferably B lymphocyte antigen function) may help to induce tumor immunity. A tumor cell transfected with a nucleic acid encoding at least one peptide of the present invention (sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma, etc.) is administered to a subject to increase the tumor-specific resistance of the subject. It is possible to overcome. If necessary, tumor cells can be transfected to express the peptide combination. For example, a peptide having only a B7-2-like activity, or an expression vector that directs the expression of a peptide related to a peptide having a B7-1-like activity and / or a B7-3-like activity is added to a tumor cell obtained from a patient. Transfect ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, gene therapy can be used to target tumor cells for transfection in vivo.

The presence of the peptides of the invention having B lymphocyte antigen activity on the surface of tumor cells provides T cells with a costimulatory signal necessary to elicit a T cell mediated immune response against transfected tumor cells. Sent. Also, MHC class I or MHC class
MHC class I α-chain protein and β2 are added to tumor cells that do not have MII class molecules or that cannot reexpress MHC class I or MHC class II molecules in sufficient quantities.
Microglobulin or MHC class II α chain protein and MHC class II β
MHC class I or MHC class II proteins can be expressed on the cell surface by transfecting a nucleic acid encoding the entire chain protein or a fragment thereof (such as a cytoplasmic domain truncated fragment). Appropriate class related to peptides having B lymphocyte antigen (B7-1, B7-2, B7-3, etc.) activity
Expression of II or class II MHC elicits a T cell mediated immune response against transfected tumor cells. Optionally, a gene encoding an antisense construct that inhibits the expression of an MHC class II-related protein, such as an invariant chain,
It is possible to co-transfect with a DNA encoding a peptide having the activity of a B lymphocyte antigen, to promote the presentation of tumor-associated antigens and to induce tumor-specific immunity. Thus, eliciting a T cell-mediated immune response in a human subject may be sufficient to overcome the tumor-specific resistance of the subject. Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary.

Example 22 (Assay for Hematopoietic Regulation Activity of Protein Expressed from cDNA or Fragment thereof) The hematopoietic regulation activity of the protein encoded by cDNA or a fragment thereof can be evaluated. For example, the effect of the protein on the differentiation of embryonic stem cells can be assessed. Many assays for such activity are familiar to those of skill in the art, and by way of example, see Johansson et al. Cellular Biology 15: 141-151.
, 1995, Keller et al., Molecular and Cellular Biology 13: 473-486, 1993,
Assays disclosed in McClanahan et al., Blood 81: 2903-2915, 1993.

The protein encoded by the cDNA or fragment thereof can also be evaluated for its effect on stem cell longevity and stem cell differentiation. Many assays for such activity are familiar to those of skill in the art, and by way of example, Fres
hney, MG Methylcellulose Colony Forming Assays, in Culture of Hematopoietic Cells.RI Freshney, et al. Eds.pp. 265-268, Wiley-Liss, Inc., N
ew York, NY. 1994, Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5.
911, 1992; McNiece, IK and Briddell, RA Primitive Hematopoietic Colo
ny Forming Cells with High Proliferative Potential, in Culture of Hemato poietic Cells .RI 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, RE Cobblestone Area Forming Cell Assay, In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.pp. 1-21, Wiley-Liss,
Inc., New York, NY. 1994, Spooncer, E., Dexter, M. and Allen, T. Long T
erm Bone Marrow Cultures in the Presence of Stromal Cells, in Culture of Hematopoietic Cells.RI Freshney, et al. Eds.pp. 163-179, Wiley-Liss
, Inc., New York, NY. 1994, Sutherland, HJ Long Term Culture Initiatin
g Cell Assay, in Culture of Hematopoietic Cells.RI Freshney, et al. E
ds. pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.

A protein exhibiting hematopoietic regulatory activity can be formulated as a formulation and used for treatment of a clinical condition that has an advantage in regulating hematopoietic activity. For example, the proteins of the invention may be useful in regulating hematopoiesis and, consequently, in treating myeloid or lymphatic cell dysfunction. Although not sufficient, the biological activities that favor colony forming cells or factor-dependent cell lines indicate that they are involved in the regulation of hematopoiesis. For example, by supporting growth and proliferation of erythroid progenitor cells alone or in combination with erythroid progenitor cells and other cytokines, such as in the treatment of various anemias, or the production of erythroid precursors and / or erythroid cells It finds use in combination with radiation / chemotherapy to stimulate cancer; granulocytes and monotherapy that are useful in combination with chemotherapy to prevent or treat the consequent myelosuppression. Helps the growth and proliferation of bone marrow cells, such as spheres / macrophages (ie, conventional CSF activity); by supporting the growth and proliferation of platelet mother cells, and consequently the growth and proliferation of platelets,
Enables the prevention or treatment of various platelet disorders, such as thrombocytopenia, and is used primarily as an alternative to or to supplement platelet transfusion; and / or Various stem cell disorders (including, but not limited to, aplastic anemia and paroxysmal nocturnal hemoglobinuria, such as those usually treated by transplantation) and radiation / chemicals to support growth and proliferation, After therapy, in-vivo or ex-vivo (i.e.,
Either bone marrow transplantation or peripheral blood progenitor cell transplantation (in combination with allogeneic or xenogeneic)
There are therapeutic uses in repopulating the stem cell compartment as healthy cells or cells that have been genetically engineered for gene therapy. Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary.

Example 23 Assay for Tissue Growth Regulation of Proteins Expressed from cDNA or Fragments thereof Proteins encoded by cDNA or fragments thereof can also be evaluated for effects on tissue growth. Many assays for such activity are familiar to those of skill in the art, and include, by way of example, assays disclosed in International Patent Application Publication Nos.WO95 / 16035, WO95 / 05846 and WO91 / 07491. Is raised.

[0248] Assays for wound healing activity, Winter, Epidermal Wound Healin g, pps. 71-112 (Maibach, H1 and Rovee, DT, eds.), Year Book Medical Publ
ishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. De
rmatol 71: 382-84 (1978), but is not limited thereto.

[0249] Proteins involved in regulating tissue growth can be formulated as pharmaceuticals and used in the treatment of clinical conditions that would benefit regulation of tissue growth. For example, the proteins of the invention may be used in compositions used for the growth or regeneration of bone, cartilage, tendons, ligaments and / or nerve tissue, as well as compositions used for wound healing and tissue repair and replacement, as well as for burns, cuts, It can be useful in treating wounds and ulcers.

The protein of the present invention induces cartilage and / or bone growth in a situation where bone is not properly formed. The protein includes, in humans and other animals,
There are uses in healing fractures and cartilage damage or defects. Preparations using the protein of the present invention may be used prophylactically in reducing subcutaneous and open fractures and improving the fixation of artificial joints. Such new bone formation is useful in cosmetic plastic surgery, as bone formation induced by osteogenic factors helps repair craniofacial defects caused by congenital, traumatic or oncological resection .

The proteins of the present invention can also be used in the treatment of periodontal disease and other tooth restoration processes. By using such a factor, an environment is obtained which induces osteogenic cells, promotes the proliferation of osteogenic cells, or induces differentiation of precursor cells of osteogenic cells. In addition, the proteins of the present invention may be used to stimulate bone and / or cartilage repair or through tissue destruction processes mediated by inflammation or inflammatory processes.
Inhibiting (collagenase activity, osteoclast activity, etc.) may be useful in treating osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can be attributed to the proteins of the present invention is tendon / ligament formation. The protein of the present invention induces the formation of tendon / ligament-like tissue or other tissue in situations where such tissue is not normally formed, and in humans and other animals, tendon or ligament tears , Deformities and other tendon or ligament defects. Preparations using the tendon / ligament-like tissue inducing protein can be used prophylactically to prevent damage to the tendon or ligament tissue, improve the fixation of the tendon or ligament to bone or other tissue, and It can be used to repair ligament tissue defects. Neoplastic tendon / ligament-like tissue formation induced by the compositions of the present invention assists in repairing other tendon or ligament defects due to congenital, traumatic or other causes, such neonatal tendon / ligament-like Histogenesis is also useful in cosmetic surgery to fix or repair tendons or ligaments. By using the composition of the present invention, it induces tendon-forming cells or ligament-forming cells, promotes the growth of tendon-forming cells or ligament-forming cells, and induces the differentiation of tendon-forming cells or ligament-forming cells. Or tendon /
An environment is provided for inducing the proliferation of ligament cells or progenitor cells ex vivo and returning them in vivo to effect tissue repair. The compositions of the present invention may also be useful in the treatment of tendinitis, Carpal tunnel syndrome and other tendon or ligament defects. The composition may include a suitable matrix and / or sequestering agent as a carrier, as is well known in the art.

The proteins of the present invention may be used for neuronal proliferation and regeneration of nerve and brain tissue, ie, diseases and disorders of the central nervous system and peripheral nervous system with degeneration, necrosis or trauma of nerve cells or nerve tissue. It may be useful for treating disorders, mechanical and traumatic dysfunction. More specifically, peripheral nerve disorders such as peripheral nerve injury, peripheral neuropathy and focal neuropathy, and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome Proteins can be used to treat diseases. Other conditions that may be treatable by the present invention include mechanical and traumatic disorders such as spinal cord dysfunction, head trauma, and cerebrovascular disorders such as seizures. Peripheral neuropathy caused by chemotherapy or other medical measures may also be treatable with the proteins of the present invention.

The proteins of the present invention may be used to improve unhealed wounds, including but not limited to pressure ulcers, ulcers associated with cerebral circulatory insufficiency, surgical wounds and trauma, etc. It may also help to enhance recovery.

The protein of the present invention can be used for organs (pancreas, liver, intestine, kidney, skin, endothelium), muscle (
It would also exhibit activity on the formation or regeneration of other tissues, such as smooth muscle, skeletal muscle or myocardium) and vascular (including vascular endothelium) tissues, or activities that promote the growth of the cells that make up such tissues. It is. Controlling or controlling fibrous scarring and producing normal tissue may also be one of the reasons for the desired effect. In addition, the protein of the present invention may exhibit angiogenic activity.

The proteins of the invention may also be useful in gut protection or in the regeneration and treatment of conditions such as pulmonary and hepatic fibrosis, reperfusion injury in various tissues and systemic damage of cytokines. .

The proteins of the present invention may be useful in promoting or inhibiting the above-mentioned tissues from differentiating from precursor tissues or cells, and in suppressing the growth of the above-mentioned tissues.

Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary. .

Example 24 Assay for Regulation of Reproductive Hormone or Cell Migration of Proteins Expressed from cDNA or Fragments Thereof Regulates proteins encoded by cDNA or fragments thereof to regulate reproductive hormones such as follicle stimulating hormone. You can also evaluate the functions that do. Many assays for such activity are familiar to those of skill in the art, and by way of example, Va
le et al., Endocrinology 91: 562-572, 1972; Ling et al., Nature 321: 779-7.
82, 1986, Vale et al., Nature 321: 776-779, 1986, Mason et al., Nature 31
8: 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 , JE Coligan et al. Eds.Green Publishing Assoc
iates 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
25: 1744-1748, Gruber et al. J. of Immunol. 152: 5860-5867, 1994, Johnst.
on et al. J. of Immunol. 153: 1762-1768, 1994.

[0260] Proteins that exhibit activity as reproductive hormones or cell migration regulators can be formulated as pharmaceuticals and used in the treatment of clinical conditions in which regulation of reproductive hormones or cell migration is advantageous. For example, a protein of the invention may exhibit activin-related activity or inhibin-related activity. Inhibin is characterized by its ability to suppress the release of follicle stimulating hormone (FSH), while activin is characterized by its ability to suppress follicle stimulating hormone (FSH).
The feature is that it stimulates the release of H). Therefore, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin β family, has a function in which inhibin suppresses the fertility of female mammals and reduces spermatogenesis in male mammals. May be useful as a birth control pill. Administering sufficient amounts of other inhibins can induce infertility in these mammals. Or,
The proteins of the present invention as homodimers or heterodimers with other protein subunits of the Inhibin-B group serve as fertility-inducing therapeutics utilizing the function of activin molecules to stimulate the release of FSH from anterior pituitary cells there is a possibility. See, for example, U.S. Patent No. 4,798,885. In addition, the proteins of the present invention may help to accelerate the time at which sexually immature mammals become fertile in order to increase the life-long fertility of livestock such as cattle, sheep and pigs.

Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary. .

Example 25 (Assay for Migration / Chemotactic Activity of Protein Expressed from cDNA or Fragment thereof) The protein encoded by cDNA or a fragment thereof can also be evaluated for migration / chemotactic activity. For example, the proteins of the present invention may have a chemotactic or chemotactic activity on mammalian cells, including monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells. It may have an activating activity (such as acting as an inflammatory cell migration factor). Migration and chemotactic proteins can be used to mobilize and bind a desired cell population to a desired site of action. Migration or chemotactic proteins offer certain advantages when treating wounds and other trauma to tissues, as well as when treating localized infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection may improve the immune response to the tumor or infectious agent.

In situations where a protein or peptide can directly or indirectly stimulate the directed sequence or migration of a particular cell population, it has chemotactic activity on such cell population.
The protein or peptide preferably has a function of directly stimulating the directed movement of cells. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in well-known assays for cell chemotaxis.

The activity of the protein of the present invention can be measured by the following method, although there are other means.

Assays for migratory activity (identifying proteins that induce or prevent chemotaxis) are based on the ability of the protein to induce translocation across the cell membrane,
It consists of assays that measure the ability of one cell population to induce adhesion to another cell population. Suitable assays for migration and adhesion include Current Protocol
s in Immunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM
Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Inter
science (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. APM
IS 103: 140-146, 1995; Mueller et al Eur. J. Immunol. 25: 1744-1748, Grube
r et al. J. of Immunol. 152: 5860-5867, 1994; Johnston et al. J. of Immun.
ol, 153: 1762-1768, 1994, but are not limited thereto.

Example 26 Assay for Coagulation Control of Proteins Expressed from cDNA or Fragments The proteins encoded by cDNA or fragments thereof can also be evaluated for their effect on coagulation. Many assays for such activity are familiar to those of skill in the art, and include, for example, Linet et al., J. Clin. Pharmacol.
26: 131-140, 1986, Burdick et al., Thrombosis Res. 45: 413-419, 1987, Hump
hrey et al., Fibrinolysis 5: 71-79 (1991), Schaub, Prostaglandins 35: 467-
474, 1988.

Proteins involved in the regulation of coagulation can be formulated as pharmaceuticals and used in the treatment of clinical conditions in which regulation of coagulation is advantageous. For example, proteins of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in treating a variety of blood clotting disorders, including genetic disorders such as hemophilia, or in treating wounds caused by trauma, surgery or other causes. And likely to enhance other hemostatic events. The protein of the present invention also dissolves thrombus or inhibits thrombus formation, and treats and prevents symptoms caused by thrombus (such as myocardial infarction and infarction of central nervous system blood vessels (such as stroke)). May help you. Alternatively, as described in detail below, a gene encoding a protein or nucleic acid that regulates the expression of these proteins may be introduced into an appropriate host cell, and the expression of the protein may be increased or decreased as necessary.

Example 27 Receptor / Ligand Interaction of Proteins Expressed from cDNA or Fragments thereof
Assay for involvement in action) The protein encoded by the cDNA or its fragment
One can also assess involvement in the interaction. Many about such activities
Assays are familiar to those skilled in the art and, by way of example, see Chapter 7.28 (Meas
urement of Cellular Adhesion under Static Conditions 7.28.1-7.28.22) in Current Protocols in Immunology , J.E.Coligan et al. Eds. Greene Publish
ing Associates and Wiley-Interscience, Takai et al., Proc. Natl. Acad. S
ci. USA 84: 6864-6868, 1987; Bierer et al., J. Exp. Med. 168: 1145-1156, 1
988, Rosenstein et al., J. Exp.Med. 169: 149-160, 1989, Stoltenborg et a.
l., J. Immunol.Methods 175: 59-68, 1994; Stitt et al., Cell 80: 661-670,
1995, Gyuris et al., Cell 75: 791-803, 1993.
Can be

For example, the protein of the present invention may be a receptor, a receptor ligand, or a receptor.
It may also show activity as an inhibitor / agonist of the / ligand interaction. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors and their ligands involved in cell-cell interactions (
Including but not limited to cell adhesion molecules (selectins, integrins and their ligands), receptor / ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses But not limited to this. Receptors and ligands are also useful for screening small molecule inhibitors or potential peptides of the relevant receptor / ligand interaction. The proteins of the present invention, including but not limited to receptor and ligand fragments, are themselves useful as inhibitors of receptor / ligand interactions.

Example 28 Assay for Anti-Inflammatory Activity of Protein Expressed from cDNA or Fragment The protein encoded by the cDNA or fragment thereof can also be evaluated for anti-inflammatory activity. For anti-inflammatory activity, it stimulates cells involved in the inflammatory response, suppresses or promotes cell-cell interactions (such as cell adhesion), suppresses or promotes chemotaxis of cells involved in the inflammatory process, It can be achieved by methods such as suppressing or promoting extravasation or stimulating or suppressing the production of other factors that more directly suppress or promote the inflammatory response. Inflammatory-related infections (e.g., septic shock, sepsis or systemic inflammatory response syndrome (SIRS), etc.), ischemia / reperfusion injury, endotoxin lethality, arthritis, hyperacute rejection by complement using proteins exhibiting such activities Response, nephritis, lung injury induced by cytokines or inflammatory cell chemoattractants, including but not limited to, inflammatory bowel disease, Crohn's disease, or overproduction of cytokines such as TNF or IL-1 It is possible to treat inflammatory conditions (including chronic or acute conditions caused by). The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigen components or substances.

Example 29 Assay for Tumor-Suppressing Activity of Protein Expressed from cDNA or Fragment The protein encoded by cDNA or a fragment thereof can also be evaluated for tumor-suppressing activity. In addition to the activities described when discussing immunological treatment or prevention of tumors, the proteins of the invention may also exhibit other anti-tumor activities. This protein can directly or indirectly direct tumor growth (e.g., ADCC
). This protein acts on tumor tissue or tumor precursor tissue, inhibits the formation of tissue necessary to support tumor growth (e.g., suppresses angiogenesis), and other factors that suppress tumor growth It can exert its tumor suppressive activity by producing an agent or cell type, or by suppressing, excluding (climinating) or inhibiting other factors, agents or cell types that promote tumor growth.

In addition, the proteins of the present invention may inhibit the following activities or effects: growth, infection or function of infectious agents including but not limited to bacteria, viruses, fungi and other parasites Or necrosis it, height, weight,
Including but not limited to hair color, eye color, skin, percent body fat or other tissue pigments or the size or shape of organs or body parts (such as breast abundance, changes in bone morphology or shape) Dietary fats, lipids, proteins, carbohydrates, to achieve (suppress or enhance) non-body characteristics, to achieve biorhythms or circadian cycles or rhythms, to achieve fertility in male or female subjects , Vitamins, minerals,
Appetite, libido, stress, cognition (to achieve metabolism, catabolism, anabolism, processing, utilization, accumulation or climination of co-factors or other trophic factors or components)
Non-hematopoietic system that achieves behavioral characteristics, provides analgesic or other pain-relieving effects, including but not limited to depressive states (including cognitive impairment), depressive states (including depressive disorders) and violent acts Promote the differentiation and proliferation of embryonic stem cells in a lineage of
Hormonal or endocrine activity, in the case of enzymes, correct enzyme deficiency, treat diseases associated with deficiency, treat hyperproliferation (such as psoriasis), immunoglobulin-like activity (such as the ability to bind antigen or complement), Act as an antigen in a vaccine composition;
It may exhibit one or more of the ability to enhance an immune response to a protein or other substance or entity that is cross-reactive with such a protein.

(Example 30) (Identification of protein interacting with polypeptide encoded by cDNA) Two such as Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech)
The -Hybrid system is used to identify proteins that interact with the polypeptide encoded by the cDNA or fragment thereof. As described in the manual attached to Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech), cD
NA or a fragment thereof is inserted into an expression vector so as to be in frame with DNA encoding the DNA binding domain of yeast transcription activator GAL4. The cDNA of a cDNA library encoding a protein that may interact with the polypeptide encoded by the cDNA or a fragment thereof is transferred to a second expression vector in frame with the DNA encoding the activation domain of GAL4. insert. The two expression plasmids are transformed into yeast, and the yeast is plated on a selective medium plate that selectively expresses a selectable marker in each expression vector and GAL4-dependent expression of the HIS3 gene. Transfectants that can grow on histidine-free media are GAL4
Screen for dependent lacZ expression. Cells that are positive in both histidine selection and the lacZ assay contain a plasmid that encodes a protein that interacts with the polypeptide encoded by the cDNA or fragment thereof.

Alternatively, the system described in Lustig et al., Methods in Enzymology 283: 83-99 (1997) may be used to identify molecules that interact with the polypeptide encoded by the cDNA. In such a system, an in vitro transcription reaction is performed on a pool of vectors containing a cDNA insert cloned downstream of a promoter that drives in vitro transcription. The resulting pool of mRNA is introduced into Xenopus laevis oocytes. The oocytes are then assayed for the desired activity.

Alternatively, pooled in vitro transcripts generated and pooled as described above can be
You may translate with ro. The pooled in vitro translation products can be assayed for the desired activity, or for interaction with a known polypeptide.

[0276] Proteins or other molecules that interact with the polypeptide encoded by the cDNA can be found in still other various ways. In one method, the cDNA
Alternatively, an affinity column containing the polypeptide encoded by the fragment can be constructed. In some forms, the affinity column contains a chimeric protein, and the protein encoded by the cDNA or a fragment thereof is fused to glutathione S-transferase. A mixture of cellular proteins or a pool of expressed proteins as described above is applied to the affinity column. As described in Ramunsen et al. Electrophoresis, 18, 588-598 (1997), proteins that interact with the polypeptide attached to the column can be isolated and analyzed on a 2D running gel. Alternatively, the protein remaining on the affinity column is purified by electrophoresis-based methods and sequenced. Using the same method, isolate antibodies, screen phage display products,
Alternatively, human antibodies can be screened by the phage display method.

[0277] Proteins that interact with the polypeptide encoded by the cDNA or fragment thereof were identified in Edwards & Leatherbarrow, Analytical Biochemistry, 246, 1-6 (19
Screening can also be performed using an optical biosensor as described in 97). The main advantage of this method is that the ratio of relevance of the protein to other interacting molecules can be determined. Therefore, it is possible to specifically select interacting molecules having a high ratio of association or interacting molecules having a low ratio of association. Generally, a target molecule is bound to the sensor surface (by a carboxymethyl dextran matrix) and a sample of the analyte molecule is placed in contact with the target molecule. The binding of the test molecule to the target molecule changes the refractive index and / or thickness. This change is detected by a biosensor. However, the above method can only be used if the change takes place in the evanescent field (a few hundred manometers from the sensor surface). In these screening assays, the target molecule may be one of the polypeptides encoded by the cDNA or fragments thereof, and the test sample may be a collection of proteins extracted from tissues or cells, a pool of expressed proteins. , Combinatorial peptides and / or peptides selected by chimeric libraries or phage display methods. The tissue or cell from which the test protein is extracted may be from any species.

In another method, the target protein is immobilized and is a collection of unique polypeptides encoded by cDNA or fragments thereof.

To study the interaction of the protein encoded by the cDNA or fragment thereof with the drug, microdialysis and Wang et al., Chromatographia, 44
, 205-208 (1997) or Busch et al., J. Chromatogr. 777:
311-328 (1997) in combination with affinity capillary electrophoresis.

The system described in US Pat. No. 5,654,150 may be used to identify molecules that interact with a polypeptide encoded by a cDNA. In this system, a pool of cDNA is
Transcribe and translate at ro and assay the reaction products for interaction with a known polypeptide or antibody.

One of skill in the art will appreciate that proteins expressed from cDNAs or fragments can be assayed for a number of activities other than those specifically set forth above. For example, the expressed protein may be evaluated for use in controlling and regulating inflammation, tumor growth or metastasis, infection or other clinical conditions. Also,
Proteins expressed from cDNA or fragments thereof may also serve as nutritional or cosmetic products.

As described below, proteins expressed from cDNA or fragments thereof can be used to generate antibodies that can specifically bind to the expressed protein or fragments thereof. The antibody may be a full-length protein encoded by one of the sequences of SEQ ID NOs: 24-73, a mature protein encoded by one of the sequences of SEQ ID NOs: 24-73, or a sequence of SEQ ID NOs: 24-73. May be capable of binding to a signal peptide encoded by one of the above. Alternatively, the antibody comprises SEQ ID NOs: 74-123
May be capable of binding to a protein fragment expressed from cDNA containing at least 10 amino acids of the sequence In some embodiments, the antibody may be capable of binding a fragment of a protein expressed from a cDNA comprising at least 15 amino acids of the sequence of SEQ ID NOs: 74-123. In another embodiment, the antibody may be capable of binding a fragment of a protein expressed from a cDNA comprising at least 25 amino acids of the sequence of SEQ ID NOs: 74-123. In yet other embodiments, the antibody may be capable of binding a fragment of a protein expressed from a cDNA comprising at least 40 amino acids of the sequence of SEQ ID NOs: 74-123.

Example 31 Production of Antibodies to Human Proteins Substantially pure proteins or polypeptides are isolated from transfected or transformed cells as described in Example 18. The protein concentration of the final preparation is adjusted to a level of a few μg / ml, for example by concentration in an Amicon filter device. In this state, a monoclonal or polyclonal antibody against the protein is prepared as follows.

A. Monoclonal Antibody Production by Hybridoma Melting Identification and singulation as described above by the classical method of Kohler, G. and Milstein, C., Nature 256: 495 (1975) or a derivative thereof. Monoclonal antibodies directed against any of the isolated peptide epitopes can be prepared from mouse hybridomas. Briefly, mice are repeatedly inoculated with a microgram of a selected protein or a peptide derived from that protein over a period of several weeks. The mouse is sacrificed and the antibody-producing cells of the spleen are isolated. Fusing spleen cells with mouse myeloma cells by polyethylene glycol,
The system is grown in selective medium containing aminopterin (HAT medium) to destroy excess unfused cells. Dilute the successfully fused cells and inoculate a diluted aliquot into the wells of a microtiter plate to continue the culture growth. The antibody in the supernatant of the well is
Engvall, E., Meth. Enzymol. 70: 419 (1980) ELISA for the first time
Antibody producing clones are identified by immunoassay or a method derived therefrom. The selected positive clones are cultured and the monoclonal antibody product is collected for use. For detailed procedures for obtaining monoclonal antibodies, see Davis, L. et al. Basic Methods in Molecular Biology Else.
vier, New York. Section 21-2.

B. Polyclonal Antibody Production by Immunization Antibodies to heterologous epitopes of a single protein can be obtained by immunizing a suitable animal with a protein expressed as described above or a peptide derived from the protein. Can be prepared.
It may be modified to increase immunogenicity or may be left unmodified. Many factors related to both the antigen and the host species affect effective polyclonal antibody production. For example, small molecules are often less immunogenic than other molecules, and may require the use of carriers or adjuvants. In addition, there is a difference in the responsiveness of the host animal to the site of inoculation and the dose, and a low titer antiserum is produced in both cases where the antigen amount is inappropriate and excessive. It appears that administering small amounts (ng level) of antigen to multiple intradermal sites gives the most reliable results. Vaitukaitis, J. et al. J. Clin. Endocrinol.Metab. 33: 988-991 (1971)
Describes an effective immunization protocol in rabbits.

Booster injections at regular intervals and collection of antisera when the antibody titer, measured semi-quantitatively, has started to decrease, for example by double immunodiffusion on agar against an antigen of known concentration. Can be. For example, Ouchterlony, O. et al., Ch
ap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (19
See (73). The plateau concentration of this antibody is usually 0.1-0 of serum (about 12 μM).
It is in the range of .2mg / ml. Fisher, D., Chap. 42 in: Manual of Clinical Immunol ogy , 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washing
Competitive binding curves are generated as described in ton, DC (1980) to determine the affinity of the antiserum for the antigen.

Antibody preparations prepared according to any of the antibody methods are useful in quantitative immunoassays for determining the concentration of an antigen producing substance in a biological sample. These preparations can also be used to semi-quantitatively or qualitatively identify the presence of an antigen in a biological sample. Furthermore, antibodies may be used in therapeutic compositions for the purpose of killing cells expressing the protein or reducing the protein concentration in the body.

(V. Utilization of cDNA or Fragment thereof as Reagent) The cDNA of the present invention can be used as a reagent in isolation methods, diagnostic assays, and forensic methods. For example, a sequence obtained from a cDNA (or a genomic DNA obtained from the cDNA) can be detectably labeled and used as a probe to isolate another sequence capable of hybridizing with the probe. In addition, the cDNA (or this c
Sequences obtained from DNA (genomic DNA) can also be used to design PCR primers for use in isolation, diagnostic or forensic methods.

(Example 32) (Preparation of PCR primers and amplification of DNA) An isolation method for cloning a nucleic acid capable of hybridizing with such a sequence using cDNA (or genomic DNA obtained from this cDNA), PCR primers can be made for various uses, including diagnostic and forensic techniques. The PCR primer is at least 10 bases in length, 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, PCR primers may be 31 bases or longer. G / G so that the melting points of the primer pairs are almost the same.
Preferably, the C ratios are also substantially the same. Various PCR techniques are well known to those skilled in the art.
For PCR technology, see Molecular Cloning to Genetic Engineering Whit
e, BA Ed. in Methods in Molecular Biology 67: Humana Press, Totowa 199
See FIG. In each of these PCR methods, one side of the nucleic acid sequence to be amplified is
The PCR primers are added to the appropriately prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase or Vent polymerase. The nucleic acid in the sample is denatured, and the PCR primers specifically hybridize to the complementary nucleic acid sequence in the sample. The primer after hybridization is extended. Thereafter, another cycle of modification, hybridization and extension is initiated. This cycle is repeated a plurality of times to create an amplified fragment having a nucleic acid sequence between the primer sites.

Example 33 Utilization of cDNA as a Probe A cDNA or a fragment thereof (or a genome obtained from this cDNA) with a detectable label familiar to those skilled in the art, including radioisotopes and non-radioactive labels. D
The probe derived from NA) can be labeled to obtain a detectable probe. Detectable probes can be single-stranded or double-stranded and can be made by techniques known in the art, including in vitro transcription, nick translation or kinase reactions. A nucleic acid sample containing a sequence hybridizable with the labeled probe is brought into contact with the labeled probe. When the nucleic acid contained in the sample is double-stranded, it may be denatured before contact with the probe. Depending on the application, the nucleic acid sample may be immobilized on a surface such as a nitrocellulose membrane or a nylon membrane. The nucleic acid sample is the genome
It may include nucleic acids obtained from various sources, including DNA, cDNA libraries, RNA or tissue samples.

[0291] Methods used to detect the presence of nucleic acids that are capable of hybridizing to a detectable probe include well-known techniques such as Southern blots, Northern blots, dot blots, colony hybridization, plaque hybridization, and the like. It is. For some applications, nucleic acids that are capable of hybridizing with the labeled probe are cloned into a vector, such as an expression vector, a sequencing vector, or an in vitro transcription vector, to facilitate characterization and expression of the hybridizing nucleic acid contained in the sample. You may. For example, as described in Example 17 above, such techniques can be used to isolate and clone sequences contained in a genomic or cDNA library that can hybridize to a detectable probe.

The PCR primers prepared as described in Example 32 above can be used for forensic analysis such as DNA fingerprinting described in Examples 34 to 38 described below. Such an analysis can utilize a detectable probe or primer based on the sequence of the cDNA or a fragment thereof (or genomic DNA obtained from the cDNA).

Example 34 Forensic Matching by DNA Sequencing In an exemplary method, a DNA sample is isolated by conventional techniques from a forensic specimen such as hair, semen, blood or skin cells. Next, according to Example 32, a large number of cDNAs (
Alternatively, a panel of PCR primers based on genomic DNA obtained from this cDNA) is used to amplify about 100-200 bases long DNA from forensic specimens. The corresponding sequence is obtained from the subject. If each of the identified DNAs is sequenced according to a conventional method, if there is a difference between the sequence obtained from the subject and the sequence obtained from the sample, this difference can be determined by a simple database comparison. . A lack of identity is conclusively demonstrated by a statistically significant difference between the suspect's DNA sequence and the DNA sequence obtained from the sample. Such a lack of identity can be demonstrated with only one sequence. On the other hand, identity using any number of sequences will be demonstrated. Preferably, at least 50 statistically identical sequences of 100 bases in length are used to prove identity between the suspect and the sample.

Example 35 Positive Identification by DNA Sequencing The techniques outlined in the above examples can be used on a large scale to identify solids in a unique fingerprint-type manner. In this technique, primers are prepared from a number of sequences shown in Table I and the attached Sequence Listing. Preferably, 20-50
Use different types of primers. Using these primers, a corresponding number of PCR generated DNA segments are obtained from the individual in question according to Example 32. Each of these DNA segments is sequenced using the method described in Example 34. A database of sequences generated using this method uniquely identifies the individual from which the sequence was obtained. Thereafter, using the same panel of primers, the individual can be absolutely correlated with a tissue or other biological specimen.

Example 36 Forensic Identification by Southern Blot The method of Example 35 is repeated to obtain a panel consisting of at least 10 amplified sequences from individuals and specimens. Preferably, the panel contains at least 50 amplified sequences. More preferably, this panel contains 100 amplified sequences. In some embodiments, the panel comprises 200 amplification sequences. This PCR-generated DNA is digested with a combination of one or preferably four base-specific restriction enzymes. Such enzymes are commercially available and are well known to those skilled in the art. After digestion, the resulting gene fragments are size-separated on an agarose gel in a plurality of duplicate wells and transferred to nitrocellulose using Southern blots well known to those skilled in the art. For an overview of Southern blots, see Davis et al. ( Basic Methods in Molecular Biology , 1986, Elsevier Press. Pp 62-65).

A panel of probes based on the cDNA (or genomic DNA derived from this cDNA) sequence, or a fragment of at least 10 bases thereof, may be labeled with radiolabels or ratios using methods well known in the art, such as nick translation or end labeling. Color-labeled and hybridized to Southern blots using techniques well known in the art (Davis et al., Supra ). Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the cDNA (or genomic DNA obtained from the cDNA). More preferably, the probe comprises at least 20-30 contiguous nucleotides from the cDNA (or genomic DNA obtained from the cDNA). In some embodiments, the probe comprises a cDNA (or genomic DNA obtained from the cDNA).
From 31 or more nucleotides. In other embodiments, the probe has cD
Consecutive at least 40, at least 50, at least 75, at least 100, at least 150 or at least 200 from NA (or genomic DNA obtained from this cDNA)
Nucleotides.

Preferably, at least 5 to 10 of these labeled probes are used, more preferably at least about 20 or 30 to generate a unique pattern. Large cDNA (
Alternatively, the band obtained by hybridization of the genomic DNA) sample obtained from this cDNA becomes a unique identifier. Cleavage by restriction enzymes will be different for each individual, and the band pattern on the Southern blot will be unique. Increasing the number of cDNA probes also increases the number of sets of bands used for identification, thus providing a statistically high level of confidence in identification.

Example 37 Dot Blot Identification Method Another technique for identifying individuals using the cDNA sequences disclosed herein utilizes a dot blot hybridization technique.

Genomic DNA is isolated from the nucleus of the subject to be identified. An oligonucleotide probe of about 30 bp length corresponding to at least 10, preferably 50 sequences obtained from cDNA or genomic DNA is synthesized. These probes are used for hybridization to genomic DNA under conditions well known to those skilled in the art. Polynucleotide kinase (Pharmaci
Use a) labeling of the oligonucleotides P ^32. Genomic DNA is inoculated into nitrocellulose or the like using a vacuum dot blot manifold (BioRad, Richmond California) to generate a dot blot. Techniques known in the prior art (Dav
Using is et al. supra ), nitrocellulose filters containing genomic sequences are baked or UV-bound to the filters, prehybridized, and hybridized with labeled probes. ^{The 32} P-labeled DNA fragments are subsequently hybridized under stringent conditions in order to detect minimal differences between the 30 bp sequence and the DNA.
Tetramethylammonium chloride is useful for identifying clones containing a small number of nucleotide mismatches (Wood et al., Proc. Natl. Acad. Sci. USA 82 (6): 1
585-1588 (1985)). The unique dot pattern allows one individual to be distinguished from the other.

A cDNA or oligonucleotide containing at least 10 contiguous bases obtained from these sequences can be used as a probe in the following selective fingerprinting method. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the cDNA (or genomic DNA obtained from the cDNA).
More preferably, the probe includes cDNA (or genomic DNA obtained from this cDNA)
At least 20 to 30 contiguous nucleotides from In some embodiments, the probe comprises 31 or more nucleotides from the cDNA (or genomic DNA obtained from the cDNA). In other embodiments, the probe comprises at least 40, at least 50, contiguous from cDNA (or genomic DNA derived from this cDNA).
At least 75, at least 100, at least 150 or at least 200 nucleotides are included.

Preferably, multiple probes having sequences from different genes are used for selective fingerprinting. A representative selective fingerprinting method for deriving a probe from cDNA is shown in Example 38 below.

Example 38 Selective "Fingerprint" Identification Techniques Using a commercially available oligonucleotide service such as Genset, Paris, France, or the like, a large number of cDNAs (or 50 From the obtained genomic DNA), a 20-mer oligonucleotide is prepared. A cell sample obtained from the subject is processed for DNA using techniques well known to those skilled in the art. Digest the nucleic acid with restriction enzymes such as EcoRI and XbaI. Following digestion, samples are applied to wells for electrophoresis. The method well-known in the prior art may be modified to adapt to polyacrylamide electrophoresis. In this example, a sample containing 5 μg (ug) of DNA is charged into a well and separated on a 0.8% agarose gel. The gel is transferred to nitrocellulose using standard Southern blot techniques.

[0303] Each oligonucleotide 10ng pooled and end-labeled with P ^32. Nitrocellulose is pre-hybridized using a blocking buffer and hybridized with a labeled probe. Following hybridization and washing, the nitrocellulose filter is exposed to X-Omat AR X-ray film. The obtained hybridization pattern is unique for each individual.

It is also contemplated within the scope of this example that the number of probe sequences used can be varied to increase accuracy or clarity.

The antibodies generated in Examples 18 and 31 above may be used to identify the tissue type or cell type from which the sample was derived as described above.

Example 39 Identification of Tissue Type or Cell Type with Labeled Tissue-Specific Antibodies Tissue-specific antigens were obtained by antibody preparations according to Examples 18 and 31 conjugated directly or indirectly to a detectable marker. To identify specific tissues. Specific antigen-binding partners in soluble protein extracts from tissue fragments, cell suspensions, or tissue samples bind to selected labeled antibody species, resulting in quantitative or semi-quantitative patterns that can be interpreted. Can be

The titer of the antiserum for these methods must be higher than that of the native preparation, so that the gamma globulin fraction can be isolated by ion exchange chromatography or ammonium sulfate fractionation, etc. Concentrate antibodies to mg / ml level.
Also, before labeling the antibody with a marker to provide the most specific antiserum,
Unnecessary antibodies against common proteins and the like must be removed from the gamma globulin fraction by using an insoluble immunoabsorbent or the like. A monoclonal antibody or a heterologous antiserum is suitable for both methods.

A. Immunohistochemical Techniques High titers of antibodies, prepared and purified as described above, can be obtained as described, for example, in Fudenberg, H.
., Chap. 26 in: Basic 503 Clinical Immunology , 3rd Ed. Lange, Los Altos,
Detectable markers as described in California (1980) or Rose, N. et al., Chap. 12 in: Methods in Immunod iagnosis , 2d Ed. John Wiley 503 Sons, New York (1980). Conjugate.

The fluorescent marker, either fluorescein or rhodamine, is preferred, but it is also possible to label the antibody with an enzyme that causes a color reaction with the support, such as horseradish peroxidase. As described below, a marker can be added to the tissue binding antibody in a second step. Alternatively, certain anti-tissue antibodies can be labeled with ferritin or other electron-dense particles, and the ferritin-binding antigen-antibody complex can be localized by electron microscopy. In yet another method, the antibody is radiolabeled, such as with ¹²⁵ I, and detected by overlaying the antibody-treated preparation with a photographic emulsion.

[0310] Preparations for performing this method can include monoclonal or polyclonal antibodies to a single protein or peptide identified as specific for a tissue type, such as brain tissue. Alternatively, antibody preparations against several antigenically distinguishable tissue-specific antigens can be used in the panel, optionally independently or as a mixture.

[0311] Tissue pieces and cell suspensions are prepared for immunohistochemical testing according to common histological techniques. Multiple cryostat sections of unknown tissue and known controls (approximately 4
μm, unfixed), and cover with a glass slide at different dilutions of the antibody preparation. Sections of known and unknown tissues must also be treated with the preparation to provide positive controls, negative controls such as pre-immune serum, and controls for non-specific staining such as buffers. There is.

The treated sections were incubated at room temperature for 30 minutes in a wet chamber, rinsed,
Then, it is washed with a buffer solution for 30 to 45 minutes. Remove excess liquid by blotting,
Develop markers.

If the first incubation did not label the tissue-specific antibody, then at this point, such as by a fluorescein-conjugated or enzyme-conjugated antibody to the immunoglobulin class of the antiserum producing species, such as a fluorescein-labeled antibody to mouse IgG. In addition, it is possible to carry out labeling by a reaction between the second antibodies. Such labeled sera are commercially available.

By measuring the color or fluorescence intensity of a tissue section and calibrating its signal using appropriate criteria, it is possible to quantify the antigens found in tissues by the methods described above.

B. Identification of Tissue-Specific Soluble Proteins Using labeled antibody reagents and detection strategies as described for immunohistology, visualization of tissue-specific proteins and identification of unknown tissues obtained by this method The sample is prepared by an electrophoretic technique so that the protein extracted from the tissue is regularly arranged and dispersed for detection based on the molecular weight.

The tissue sample is homogenized using the Virtis device. The cell suspension is disrupted by Dounce homogenous or osmotic lysis (in each case using detergent if necessary) and the cell membrane is disrupted as practiced in the prior art. Insoluble components of the cells, such as nuclei, microsomes, and membrane fractions, are removed by ultracentrifugation, and if necessary, the soluble protein-containing fraction is concentrated and stored for analysis.

By conventional SDS polyacrylamide electrophoresis, Davis, L. et al., Section
19-2 in: Basic Methods in Molecular Biology (P. Leder, ed), Elsevier, N
ew York (1986) and the like, using a range of amounts of polyacrylamide in a set of gels to resolve the entire molecular weight range of proteins detected in a sample, Separate samples into individual protein species. A size marker is used in combination for the purpose of estimating the molecular weight of the constituent protein. A convenient volume of about 5-55 μl containing about 1-100 μg of protein is taken as the sample size for analysis.
Aliquots of each separated protein are blotted to nitrocellulose filter paper. At this time, the decomposition pattern is maintained. Prepare multiple copies. For a method known as Western blot analysis, see Davis, L. et al., (Supra).
This is fully described in section 19-3. A set of nitrocellulose blots is stained with Coomassie blue dye to visualize all proteins so that they can be compared to antibody-bound proteins. The remaining nitrocellulose filters were prepared as in Example 18 and
Incubate with a solution of one or more specific antisera against the tissue specific protein prepared as described in 31. In this method, similarly to the above-mentioned method A, an appropriate positive sample, a negative sample, and a reagent control are used in combination.

In either method A or B, according to various strategies and variants thereof,
A detectable label can be associated with the primary tissue antigen-primary antibody complex. In a direct method, the primary specific antibody can be labeled, or the unlabeled conjugate can be bound to a labeled secondary anti-IgG antibody. In another method, either the primary or secondary antibody is conjugated to a biotin molecule. Biotin can bind to the avidin conjugate marker in a subsequent step. According to yet another strategy, an enzyme-labeled or radioactive protein A having the property of binding any IgG is bound in a final step to either a primary or a secondary antibody.

Visualizing tissue-specific antigen binding to one or more tissue-specific antibodies prepared from a gene sequence identified from a cDNA sequence at levels above those seen in control tissues, such as forensic samples It is possible to identify tissues of unknown origin or differentiated tumor tissues that have metastasized to foreign body sites.

In addition to uses in forensics and identification, cDNA (or genomic DNA derived from this cDNA) may be mapped to its chromosomal location. In Example 40 below, the cDNA
The radiation hybrid (RH) mapping of the human chromosome region using is described. Example 41 below describes a representative method for mapping cDNA (or genomic DNA obtained from this cDNA) to a location on a human chromosome. In Example 42 below, cDNA (or genomic DNA obtained from this cDNA) was
The mapping to metaphase chromosomes by itu Hybridization (FISH) will be described.

Example 40 Radiation Hybrid Mapping of cDNA to the Human Genome Radiation hybrid (RH) mapping is a somatic genetic method that can be used for high-resolution mapping of the human genome. In this method, a cell line containing one or more human chromosomes is lethally exposed to light and each chromosome is fragmented. The size of the fragments depends on the irradiation dose. These fragments are rescued by fusing cultured rodent cells to generate subclones containing different fragments of the human genome. This technique is described in Benham et al. (Genomics 4: 509-517, 1989) and Cox et a
l., (Science 250: 245-250, 1990). Since these subclones are of random and independent nature, any human genomic marker can be efficiently mapped. Human DNA isolated from a panel of 80-100 cell lines serves as a mapping reagent for sequencing the cDNA (or genomic DNA obtained from this cDNA). In this method, distance is measured by using the frequency of fragmentation between markers, and a conventional EST (Schuler et al., Science 274: 540-546, 1996)
Can be used to construct a high resolution map as obtained using

[0324] RH mapping was performed using growth hormone (GH) and thymidine kinase (TK) (Foster et a
l., Genomics 33: 185-192, 1996), the region surrounding the Gorlin syndrome gene (Obermayr
et al., Eur. J. Hum.Genet. 4: 242-245, 1996), covering the entire short arm of chromosome 12.
Locus (Raeymaekers et al., Genomics 29: 170-178, 1995), neurofibromatosis 2
Human chromosome 22 region containing the type locus (Frazer et al., Genomics 14: 574-584, 1
992) and 13 loci on the long arm of chromosome 5 (Warrington et al., Genomics 11: 7
01-708, 1991) has been used to generate high-resolution whole-genome radiation hybrid maps of human chromosome 17q22-q25.3 on genes.

Example 41 Mapping of cDNA to Human Chromosome Using PCR Techniques Using PCR-based methodology, cDNA (or genomic DNA obtained from this cDNA)
Can be assigned to human chromosomes. In such methods, oligonucleotide primer pairs are designed from the cDNA sequence (or the sequence of the genomic DNA obtained from the cDNA sequence) to minimize the potential for amplification by introns. Preferably, these oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification. Creating PCR primers from known sequences is well known to those skilled in the art. For a review of PCR technology, see Erlich,
HA, PCR Technology; Principles and Applications for DNA Amplification . 1992. WH Freeman and Co., New York.

[0324] These primers are utilized in the polymerase chain reaction (PCR) to generate the entire human genome D
Amplify template from NA. The PCR conditions are as follows. 60ng of genomic DNA to PC
Using 80 ng of each oligonucleotide primer, 0.6 units of Taq polymerase and ³² P-labeled deoxycytidine triphosphate 1-Cu, used as R template, with each oligonucleotide primer. 94
After 30 cycles of 1.4 minutes at ℃, 2 minutes at 55 ° C, and 2 minutes at 72 ° C, PCR was performed in a microplate thermocycler (Techne) under the conditions of final extension at 72 ° C for 10 minutes.
Is carried out. The amplification products are analyzed on a 6% polyacrylamide sequencing gel and visualized by autoradiography. If the length of the resulting PCR product is equal to the distance between the ends of the primer sequence contained in the cDNA sequence from which the primer was derived,
Two panels of human-rodent somatic cell hybrids: BIOS PCRable DNA (BIO
The PCR reaction is repeated using DNA templates obtained from S) and NIGMS human-rodent somatic cell hybrid mapping panel No. 1 (NIGMS, Camden, NJ).

[0324] PCR is used to screen a series of somatic cell hybrid cell lines that contain a defined population of human chromosomes for the presence of a particular cDNA (or genomic DNA derived from this cDNA). DNA is isolated from somatic cell hybrids and used as a staining template for PCR reactions using primer pairs from cDNA (or genomic DNA obtained from this cDNA). Only somatic cell hybrids having a chromosome containing the human gene corresponding to the cDNA (or genomic DNA obtained from this cDNA) produce an amplified fragment. By analyzing the separation pattern of the PCR product from the somatic cell hybrid DNA template,
The cDNA (or genomic DNA obtained from this cDNA) is assigned to a chromosome. One human chromosome present in all of the cell hybrids producing the amplified fragment is the chromosome containing that cDNA (or genomic DNA obtained from this cDNA). For a review on somatic gene mapping experiments techniques and results analysis, see (Ledbetter
et al., Genomics 6: 475-481 (1990)).

Alternatively, cDNA (or genomic DNA obtained from this cDNA) can be mapped to individual chromosomes using FISH as described in Example 42 below.

(Example 42) (Mapping of cDNA to chromosome using Fluorescence in situ Hybridization) Using Fluorescence in situ Hybridization, cDNA (or genomic DNA obtained from this cDNA) can be converted to a specific chromosome on a specific chromosome. It is possible to map to a location. Chromosomes used in the Fluorescence in situ Hybridization technique can be obtained from a variety of sources, including cell culture, tissue or whole blood.

In a preferred embodiment, Cherif et al. (Proc. Natl. Acad. Sci. USA, 87
: 6639-6643, 1990).
The chromosomal localization position of genomic DNA obtained from cDNA) is obtained. Plant hemagglutinin (PHA)
Metaphase chromosomes are prepared from stimulated blood cell donors. PHA-stimulated lymphocytes obtained from healthy males are cultured for 72 hours in RPMI-1640 medium. For synchronization, methotrexate (10 μM) was added over 17 hours, followed by 5-bromodeoxyuridine (5-BudR).
, 0.1 mM) over 6 hours. The cells are harvested after the addition of colcemide (1 μg / ml) in the last 15 minutes. The cells are collected, washed with RPMI, and KCl (75 mM
) And fix with three changes of methanol: acetic acid (3: 1). The cell suspension is dropped on a glass slide and air dried. Label the cDNA (or genomic DNA obtained from this cDNA) with biotin-16 dUTP by nick translation according to the manufacturer's instructions (Bethesda Research Laboratories, Bethesda, Md.) And use a Sephadex G-50 column (Pharmacia, Upssala, Sweden). ) And sediment. Immediately prior to hybridization, the DNA pellet was dissolved in hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, sonicated salmon sperm DNA 1 mg / ml, pH 7) and incubated at 70 ° C. Denature the probe in 10 minutes.

A slide glass maintained at −20 ° C. is treated with RNAse A (100 μg / ml) at 37 ° C. for 1 hour, washed three times with 2 × SSC, and dehydrated with an ethanol series. 70% formamide,
After denaturing the chromosome preparation in 2 × SSC at 70 ° C. for 2 minutes, dehydrate at 4 ° C. Slide the glass slide with proteinase K (10 μg / 100 ml in 20 mM Tris-HCl, 2 mM CaCl ₂ ).
Treat at 37 ° C for 8 minutes and dehydrate. The hybridization mixture containing the probe is mounted on a glass slide, covered with a coverslip, sealed with rubber cement, and incubated overnight at 37 ° C. in a wet chamber. After hybridization and post-hybridization washes, the biotinylated probe is detected by avidin-FITC and amplified using another layer of biotinylated goat anti-avidin and avidin-FITC. For chromosomal localization, fluorescent R-bands are obtained as described above (Cherif et al., Supra). Observe the slide glass under a LEICA fluorescence microscope (DMRXA). When the chromosomes were counterstained with propidium iodide, the fluorescent signal of the probe appears as symmetric yellow-green dots on both chromatids of the fluorescent R-band chromosome (red). Therefore, a specific cDNA (or genomic DNA obtained from this cDNA) can be localized to a specific cell-generated R band on a specific chromosome.

(Example 43) (Use of cDNA in Construction or Expansion of Chromosome Map) cDNA (or genomic DNA obtained from this cDNA) is assigned to a specific chromosome by the method described in Examples 40 to 42 above. After that, using this to cDNA (or this cDNA
High-resolution map of the chromosome where the genomic DNA obtained from) is located, and the chromosome contained in the sample can be identified.

In chromosome mapping, it is necessary to assign a specific unique sequence to a specific chromosome as described above. After mapping the unique sequence to a particular chromosome, it is ordered relative to other unique sequences located on the same chromosome. One method for chromosome mapping utilizes a series of yeast artificial chromosomes (YACs) containing thousands of inserts from the chromosome of the organism from which the cDNA (or genomic DNA obtained from this cDNA) is obtained. . This method is described in Ramaiah Nagaraja et al. Genome Research 7:
210-222, March 1997. Briefly, in this method, each chromosome is broken into overlapping fragments, which are inserted into a YAC vector. The YAC insert is screened using PCR or other methods to determine whether the cDNA whose position is to be determined (or genomic DNA obtained from this cDNA) is included. After finding an insert containing the cDNA (or genomic DNA from this cDNA), the insert was analyzed by PCR or other methods to derive the chromosomal or cDNA (or genomic DNA from this cDNA). It is possible to determine whether other sequences known to be in the region are also included in the insert. This process is called YA
Iteratively, for each insert of the C library, it is possible to determine the position of each cDNA (or genomic DNA obtained from this cDNA) relative to other cDNAs and relative to other known chromosomal markers. In this way, a high-resolution map of the distribution of a large number of unique markers along each of the chromosomes of the organism is obtained.

As described in Example 44 below, cDNA (or genomic DNA obtained from this cDNA) can also be used to identify genes associated with a particular phenotype, such as a genetic disease or drug response. .

Example 44 Identification of Genes Associated with Genetic Diseases or Drug Responses This example demonstrates that cDNA (or genomic DNA obtained from this cDNA) can be associated with particular phenotypic features. This is to explain a useful method. In this example, a specific cDNA (or genomic DNA obtained from this cDNA) is used as a test probe to associate the cDNA (or genomic DNA obtained from this cDNA) with a specific phenotypic characteristic.

The cDNA (or genomic DNA obtained from the cDNA) is mapped to a specific location on the human chromosome using techniques such as those described in Examples 40 and 41 or other techniques well known in the art. Mendelian Inheritance in Man (V. McKusick , Mend elian Inheritance in Man ( When you search for online accessible) through Johns Hopkins University Welch Medical Library, the human chromosome region containing the cDNA (or genomic DNA obtained from the cDNA), It turns out to be a highly gene-rich region, including known genes and diseases or phenotypes for which the genes have not been identified.
Thus, the gene corresponding to this cDNA (or genomic DNA obtained from this cDNA) is a direct candidate for each of these genetic diseases.

[0335] Cells isolated from patients suffering from or having a phenotype of these diseases are isolated and spread in culture. Genomic DNA, mRNA or cDNA obtained from a patient is screened using PCR primers obtained from cDNA (or genomic DNA obtained from this cDNA). Further analysis will reveal that the cDNA (or this cDNA
Genomic DNA from A) can be positively associated with a particular disease. Alternatively, when a sample is derived from an individual having a disease-related phenotype by PCR analysis, a fragment having a different length than when derived from a healthy individual may be obtained. It turns out that it may be the cause of the disease.

(VI. Use of cDNA (or Genomic DNA Obtained from This cDNA) in Vector Construction) Using the present cDNA (or genomic DNA obtained from this cDNA), a protein encoded by a gene inserted into a vector A secretion vector capable of directing the secretion of E. coli can also be constructed. The use of such a secretion vector reduces the number of background proteins for which the desired protein must be purified or concentrated, and facilitates the purification or concentration of the protein encoded by the gene inserted into the vector. There is. Examples of secretion vectors will be described later.

Example 45 (Construction of Secretory Vector) The secretory vector of the present invention contains a promoter capable of directing the expression of a gene in a target host cell, tissue or organism. Such promoters include the Rous sarcoma virus promoter, the SV40 promoter, the human cytomegalovirus promoter, and other promoters familiar to those of skill in the art.

The cDNA (such as one of the signal sequences of SEQ ID NOs: 24-73 listed in Table I above,
Alternatively, a signal sequence obtained from the genomic DNA obtained from the cDNA is operably linked to the promoter such that the mRNA transcribed from the promoter directs the translation of the signal peptide. The host cell, tissue or organism is a cDNA
Any cell, tissue, or organism that recognizes the signal peptide encoded by the signal sequence contained in (or the genomic DNA obtained from this cDNA) may be used. Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues or organisms, insect cells, tissues or organisms, or yeast.

The secretion vector contains a cloning site for inserting a gene encoding a protein to be secreted. This cloning site facilitates cloning of the inserted 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 mRNA transcribed from the promoter. The signal peptide directs extracellular secretion of the fusion protein.

The secretion vector may be DNA or RNA, may be integrated into the host chromosome or may be maintained in a stable manner as an extrachromosomal replicon in the host, or may be an artificial chromosome, or May temporarily exist in the host. Preferably, the secretory vector is maintained in multiple replicates in each host cell. As used herein, multiple replication means at least 2, 5, 10, 20, 25, 50 or more replications per cell. In some embodiments, multiple copies are maintained extrachromosomally. In other embodiments, the chromosomal sequence is amplified to obtain multiple copies.

Many nucleic acid backbones suitable for use as secretion vectors are well known to those of skill in the art and include, by way of example, retrovirus vectors, SV40 vectors, bovine papilloma virus vectors, yeast plasmids, yeast episomal plasmids, yeast artificial chromosomes , Human artificial chromosome, P element vector, baculovirus vector,
Alternatively, bacterial plasmids that can be temporarily introduced into a host can be used.

The secretion vector may include a polyA signal such that the polyA signal is located downstream of the gene to be inserted into the secretion vector.

After inserting the gene encoding the protein desired to be secreted into the secretion vector,
The secretion vector is introduced into host cells, tissues or organisms using calcium phosphate precipitation, DEAE dextran, electroporation, liposome-mediated transfection, virus particles, or as naked DNA. next,
The protein encoded by the inserted gene is purified or concentrated from the supernatant by conventional techniques such as ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, HPLC and the like. Or,
The secreted protein may be made sufficiently concentrated or pure in the growth medium or supernatant of the host and made available for its intended purpose without further concentration.

[0344] Alternatively, a signal sequence may be inserted into a vector designed for gene therapy.
In such vectors, the signal sequence is operably linked to the promoter such that the mRNA transcribed from the promoter encodes a signal peptide. The cloning site is located downstream of the signal sequence so that the gene encoding the protein desired to be secreted can be easily inserted into the vector and fused to the signal sequence. The vector is introduced into a suitable host cell. Extracellular secretion of the protein expressed from the promoter produces a therapeutic effect.

The cDNA or 5 ′ EST can be used to sequence a gene-expressible cDNA or a sequence located upstream of the 5 ′ EST, including promoter sequences, enhancer sequences, and other upstream sequences that affect the level of transcription or translation. It may be cloned. Once identified and cloned, these upstream control sequences can be used in expression vectors designed to direct the expression of the inserted gene in the desired spatial, temporal, developmental, or quantitative manner. . The following example describes a method for cloning cDNA or sequences upstream of the 5 'EST.

(Example 46) (Use of cDNA or its fragment in cloning of upstream sequence from genomic DNA) Using cDNA or a sequence derived from 5'EST, a promoter of a corresponding gene is isolated by a chromosome walking method. be able to. GenomeWalk available from Clontech
In the chromosome walking method using the er (registered trademark) kit, five complete genomic DNA samples are digested with different restriction enzymes having a 6-base recognition site and remaining blunt ends. After digestion, an oligonucleotide adapter is ligated to each end of the resulting genomic DNA fragment.

[0347] For each of the five genomic DNA libraries, a first PCR reaction is performed using the outer adapter primer and outer gene-specific primer provided in the kit, according to the manufacturer's instructions. For this gene-specific primer,
It should be chosen to be specific for cDNA or 5 'EST, and its melting point, length, location in the cDNA or 5' EST should be appropriate for use in the PCR reaction. For the first PCR reaction, 5 ng of genomic DNA, 5 μl of 10 × Tth reaction buffer, 0.2 mM of each dNTP, 0.2 μM each of the outer adapter primer and the outer gene-specific primer, and Mg
(OAc) ₂ utilizes 1.1 mM and 1 μl of Tth polymerase 50 × mixture in a total volume of 50 μl. The reaction cycle of the first PCR reaction was 1 minute at 94 ° C / 2 seconds at 94 ° C, and 3 minutes at 72 ° C (7 minutes).
Cycle) / 94 ° C for 2 seconds, 67 ° C for 3 minutes (32 cycles) / 67 ° C for 5 minutes.

The product of the first PCR reaction is diluted and, according to the manufacturer's instructions, a template for the second PCR reaction using a pair of nested primers located inside the amplicon obtained in the first PCR reaction Use as For example, reaction product 5 of the first PCR reaction mixture
The μl may be diluted 180 times. First P except for using nested primers
The reaction is performed in a 50 μl volume of the same composition as the CR reaction. The first nested primer is adapter specific and comes with a GenomeWalker® kit.
The second nested primer must be specific for the particular cDNA or 5 'EST cloning the promoter, and its melting point, length, location in the cDNA or 5' EST must be appropriate for use in the PCR reaction . The reaction parameters for the second PCR reaction were 94 ° C for 1 minute / 94 ° C for 2 seconds, 72 ° C for 3 minutes (6 cycles) / 94 ° C for 2 seconds, 67 ° C for 3 minutes (25 minutes). (Cycle) / 67 ° C for 5 minutes.

The products of the second PCR reaction are purified, cloned, and sequenced by standard methods. Alternatively, two or more human genomic DNA libraries can be constructed using two or more types of restriction enzymes. The digested genomic DNA is cloned into a vector that can be converted into single-stranded DNA, circular DNA or linear DNA. A biotinylated oligonucleotide containing at least 15 nucleotides from the cDNA sequence or 5 ′ EST sequence is converted to a single-stranded DN
Hybridize to A. A hybrid of a biotinylated oligonucleotide and a single-stranded DNA containing a cDNA or EST sequence is isolated as described in Example 17 above. Thereafter, the single-stranded DNA containing the cDNA sequence or EST sequence is released from the beads,
The DNA is converted to double-stranded DNA using a primer specific to the cDNA sequence or 5 ′ EST sequence or a primer corresponding to the sequence contained in the cloning vector. The resulting double-stranded DNA is transformed into bacteria. DNA containing the 5 'EST sequence or cDNA sequence is identified by colony PCR or colony hybridization.

After cloning and sequencing the upstream genomic sequence as described above,
By comparing the sequence upstream of the cDNA or 5 'EST with a database containing known transcription start sites, transcription factor binding sites or promoter sequences,
Potential promoters and transcription initiation sites within this upstream sequence may be identified.

In addition, as described below, a promoter of an upstream sequence can be identified using a promoter reporter vector.

Example 47 Identification of Promoter in Cloned Upstream Sequence The genomic sequence upstream of the cDNA or its fragment was obtained from pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer available from Clontech. Or pEGF
It is cloned into a suitable promoter reporter vector such as a P-1 promoter reporter vector. Briefly, each of these promoter reporter vectors contains a plurality of cloning sites located upstream of a reporter gene encoding an easily assayable protein such as secreted alkaline phosphatase, β-galactosidase or green fluorescent protein. cDNA or 5'EST
Insert the upstream sequence in both directions into the cloning site upstream of the reporter gene,
Introduce into a suitable host cell. The level of the reporter protein is assayed and compared to the level in a vector without an insert at the cloning site. The expression level in the vector containing the insert is higher than in the control vector, indicating the presence of the promoter in the insert. If necessary, the upstream sequence may be cloned into a vector containing the enhancer to increase the level of transcription from a weak promoter sequence. Expression levels are significantly higher than those observed in the vector without the insert, indicating the presence of the promoter sequence in the inserted upstream sequence.

For host cells suitable for promoter reporter vectors, see the cDNAs described above.
And EST expression patterns. For example, if expression pattern analysis reveals that mRNA corresponding to a particular cDNA or a fragment thereof is expressed in fibroblasts, a promoter reporter vector may be introduced into the human fibroblast cell line.

The promoter sequence in the upstream genomic DNA may be further defined by constructing a nested deletion in the upstream DNA using conventional techniques such as digestion with exonuclease III. The deletion fragment thus obtained is inserted into a promoter reporter vector, and it can be determined whether the deletion has been reduced or not, and whether the promoter activity has been eliminated. In this way, the boundaries of the promoter can be defined. If necessary, individual potential regulatory sites in the promoter may be identified by site-directed mutagenesis or linker scanning, and potential transcription factor binding sites in the promoter may be eliminated individually or in combination. . The effect of these mutants on the transcription level can be determined by inserting the mutant into the cloning site of the promoter reporter vector.

Example 48 (Cloning and Identification of Promoter) Using the method described in Example 47 above in 5′EST, sequences upstream of several genes were obtained. Primer pair GGG AAG ATG GAG ATA GTA TTG CCT G (SEQ ID NO: 15
) And CTG CCA TGT ACA TGA TAG AGA GAT TC (SEQ ID NO: 16) to obtain a promoter with in-house reference number P13H2 (SEQ ID NO: 17).

Primer pair GTA CCA GGGG ACT GTG ACC ATT GC (SEQ ID NO: 18) and CTG TGA C
Using CA TTG CTC CCA AGA GAG (SEQ ID NO: 19), in-house reference number P15B4 (SEQ ID NO:
20) promoter was obtained.

Primer pair CTG GGA TGG AAG GCA CGG TA (SEQ ID NO: 21) and GAG ACC ACA CA
Using G CTA GAC AA (SEQ ID NO: 22), the promoter of in-house reference number P29B6 (SEQ ID NO: 23) was obtained.

FIG. 4 is a diagram schematically illustrating isolated promoters and a method for assembling these promoters into corresponding 5 ′ tags. Computer program Ma
Using tInspector release 2.0, August 1996, upstream sequences were screened for the presence of motifs resembling transcription factor binding sites or known transcription start sites.

FIG. 5 is a diagram showing transcription factor binding sites present in each of these promoters. The matrix column shows the matrix name of the used MatInspector. The position column indicates the 5 'position of the promoter site. The sequence is counted from the transcription site determined by matching the genome sequence with the 5 ′ EST sequence. The “orientation” column indicates the DNA strand at which the site was found, and the + strand is the coding strand determined by matching the genomic sequence with the 5′EST sequence. The "Score" column shows the MatInspector score found for this site. "
In the "Length" column, the length of the site is shown as a nucleotide length. The “sequence” column shows the sequence of the found site.

[0360] Promoters and other regulatory sequences located upstream of the cDNA or 5'EST can be used to direct the expression of the inserted gene in the desired spatial, temporal, developmental or quantitative manner. An expression vector can be designed. Using the results of the expression analysis described in Example 10 above, a promoter capable of directing a desired spatial, temporal, developmental or quantitative pattern can be selected. For example, when a promoter that gives high levels of expression in muscle is desirable, a cDNA derived from mRNA expressed at high levels in muscle or a promoter sequence upstream from 5′EST determined by the method of Example 10 is used. May be used as an expression vector.

Preferably, the desired promoter is located near a plurality of restriction sites so that the promoter can drive expression of the inserted gene, to facilitate cloning of the desired insert downstream from the promoter. . The promoter may be inserted into a conventional nucleic acid backbone designed for extrachromosomal replication, integration into the host chromosome, or transient expression. Suitable scaffolds for existing expression vectors include retroviral scaffolds, scaffolds from eukaryotic episomes such as SV40 or bovine papilloma virus, scaffolds from bacterial episomes or artificial chromosomes.

To direct polyadenylation of mRNA transcribed from a gene inserted into the expression vector, the expression vector preferably also contains a polyA signal downstream of a plurality of restriction sites.

Following identification of the promoter sequence using the methods of Examples 46-48, the following examples
Proteins that interact with the promoter can be identified as described in 49.

Example 49 Identification of a Protein That Interacts with a Promoter Sequence, Upstream Regulatory Sequence or mRNA Homology to a well-known transcription factor binding site or conventional mutagenesis to a reporter plasmid containing a promoter sequence By the deletion analysis, a sequence in the promoter region that is easily bound to a transcription factor can be identified. For example, a deletion is created in a reporter plasmid containing a promoter sequence of interest operably linked to an assayable reporter gene. Reporter plasmids with various deletions in the promoter region are transfected into appropriate host cells and the effect of the deletion on expression levels is assayed. Site-directed mutagenesis, linker scanning analysis, or other techniques familiar to those skilled in the art can be used to further localize the transcription factor binding site in the region where the deletion reduces expression levels. Encode a protein that interacts with the promoter sequence using a one-hybrid system, such as that described in the manual accompanying the Matchmaker One-Hybrid System kit available from Clontech (Cat.No.K1603-1) Nucleic acids can be identified. Briefly, Matchmaker One-hybrid
The system is as follows. The target sequence to be identified for the binding protein is cloned upstream of a selectable reporter gene and integrated into the yeast genome. Preferably, multiple copies of the target sequence are inserted into the reporter plasmid in tandem.

A library consisting of a fusion between the cDNA to be evaluated for its ability to bind to a promoter and an activation domain of a yeast transcription factor such as GAL4 is transformed into a yeast strain containing an integrated reporter sequence. The yeast is inoculated into a selection medium and cells expressing a selectable marker linked to a promoter sequence are selected. Colonies growing on the selection medium contain genes encoding proteins that bind to the target sequence. Sequencing is performed to further characterize the insert within the gene encoding the fusion protein. Alternatively, the insert can be inserted into an expression vector or
It may be inserted into the ro transcription vector. The binding of the polypeptide encoded by the insert to the promoter DNA is confirmed by techniques familiar to those skilled in the art, such as gel shift analysis or DNAse protection analysis.

VII. Use of cDNA (or Genomic DNA Obtained From This cDNA) in Gene Therapy The present invention includes antisense and triple helix strategies, as described in Examples 50 and 51 below. The use of cDNA (or genomic DNA obtained from this cDNA) in gene therapy strategies that include: In the antisense method, a nucleic acid sequence complementary to the mRNA is hybridized intracellularly with the mRNA, thereby blocking the expression of the protein encoded by the mRNA. Antisense sequences prevent gene expression by a variety of mechanisms. For example, by the antisense sequence,
The function of the ribosome to translate mRNA may be inhibited. Antisense sequences also block mRNA transport from the nucleus to the cytoplasm and can be used for translation
In some cases, the amount of mRNA is limited. Another mechanism by which antisense sequences inhibit gene expression is interference with mRNA splicing. In yet another strategy, the antisense nucleic acid may be incorporated into a ribozyme that has the ability to specifically cleave the target mRNA.

Example 50 (Preparation and Use of Antisense Oligonucleotide) An antisense nucleic acid molecule used for gene therapy may be either a DNA sequence or an RNA sequence. These molecules may include a sequence that is complementary to the sequence of the cDNA (or genomic DNA obtained from the cDNA). Antisense nucleic acids are
It must be of a length and melting point such that an intracellular duplex is formed that is sufficiently stable to suppress mRNA expression in the duplex. For a strategy for designing antisense nucleic acids suitable for use in gene therapy, see Green et al., Ann. Rev. Bi.
ochem. 55: 569-597 (1986) and Izant and Weintraub, Cell 36: 1007-1015 (19
84).

In some strategies, the orientation of the coding region is reversed from that of the promoter so that the opposite strand is transcribed from that normally transcribed in the cell, thereby reducing the nucleotide sequence encoding the protein. Obtain an antisense molecule. The antisense molecule can be transcribed using an in vitro transcription system, such as one that uses T7 or SP6 polymerase to generate the transcript. Another method involves operably linking a DNA having an antisense sequence to a promoter with an expression vector.
It transcribes an antisense nucleic acid in vivo.

Alternatively, an oligonucleotide complementary to the strand normally transcribed in the cell may be used in vitro.
May be combined. Therefore, the antisense nucleic acid is complementary to the corresponding mRNA, and can be hybridized with the mRNA to obtain a duplex. In some embodiments, the antisense sequence may have a modified sugar phosphate backbone to increase stability and reduce susceptibility to RNase activity. Examples of suitable variants for use in antisense strategies include 2'O-methyl RNA oligonucleotides and protein-nucleic acid (PNA) oligonucleotides. Still other examples are described in Rossi et al., Pharmacol. Ther., 50 (2): 245-254, (1991).

[0370] Various types of antisense oligonucleotides complementary to the sequence of the cDNA (or genomic DNA obtained from the cDNA) can be utilized. In one preferred embodiment, the stable and semi-stable antisense oligonucleotides described in WO 94/23026 are used. In these molecules, the 3 ′ end or both the 3 ′ end and the 5 ′ end are linked to complementary base pairs by intramolecular hydrogen bonds. More preferably, these molecules are able to withstand exonuclease attack and exhibit greater stability than conventional antisense oligonucleotides.

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

In yet another preferred embodiment, the covalently crosslinked antisense oligonucleotides described in WO 96/31523 are used. These double-stranded or single-stranded oligonucleotides contain one or more covalent crosslinks between oligonucleotides or covalent crosslinks within oligonucleotides. Here, the chain consists of an amide bond between a primary amine group of one chain and a carboxyl group of the other chain or the same chain, and the primary amine group is located at the 2′-position of the monosaccharide ring of the chain nucleotide. And the carboxyl group is carried by an aliphatic spacer group substituted with another chain or a nucleotide or nucleotide analog of the same chain.

The antisense oligodeoxynucleotides and oligonucleotides disclosed in International Patent Application Publication No. WO 92/18522 may be used. These molecules have stability against degradation, have a transcriptional regulatory recognition sequence that binds to at least one regulatory protein, and are effective as decoys. These molecules have a “hairpin” structure, a “dumbbell” structure, a “modified dumbbell” structure, a “bridged” decoy structure, a “loop” structure, and the like.

In another preferred embodiment, use is made of a cyclic double-stranded oligonucleotide as described in EP-A-0 572 287 A2. These ligated oligonucleotide "dumbbells" contain binding sites for transcription factors that sequester the transcription factor and thereby repress gene expression under the control of these factors.

The closed antisense oligonucleotides disclosed in International Patent Application Publication No. WO 92/19732 are also included. Since these molecules do not contain free ends, they are more resistant to degradation by exonucleases than conventional oligonucleotides. These oligonucleotides may be multifunctional and interact in regions distant from the target mRNA.

[0376] In vitro expression analysis may be used to determine the appropriate concentration of antisense nucleic acid required to suppress gene expression. Antisense molecules may be introduced into cells by diffusion, injection, infection or transfection, etc. using procedures well known in the art. For example, a naked oligonucleotide as it is, an oligonucleotide encapsulated in a lipid, an oligonucleotide sequence encapsidated by a viral protein, or an oligonucleotide operably linked to a promoter contained in an expression vector. An antisense nucleic acid can be introduced. The expression vector may be any of a variety of expression vectors known in the art, such as a retroviral or viral vector, a vector capable of extrachromosomal replication or an integration vector.

The antisense molecules can be introduced into the cell sample at various concentrations, preferably at a concentration of 1 × 10 ⁻¹⁰ M to 1 × 10 ⁻⁴ M. After identifying the lowest concentration that allows for adequate control of gene expression, the optimal dose is converted to a dose suitable for in vivo use. For example, when the inhibitory concentration of 1 × 10 ⁻⁷ in culture is converted into a dose, it becomes about 0.6 mg / kg of body weight. After testing for toxicity of oligonucleotides in laboratory animals, oligonucleotide concentrations of around 100 mg / kg body weight or even higher can occur. It is also contemplated that cells harvested from vertebrates are removed, treated with antisense oligonucleotides, and re-introduced into vertebrates.

It is also contemplated that the antisense oligonucleotide sequence is incorporated into a ribozyme sequence so that the antisense can specifically bind to and cleave its target mRNA. See Rossi et al., Supra, for technical applications of ribozymes and antisense oligonucleotides.

In a preferred use of the present invention, the polypeptides encoded by the genes are first identified and techniques including, but not limited to, antibody-mediated tests such as RIA and ELISA, functional assays or radiolabels. To allow the efficiency of antisense inhibition to translation to be monitored.

The cDNA of the present invention (or genomic DNA obtained from the cDNA) may be used for gene therapy mainly based on triple helix formation in cells. Triple helix oligonucleotides are used to suppress transcription from the genome. When these oligonucleotides are associated with a particular gene, they are particularly useful for studying changes in cellular activity. The cDNA of the present invention (or genomic DNA obtained from this cDNA), or more preferably, a fragment of these sequences is used to express a gene in an individual suffering from a disease associated with the expression of a specific gene. It is possible to inhibit. Similarly, using fragments of cDNA (or genomic DNA obtained from this cDNA),
It is possible to study the effect of inhibiting the transcription of specific genes in cells. Heretofore, homopurine sequences have been considered most useful for triple helix strategies. However, homopyrimidine sequences can also suppress gene expression. Such homopyrimidine oligonucleotides bind to the major groove of the homopurine: homopyrimidine sequence. Thus, types of therapy of sequences obtained from the cDNA or sequences obtained from the gene corresponding to the cDNA are included within the scope of the present invention.

Example 51 (Preparation and Use of Triple Helix Probe) The sequence of cDNA (or genomic DNA obtained from this cDNA) was scanned to identify a 10- to 20-mer homopyrimidine or homopurine fragment, This can be used in a triple helix-based strategy to suppress gene expression. After identification of the candidate fragment of homopyrimidine or homopurine, the gene expression-suppressing efficiency is evaluated by introducing an oligonucleotide containing the candidate sequence into tissue culture cells, which usually express the target gene, in various amounts. This oligonucleotide can be prepared on an oligonucleotide synthesizer or purchased from companies that support the synthesis of proprietary oligonucleotides, such as GENSET, Paris, France.

The calcium phosphate precipitation method, DEAE dextran method, electroporation method,
Using various methods well known to those skilled in the art, including but not limited to liposome-mediated transfection methods or uptake in the native state,
Oligonucleotides can be introduced into cells.

[0393] Using techniques such as Northern blots, RNase protection assays, and PCR-based strategies, the reduced cell function or gene expression of the treated cells is monitored and the target gene in the cells treated with the oligonucleotide is monitored. Monitor transcript levels. The cell function to be monitored is predicted based on the homology between the target gene corresponding to the cDNA from which the oligonucleotide was derived and the known gene sequence related to the specific function. In addition, particularly when the cDNA is associated with a disease, the method described in Example 44 may be used to determine whether a cell from an individual suffering from a particular genetic disease has abnormal physiology. It is also possible to predict the function.

Next, the procedure of Example 50 was performed using the method described above and described in Example 50.
A dose may be calculated based on the results in vitro, and an oligonucleotide effective for suppressing gene expression in tissue culture cells may be introduced at this dose in vivo.

In some embodiments, α-anomers can be used in place of the natural (β) anomer of the oligonucleotide unit to increase the nuclease resistance of the oligonucleotide. Furthermore, an intercalating agent such as ethidium bromide can be added to the 3 'end of the α oligonucleotide to stabilize the triple helix. For methods of generating oligonucleotides suitable for triple helix formation, see Griffin et al. (Scie
nce 245: 967-971 (1989).

Example 52 Use of cDNA in Expressing Encoded Protein in Host Organism The cDNA of the present invention can be used to express an encoded protein in a host organism to produce beneficial effects. Can spawn. In such procedures, the encoded protein can be expressed transiently in the host organism or can be stably expressed in the host organism. The encoded protein may have any of the activities described above. The encoded protein may be a protein lacking in the host organism, or the encoded protein may enhance an existing level of the protein in the host organism.

A full-length cDNA encoding a signal peptide and a mature protein, or a cDNA encoding only a mature protein, is introduced into a host organism. At this time, the cDNA can be introduced into the host organism using various techniques well known to those skilled in the art. For example, if the encoded protein is expressed in the host organism, the naked DN
Inject the cDNA into the host organism as A, producing a beneficial effect.

Alternatively, the expression vector downstream of the promoter active in the host organism may contain cD
NA may be cloned. The expression vector may be any expression vector designed for gene therapy, including viral or retroviral vectors.

An expression vector can be introduced directly into a host organism so that the encoded protein is expressed in the host organism and has a beneficial effect. In another method, the expression vector can be introduced into cells in vitro. Cells containing the expression vector are then selected and introduced into a host organism, where the encoded protein is expressed to obtain a beneficial effect.

Example 53 Use of Signal Peptide in Transferring Protein to Cells The short hydrophobic core region (h) of the signal peptide encoded by the cDNA of the present invention or a fragment thereof was used as a carrier. The peptide or protein of interest, the so-called cargo, can be transferred to tissue culture cells (Lin et al., J.
Biol. Chem., 270: 14225-14258 (1995), Du et al., J. Peptide Res., 51: 23.
5-243 (1998), Rojas et al., Nature Biotech., 16: 370-375 (1998)).

When translocating cell permeable peptides of limited size (up to about 25 amino acids) through the cell membrane, chemical synthesis may be used to add the h-region to the C-terminus or N-terminus to the cargo peptide of interest. You may. Alternatively, if a longer peptide or protein is to be transferred to the cell, the cDNA sequence encoding the h region or a fragment thereof may be replaced with the 5 ′ end or 3 ′ of the DNA sequence encoding the cargo polypeptide.
'The nucleic acid can be genetically modified to link with the termini using techniques familiar to those skilled in the art. Such genetically modified nucleic acids are translated in vitro or in vivo by conventional techniques after transfection into appropriate cells to produce the resulting cell permeable polypeptide. Next, a suitable host cell is simply incubated with the cell permeable polypeptide and translocated through the membrane.

The method can be applied to the study of various intracellular functions and cellular processes. For example, it has been used to probe functionally related domains of intracellular proteins and to study protein-protein interactions involved in signal transduction pathways (Lin et al., Supra; Lin et al., Supra). , J. Biol. Chem
., 271: 5305-5308 (1996), Rojas et al., J. Biol. Chem., 271: 27456-27461.
(1996), Liu et al., Proc. Natl. Acad. Sci. USA, 93: 11819-11824 (1996).
Rojas et al., Bioch. Biophys. Res. Commun., 234: 675-680 (1997)).

[0393] Such techniques can be used in cell therapy to transfer proteins that produce a therapeutic effect. For example, cells isolated from a patient may be treated with the transferred therapeutic protein and then re-introduced into the host organism.

Alternatively, the h region of the signal peptide of the present invention can be used in combination with a nuclear localization signal to send the nucleic acid to the cell nucleus. Such oligonucleotides were used to inhibit the processing and maturation of target cell RNA as described in Examples 50 and 51, respectively.
Antisense oligonucleotides or oligonucleotides designed to form triple helices as described in, supra.

Example 54 (Computer Embodiment) As used herein, the expression “cDNA code of SEQ ID NOS: 24-73” refers to a nucleotide sequence of SEQ ID NOs: 24-73, a fragment of SEQ ID NOs: 24-73, SEQ ID NOs: 24-73
And nucleotide sequences complementary to the fragments of SEQ ID NOs: 24-73, and sequences complementary to all of the above sequences. As fragments, SEQ ID NOS: 24 to
73 consecutive at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75
, 100, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides. Preferably, the fragment is a new fragment.
Preferably, the fragments include the polynucleotides shown in Table III or at least 8, 10, 12, 15, 18 contiguous of the polynucleotides shown in Table III.
, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides. Homologous sequences and fragments of SEQ ID NOs: 24-73 are at least 99%, 98%, 97%, 96%, 95%
, 90%, 85%, 80% or 75% homologous. Homology can be determined using any of the computer programs and parameters described in Example 17, including those using BLAST2N with default parameters or using any of the parameters changed. . Homologous sequences also include RNA sequences in which uridine in the cDNA code of SEQ ID NOS: 24 to 73 has replaced thymidine. Homologous sequences can be obtained using any of the methods described herein or as a result of correcting for sequencing errors as described above. Preferably, the homologous sequence and fragments of SEQ ID NOs: 24 to 73 include the polynucleotide shown in Table III or at least 8, 10
, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 5
Fragments containing 00, 1000 or 2000 nucleotides are mentioned. SEQ ID NO: 24
~ 73 cDNA code in conventional one-letter format (Styer, Lubert. Biochemist
ry, 3rd ^edition.See inside the back cover of W. H Freeman & Co., New York.)
It will be understood that they can be expressed in any other form that records the homology of the nucleotides in the sequence.

As used herein, the expression “polypeptide code of SEQ ID NOs: 74 to 123” refers to the polypeptide sequence of SEQ ID NOs: 74 to 123 encoded by the cDNA of SEQ ID NOs: 24 to 73, Includes polypeptide sequences complementary to the polypeptide, or fragments of any of the above sequences. A homologous polypeptide sequence is at least 99%, 98%, 97% with one of the polypeptide sequences of SEQ ID NOs: 74-123.
, 96%, 95%, 90%, 85%, 80% or 75% homologous polypeptide sequences.
Using FASTA with default parameters, or using any of the computer programs and parameters described herein, including those using any of the parameters changed, it is possible to determine homology. it can.
Homologous sequences can be obtained using any of the methods described herein or as a result of correcting for sequencing errors as described above. The polypeptide fragment comprises at least 5, 8, 10, 1 contiguous of the polypeptide of SEQ ID NOs: 74-123.
Including 2, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 amino acids.
Preferably, this fragment is a novel fragment. Preferably, the fragment is a polypeptide encoded by the polynucleotide shown in Table III, or at least 5, 10, 15, 20, 25, 30, 35, or more continuous polypeptides encoded by the polynucleotide shown in Table III. , 40, 50, 75, 100 or 150 amino acids thereof. For the polypeptide codes of SEQ ID NOs: 74-123, conventional one-letter or three-letter formats (Starrier, Lubert.
^{try, 3 rd edition. W. H} Freeman & Co., see inside the back cover of New York
) Or any other form of polypeptide homology in sequence.

The cDNA code for SEQ ID NOs: 24-73 and the SEQ ID NOs: 74-123 polypeptide code are:
One of ordinary skill in the art will appreciate that it can be stored, recorded, and manipulated on any medium that can be read and accessed by a computer. As used herein, the terms "recorded" and "stored" refer to the process of storing information on a computer medium. One of ordinary skill in the art will employ any of the well-known methods for recording information on a computer-readable medium, and may include one or more of the cDNA codes of SEQ ID NOs: 24-73, or SEQ ID NOs: 74-73. Products having one or more of the 123 polypeptide codes can be readily generated. Another embodiment of the present invention provides that the cDNA code of SEQ ID NOs: 24-73 is at least 2, 5, 1
0, 15, 20, 25, 30, or 50 recorded computer readable media. Another embodiment of the present invention provides that the polypeptide code of SEQ ID NOs: 74-123 is at least 2,5.
, 10, 15, 20, 25, 30, or 50 recorded computer-readable media.

Computer readable media include magnetically readable media, optically readable media, electronically readable media, and magnetic / optical media. For example, computer readable media include hard disks,
Floppy disks, magnetic tapes, CD-ROMs, digital versatile disks (DVDs), random access memories (RAMs) or read only memories (ROMs) and other media well known to those skilled in the art can be used.

Embodiments of the present invention include systems, and in particular, computer systems that record and manipulate the sequence information described herein. One example of the computer system 100 is shown in block diagram form in FIG. As used herein, "computer system" refers to the nucleotide sequence of the cDNA code of SEQ ID NOs: 24-73, SEQ ID NO: 74.
Means hardware components, software components, and data storage components used in the analysis of the amino acid sequence of the polypeptide code of ~ 123. In one embodiment, computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, CA). Preferably, computer system 100 includes a processor for processing, accessing, and manipulating the sequence data. The processor 105 has Penti from Intel
In addition to the um III, any known type of central processing unit can be utilized, such as Sun, Motorola, Compaq or similar processors from IBM.

[0400] Computer system 100 includes a processor 105, one or more internal data storage components 110 for storing data, and one or more internal data storage components 110 for retrieving data stored in the data storage components. It is preferably a general-purpose system including a data search device. Those skilled in the art will readily appreciate that any currently available computer system is suitable.

In one particular embodiment, the computer system 100 has a processor 105 connected to a bus connected to main storage 115 (preferably implemented as RAM), a hard drive, and / or data. One or more internal data storage devices 110, such as other computer-readable media, are included. In some embodiments, the computer system 100 further comprises an internal data storage device 1
It includes one or more data retrieval devices 118 for reading data stored in 10.

[0402] The data retrieval device 118 may be, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or the like. In some embodiments, the internal data storage device 110 is a computer-readable removable medium with control logic and / or data recorded thereon, such as a floppy disk, compact disk, magnetic tape, and the like. The computer system 100 advantageously includes or is programmed with appropriate software to read control logic and / or data from the data storage component after insertion into the data retrieval device. There is.

The computer system 100 includes a display 120 used to display output and show to a computer user. Also, the computer system 100 may be connected to other computer systems 125a-c in a network or wide area network.
Note that it is also possible to provide central access to computer system 100 by linking to

The nucleotide sequence of the cDNA encoding of SEQ ID NOS: 24-73, or
Software (search tool, comparison tool, modeling tool, etc.) for accessing and processing the amino acid sequence of the polypeptide code may be resident in the main storage device 115 at the time of execution.

In some embodiments, the computer system 100 further comprises the cDNA code of SEQ ID NOS: 24-73 or SEQ ID NO: 74 above stored on a computer readable medium.
~ 123 polypeptide code with a reference nucleotide sequence or polypeptide sequence stored on a computer readable medium. A “sequence comparer” is implemented on the computer system 100 to combine a nucleotide sequence or polypeptide sequence with other nucleotide or polypeptide sequences and / or peptides, peptides, or sequences whose sequences or structures are stored in data storage means. By one or more programs that compare compounds, including but not limited to mimics and chemicals. For example, the sequence comparer may comprise the nucleotide sequence of the cDNA code of SEQ ID NOS: 24-73 or the amino acid sequence of the polypeptide code of SEQ ID NOs: 74-123 stored on a computer readable medium and the reference sequence stored on a computer readable medium. To identify motifs or structural motifs that are related to homology, biological function. The various sequence comparer programs described elsewhere in this patent specification are specifically intended for use in aspects of the present invention.

FIG. 7 shows a process 200 for comparing a new nucleotide or protein sequence to a sequence database to determine the level of homology between the new sequence and the sequence stored in the database. 4 is a flowchart illustrating an embodiment. The sequence database can be a private database stored in the computer system 100, or a GENBANK, PIR, or SW available via the Internet.
It may be a public database such as ISSPROT.

After starting in the start state 201, the process 200 moves to a state 202 in which a new array to be compared is stored in the memory of the computer system 100. As mentioned above, the memory may be any type of memory, including a RAM or an internal storage device.

Process 200 opens state database 204 for analysis and comparison 204.
Move on to Process 200 moves to state 206 where the first sequence stored in the database is read into memory on the computer. Subsequently, a comparison is performed in state 210 to determine whether the first array is identical to the second array. It is important to note that this step is not limited to an exact comparison of the new sequence with the first sequence in the database. Well-known methods for comparing two nucleotides or protein sequences, even when the sequences are not identical, are well known to those skilled in the art. For example, a gap can be introduced into one of the sequences to increase the level of homology between the two test sequences. The parameters that control whether gaps or other features are introduced into the sequence during the comparison are generally those entered by the user of the computer system.

After comparing the two sequences in state 210, a determination is made in decision state 210 as to whether the two sequences are identical. Of course, the term "identical" is not limited to sequences that are completely identical. Sequences within the homology parameters entered by the user are considered "identical" in process 200.

If a determination is made that the two sequences are identical, the process 200 moves to a state 214 where the sequence names obtained from the database are displayed and presented to the user. In this state,
The user is notified that the sequence whose name is displayed satisfies the input homology constraint. When the name of the stored array is displayed to the user, the process
200 moves to a decision state 218 where it is determined whether other sequences remain in the database. If no other arrays remain in the database, process 200 exits to status 2
End with 20. However, if there are still arrays in the database, the process 200 moves the pointer to the next array in the database and moves to a state 224 where it can be compared to the new array. In this way, the new sequences are aligned and compared to each sequence in the database.

If a determination is made in decision state 212 that the sequences were not homologous, the process 200
Immediately moves to decision state 218 to determine if other sequences in the database are available for comparison.

Accordingly, one aspect of the invention is a processor, a data storage device having stored thereon the nucleic acid code of SEQ ID NOS: 24-73 or the polypeptide code of SEQ ID NOS: 74-123, and the nucleic acid code of SEQ ID NOS: 24-73. Alternatively, a computer system comprising a data storage device in which a reference nucleotide sequence or polypeptide sequence to be compared with the polypeptide codes of SEQ ID NOs: 74 to 123 is stored in a searchable state, and a sequence comparer for performing the comparison. . The sequence comparer may indicate the level of homology between the sequences to be compared, or the structural motif of the nucleic acid code of SEQ ID NOS: 24 to 73 and the polypeptide code of SEQ ID NOS: 74 to 123 described above. It may be specified. The sequence comparer may also identify a structural motif in the sequence compared to these cDNA and polypeptide codes. In some embodiments, at least 2, 5, 10, 15, 20, 25, 3 of the cDNA code of SEQ ID NOs: 24-73 or the polypeptide code of SEQ ID NOs: 74-123.
An array of 0 or 50 can be stored on the data storage device.

[0413] Another embodiment of the present invention is a method for determining the level of homology between the nucleic acid codes of SEQ ID NOS: 24-73 and a reference nucleotide sequence, using a computer program for determining the level of homology. A method comprising reading a nucleic acid code and a reference nucleotide sequence, and using a computer program to determine homology between the nucleic acid code and the reference nucleotide sequence. This computer program may be any of a variety of computer programs for determining the level of homology, such as using BLAST2N with default parameters or using any of the parameters changed, such as This includes those specifically listed in the present specification. This method can be realized using the computer system described above. In addition, using a computer program, the cDNA code of SEQ ID NOS: 24 to 73 was read as 2, 5, 10, 15, 20, 25, 30, or 50, and the homology between the cDNA code and the reference nucleotide sequence was determined. May be implemented.

FIG. 8 is a flowchart illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. After starting in the start state 252, the process 250 moves to a state 254 in which the first array to be compared is stored in the memory. Next, the second array to be compared is stored in memory in state 256. Then process 25
A 0 transitions to a state 260 of reading the first character of the first array, followed by a state 262 of reading the first character of the second array. If the sequence is a nucleotide sequence, it will be understood that this letter is usually any one of A, T, C, G or U. If the sequence is a protein sequence, this would be a single letter amino acid code so that the first and second sequences can be easily compared.

Subsequently, in a determination state 264, it is determined whether or not these two characters are the same. If so, the process 250 moves to the state 268 where the next character of the first and second arrays is read. Subsequently, it is determined whether the next character is the same.
If so, the process 250 continues the loop until the two characters are different. If a determination is made that the next two characters are different, process 250 moves to state 274 to determine if there are any other characters to read in either array.

If there are no characters to read, the process 250 moves to a state 276 that indicates to the user the level of homology between the first and second sequences. The homology level is determined by calculating the ratio of letters between identical sequences to the total number of sequences in the first sequence. Thus, if all letters of the first 100 nucleotide sequence are aligned with all letters of the second sequence, the homology level will be 100%.

Alternatively, the computer program compares the nucleotide sequence of the cDNA encoding the invention to a reference nucleotide sequence and, at one or more positions, SEQ ID NO: 24
~ 73 may be a computer program for determining whether the nucleic acid code differs from the reference nucleotide sequence. Optionally, such a program records the length and identity of the inserted, deleted or substituted nucleotides relative to the sequence of the reference polynucleotide or the sequence of the nucleic acid code of SEQ ID NOs: 24-73. In one embodiment, the computer program is a program that determines whether the nucleotide sequence of the cDNA encoding SEQ ID NOs: 24-73 comprises a biallelic marker or single nucleotide polymorphism (SNP) relative to a reference nucleotide sequence. There may be. These single nucleotide polymorphisms may have single base substitutions, insertions, or deletions, while biallelic markers have about 1-10 contiguous base substitutions, insertions or deletions. May be included.

[0418] Another embodiment of the present invention is a method for determining the level of homology between a polypeptide code of SEQ ID NOs: 74-123 and a reference polypeptide sequence, comprising using a computer program to determine the level of homology. Reading the polypeptide code of SEQ ID NOs: 74-123 and the reference polypeptide sequence, and determining the homology between the polypeptide code and the reference polypeptide sequence using a computer program. is there.

Accordingly, another embodiment of the present invention is a method for determining whether the nucleic acid code of SEQ ID NOs: 24-73 differs from a reference nucleotide sequence by one or more nucleotides, Reading the nucleic acid code and the reference nucleotide sequence using a computer program that identifies differences between the nucleic acid sequences, and using the computer program to identify differences between the nucleic acid code and the reference nucleotide sequence It is. In some embodiments, the computer program is a program for identifying single nucleotide polymorphisms. This method
This can be realized by the computer system described above and the method shown in FIG.
In addition, using a computer program, at least the cDNA code of SEQ ID NOS: 24-73.
Performing the above method by reading 2, 5, 10, 15, 20, 25, 30, or 50 and the reference nucleotide sequence and using a computer program to identify differences between the cDNA code and the reference nucleotide sequence Is also good.

In another embodiment, the computer-based system comprises the cDNA of SEQ ID NOs: 24-73.
It may further comprise an identifier for identifying characteristics of the nucleotide sequence of the code or the amino acid sequence of the polypeptide code of SEQ ID NOs: 74 to 123.

“Identifiers” are one or more programs that identify particular features of the nucleotide sequence of the cDNA encoding SEQ ID NOs: 24-73 or the amino acid sequences of the polypeptide encoding SEQ ID NOs: 74-123, described above. Means In one embodiment, the identifier may include a program that identifies the open reading frame of the cDNA code of SEQ ID NOs: 24-73.

FIG. 9 shows an identifier process 300 for detecting the presence of features in an array.
4 is a flowchart showing an embodiment of the present invention. The process 300 begins at a start state 302 and transitions to a state 304 where the first sequence identified for a feature is stored in the memory 115 of the computer system 100. Thereafter, the process 300 moves to the state 306 where the sequence feature database is opened. Such a database includes, in addition to the name of each feature, a list of attributes for that feature. For example, the feature name could be "Start Codon" and the attribute would be "ATG". Another example is a feature name “TAATAA Box” and a feature attribute “TAATAA”. One example of such a database is created by the University of Wisconsin Genetic Computer Group (www.gcg.com).

After the feature database has been opened in state 306, process 300 moves to state 308, where the first feature is read from the database. Thereafter, in state 310, the attribute of the first feature is compared with the first array. Next, in decision state 316, it is determined whether the attribute of the feature was in the first array. If the attribute is found, the process 300 moves to a state 318 that displays the name of the found feature and presents it to the user.

Thereafter, the process 300 moves to the decision state 320 where it is determined whether the movement feature is present in the database. If there are no more features, the process 300 ends at an end state 324. However, if other features exist in the database,
The process 300 reads the next array of features in state 326 and returns to state 310 to compare the next feature attribute to the first array.

If the attribute of the feature is not found in the first array in decision state 316, the process 30
Note that 0 goes directly to decision state 320 to determine if the feature is still in the database.

In another embodiment, the identifier may comprise a molecular modeling program that determines the three-dimensional structure of the polypeptide code of SEQ ID NOs: 74-123. In some embodiments, molecular modeling programs identify target sequences that are closest to a profile that represents the structural environment of residues of a known three-dimensional protein structure (e.g., published July 25, 1995, Eisenberg et al. U.S. Patent No. 5,43
See 6,850). In another approach, the well-known three-dimensional structure of a particular family of proteins is superimposed to define structurally conserved regions in that family. This protein modeling technique also uses a well-known three-dimensional structure of a homologous protein, and obtains a structure close to the structure of the polypeptide code of SEQ ID NOS: 74 to 123. (For example, 1996
See U.S. Patent No. 5,557,535 to Srinivasan, et al., Issued September 18, 1998).
Conventional homology modeling techniques were routinely used to construct protease and antibody models (Sowdhamini et al., Protein Engineering 10: 207, 215 (1997)). If the sequence identity between the protein of interest and the template protein is low, a three-dimensional protein model can be developed using comparative methods. In some cases, these proteins fold into similar three-dimensional structures, despite the very low sequence homology of the proteins. For example, the three-dimensional structure of many helical cytokines folds into a similar three-dimensional topology, albeit with low sequence homology.

With the recent development of the Threading method, similar folding patterns can be identified in a variety of situations when the structural association between the target and the template cannot be detected at the sequence level. In the hybrid method that recognizes folds using Multiple Sequence Threading (MST), the distance geometry program DRAGON
Infer the structural equivalence from the output of ading, construct a low-resolution model, and construct an all-atom representation using a molecular modeling package such as QUANTA.

According to this three-step method, using a novel fold recognition algorithm MST, which can simultaneously perform the three-dimensional structure of a plurality of aligned arrangements in one or more 3D structures,
First, a candidate template is identified. In the second step, the structure equivalent obtained from the MST output is converted into an inter-residue distance suppression value, which is supplied to the distance geometry program DRAGON together with the auxiliary information obtained in the secondary structure prediction. This program combines suppression values in a fair way and quickly generates a large number of low-resolution model conformations. No.
In step 3, these low-resolution model conformations are converted to all-atom models, and energy minimization is performed using the molecular modeling package QUANTA (Aszod
i et al., Proteins: Structure, Function, and Genetics, Supplement 1: 38-42
(1997).

Using the results of the molecular modeling analysis in a rational drug design procedure, SEQ ID NO: 74
Agents that modulate the activity of the ~ 123 polypeptide code can be identified.

Accordingly, another aspect of the present invention is a method for identifying a feature of the cDNA code of SEQ ID NOs: 24-73 or the polypeptide code of SEQ ID NOs: 74-123, comprising a computer program for identifying features in the code. Reading the nucleic acid or polypeptide code, and identifying features in the nucleic acid or polypeptide code with a computer program. In one embodiment,
The computer program includes a computer program for identifying a reading frame.
In yet other embodiments, the computer program identifies structural motifs of the polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. Using a computer program, read a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cDNA code of SEQ ID NOs: 24-73 or the polypeptide code of SEQ ID NOs: 74-123, The above methods may be performed by using a program to identify features of the cDNA or polypeptide code.

[0431] The cDNA codes of SEQ ID NOS: 24-73 or the polypeptide codes of SEQ ID NOs: 74-123 can be stored and manipulated in various formats in various data processor programs. For example, the cDNA code of SEQ ID NOS: 24-73 or the polypeptide code of SEQ ID NOS: 74-123 can be stored in text format as a file for a word processor such as Microsoft WORD or WORDPERFECT. Alternatively, DB2
, SYBASE, ORACLE etc. with various database programs well known to those skilled in the art
It may be stored as an ASCII file. Also, a number of computer programs and sources, such as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences used for comparison with the cDNA code of SEQ ID NOs: 24-73 or the polypeptide codes of SEQ ID NOs: 74-123. Database available. Hereinafter, the present invention is not limited, but is listed as a reference for programs and databases useful for use in combination with the cDNA codes of SEQ ID NOS: 24 to 73 or the polypeptide codes of SEQ ID NOS: 74 to 123. Available programs and databases include MacPattern (EMBL), DiscoveryBase (Molecular Applic
ations Group), GeneMine (Molecular Applications Group), Look (Molecular
Applications Group), MacLook (Molecular Applications Group), BLAST and B
LAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403
(1990)), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444).
(1988)), FASTDB (Brutlag et al. Comp.App.Biosci. 6: 237-245, 1990), Ca
talyst (Molecular Simulations Inc.), Catalyst / SHAPE (Molecular Simulatio
ns Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecu
lar Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discove
r (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Fel
ix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), Q
uanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations I
nc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations
Inc.), Quanta / Protein Design (Molecular Simulations Inc.), WebLab (Molec
ular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations
Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular S
imulations Inc.), EMBL / Swissprotein Database, MDL Available Chemicals Di
rectory Database, MDL Drug Data Report Database, Comprehensive Medicinal
Chemistry Database, Derwents's World Drug Index Database, BioByteMaster
Examples include, but are not limited to, the File Database, Genbank Database, and Genseqn Database. From the disclosure herein, one of ordinary skill in the art will recognize many other programs and databases.

Motif detectable by the above programs include leucine zipper, helical turn helix motif, glycosylation site, ubiquitin binding site, sequences encoding α-helix and β-sheet, signals indicating secretion of the encoded protein. Sequences involved in the regulation of transcription, such as a peptide-encoding signal sequence, homeobox, acidic extension, enzyme activation site, substrate binding site and enzyme cleavage site, may be mentioned.

Example 55 (Method for Generating Nucleic Acid) The present invention also includes a method for producing cDNAs of SEQ ID NOS: 24 to 73, genomic DNA obtained from such cDNAs, or fragments thereof. The method involves sequentially joining nucleotides to produce a nucleic acid comprising the sequence described above. Various nucleic acid synthesis methods are well known to those skilled in the art.

In many of these methods, the synthesis is performed on a solid support. This includes the 3 'phosphoramidite method in which the 3' terminal base of the desired oligonucleotide is immobilized on an insoluble carrier. The nucleotide base to be added is blocked with a 5 'hydroxyl and activated with a 3' hydroxyl to couple with the immobilized nucleotide base. Unblocking the new immobilized nucleotide compound and repeating this cycle produces the desired polynucleotide. Alternatively, U.S. Pat.No. 5,049,65
Polynucleotides may be prepared as described in No. 6. In some embodiments, several polynucleotides prepared as described above are ligated to produce a longer polynucleotide having the desired sequence.

(Example 56) (Method for Producing Polypeptide) The present invention also relates to a method for producing a polynucleotide encoded by the cDNA of SEQ ID NOS: 24 to 73, a genomic DNA obtained from such a polynucleotide, or a fragment thereof. , SEQ ID NOs: 74 to 123 or methods for producing fragments thereof. These methods involve sequentially joining amino acids to produce a nuclear polypeptide having the sequence described above. In some embodiments, the polypeptides produced by these methods are less than 150 amino acids in length. In another embodiment, the polypeptides produced by these methods are 120 amino acids or less.

[0436] Methods for producing a variety of polypeptides are well known to those of skill in the art, and include, for example, methods of linking a carboxyl-terminal amino acid with polyvinylbenzene or other suitable resin. The amino acid to be added contains a blocking group for the amino moiety and a side-chain reactive group so that only the carboxyl moiety can react. The carboxyl group can be activated with a carbodiimide or other activator to bind to the immobilized amino acid. After removal of the blocking group, the cycle is repeated to produce a polypeptide having the desired sequence. Alternatively, the method described in US Pat. No. 5,049,656 may be used.

Example 57 Functional Analysis of Predicted Protein Sequence Following double sequencing, contigs were assembled for each of the cDNAs of the invention and compared to known sequences, respectively, available at the time of filing. These sequences are from the following databases: Genbank (Release 108), EMBL (Release 58 and Daily Update), Genseq (Release 35.3), Swissprot (Release 37), Ge
nbank (Release 108 and daily updates until October 15, 1998), Genseq
(Release 32), PIR (Release 53) and Swissprot (Release 35). In some cases, new reading frames other than those previously selected were selected based on homology with other proteins. For example, the new reading frame of SEQ ID NO: 27 no longer contains any signal peptide.

Next, predicted proteins of the invention that correspond to known proteins were further classified into three categories according to the level of homology.

The first category includes the total length of the protein with at least 8
Proteins of the invention that are 0% identical are included. These proteins are apparently close homologs, most likely having the same or very similar function to the matched protein.

The second category includes proteins of the present invention that have further homology (35-80% of the total protein), and therefore have a function similar to that of the matched protein of the present invention. It is indicated that there is a possibility of having

The third category includes proteins that exhibit homology to domains of known proteins, so that matched proteins and proteins of the invention share similar features, such as functional domains. It turns out that there are cases.

Note that in numbering amino acids in the protein sequences described below in FIGS. 10-13 and Table V, the first encountered methionine is indicated by amino acid number 1. In the attached sequence listing, the first amino acid of the mature protein resulting from cleavage of the signal peptide is indicated by amino acid No. 1 and the first amino acid of the signal peptide is indicated by an appropriate minus number in accordance with the rules for the sequence listing.

Also, all amino acid sequences (SEQ ID NOs: 74-123) were scanned for known protein signatures and motifs. This exploration was performed against the Prosite 15.0 database using the following GCG packaged Proscan software.

The polypeptide encoded by the cDNA was screened for the presence or absence of known structural or functional motifs, or for the presence of signatures, small amino acid sequences that are well conserved among members of the protein family. The storage area is the PROSITE databank, especially http://expasy.hcuge.ch/sprot/prosite.html
Used to derive the consensus patterns or matrices contained in the prosite.dat file at prosite_convert program and prosite_s
cD using the can program (http://ulrec3.unil.ch/ftpserveur/prosite_scan)
I found a sign on NA.

For each pattern obtained using the prosite_convert program contained in the prosite.dat file, the frequency of irrelevant hits for the population of human secreted proteins contained in the SWISSPROT databank was evaluated to determine the new protein The detection accuracy for the sequence was tested. The ratio of the number of hits for the shuffled protein (window size 20 amino acids) to the number of hits for the native (unshuffled) protein was used as an index. Searches using prosite_scan skipped patterns where the above ratio exceeded 20% (1 hit for shuffled proteins, 5 hits for unshuffled proteins). A program used to shuffle protein sequences (db_shuffled) and a program used to determine statistical values for each pattern included in the protein data bank (prosite_statistics) are ftp sites http://ulrec3.unil.ch/ftpserveur/ pr
Available at osite_scan.

A) Protein highly relevant to known proteins (protein of SEQ ID NO: 76 (in-house identification number 105-095-1-0-D10-FLC)) As is clear from the alignment in FIG. The protein of SEQ ID NO: 76 encoded by the 26 cDNA is a human parotid secretory protein HPSP (Genseq accession number W60).
682 and SEQ ID NO: 124). Antagonists of this protein may be used to treat cancers and autoimmune diseases, especially of secretory or gastrointestinal tissues.

Taken together, these data suggest that the protein of SEQ ID NO: 76 or a portion thereof may play a role in cell differentiation and / or proliferation. Therefore, the diagnosis of some dysfunctions, including but not limited to cancer and autoimmune diseases, of this protein or portions thereof
It may be helpful for treatment.

(Protein of SEQ ID NO: 93 (Company No. 117-007-2-0-C4-FLC)) As is clear from the alignment in FIG. 11, the protein of SEQ ID NO: 93 encoded by the cDNA of SEQ ID NO: 43 Exhibits homology to human proteins that appear to be transmembrane (Genseq accession number W88491 and SEQ ID NO: 125). This protein is
1 shows homology to human and porcine α-2-HS glycoprotein precursor (fetuin) belonging to the cystatin superfamily. The protein having a length of 382 amino acids of SEQ ID NO: 93 is close to fetuin in size, and has 12 cysteines (positions 36, 93, 104, 117, 137, 151, 154, and 154 shown in bold in FIG. 216th, 224th, 237th, 25th
(Positions 4 and 368) are conserved, showing a cystatin-like domain with a conserved region around the second cysteine (positions 89-96 underlined in FIG. 11), but there is a common PROSITE signature for fetuin do not do. The protein of the present invention also has a potential active site, QxVxG (positions 198 to 202 shown in italics in FIG. 11). The cystatin superfamily includes evolutionarily related proteins with different functions, such as cysteine protease inhibitors, stepins, fetuins and kininogens (reviewed by Brown and Dziegielewska, Prot. Science, 6 : 5-12 (1997)). See).

Taken together, these data suggest that the protein of SEQ ID NO: 93 or a portion thereof may play some role in cell proteolysis, possibly as a protease inhibitor. Thus, this protein or a portion thereof may include some but not limited to cancer, especially tumor progression and metastasis, chronic inflammation, neurodegenerative disorders such as Alzheimer's disease, diabetes, hypertension and immune disorders. May help diagnose and / or treat dysfunction of
Modulating the clotting properties may also help treat patients with cardiovascular disorders.

(Protein of SEQ ID NO: 75 (Company No. 105-031-3-0-D6-FLC)) As is clear from the alignment in FIG. 12, the sequence encoded by the cDNA of SEQ ID NO: 25
The protein in column 75 is the predicted mouse sialyltransferase protein (TREMBL recipient).
Accession number O88725 and SEQ ID NO: 126). Sialyltransferase
Of serum glycoproteins in cell-cell communication, cell-matrix interaction, and circulation
Biosynthesis of sialosides important for various biological processes such as maintenance
Is a type II transmembrane protein involved in growth (Sjoberg et al., J. Biol. Chem. 271 : 7450-7459 (1996), Tsuji, J. Biochem.1201-13 (1996)). SEQ ID NO: 75
Proteins are the two conserved motifs of the sialyltransferase protein family.
In other words, it is involved in the recognition of sugar nucleotide donors common to all sialyltransferases
The sialyl motif L located at the center (positions 73 to 120 shown in bold in FIG.
) And the sialyl mochi located at the C-terminus of the protein
12 (positions 211 to 233 shown in italics in FIG. 12). Further, 302 of SEQ ID NO: 75
Amino acid-long proteins are one of the members of the sialyltransferase family
The size is close. In addition, the protein of the present invention has a predicted transmembrane structure. Special
In addition, the software TopPred II (Claros and von Heijne, CABIOS applic. Ten : 685-686 (1994)), two potential transmembrane segments (Fig.
7 to 27 and 206 to 226 underlined).

Taken together, these data suggest that the protein of SEQ ID NO: 75, or a portion thereof, may play a role in sialyl-glycoconjugate biosynthesis, possibly as a sialyltransferase. . Thus, this protein or a portion thereof may be useful in diagnosing and / or treating some dysfunctions, including but not limited to cancer, cystic fibrosis and hypothyroidism .

(Protein of SEQ ID NO: 104 (Internal Reference No. 108-008-5-O-C5-FL)) The protein of SEQ ID NO: 104 encoded by the cDNA of SEQ ID NO: 54 is a murine recomb
It exhibits extensive homology to the entire length of ination activating gene 1 inducing protein (Genbank accession number X96618 and SEQ ID NO: 177). As is evident from the alignment in FIG. 13, the 6th, 7th, and 10th positions
13th, 17th, 25th, 34th to 35th, 42nd, 51st, 56th, 62nd, 68th, 71st, 74th,
78, 91, 93, 95-96, 106, 121-122, 151-152, 159, 162-1
63rd, 170-171, 176-177, 188, 190, 192, 196, 199, 202-20
The amino acid residues except for positions 3, 206, 210, 215 and 217 are the same amino acid residues. This protein, which contains four potential transmembrane segments, has been implicated in the induction of recombination of the V (D) J segment in T cells (Muraguchi et al, Leuk Lymphoma, 30: 73-85
(1998)).

Taken together, these data suggest that the protein of SEQ ID NO: 104 may play some role in the formation of the lymphocyte repertoire. Thus, the protein or a portion thereof may be useful in diagnosing and / or treating some dysfunctions, including but not limited to cancer, immune disorders and infectious disorders. It may also be useful for modulating the infectious or immune response to an infectious agent such as HIV.

B) A protein having a low association with a protein having a known function (protein of SEQ ID NO: 87 (company identification number: 116-073-4-0-C8-FLC)) Sequence encoded by cDNA of SEQ ID NO: 37 Some of the proteins of number 87 show homology to the full length of a widely (fish, bird and mammal) conserved family of lysozyme C precursors. This protein also displays the α-lactalbumin / lysozyme C PROSITE signature that is characteristic of the glysosyl hydrolase or family 22 (positions 162 to 180, see Table V). Lysozyme C is a lytic defense enzyme and α-lactalbumin is a regulatory subunit of lactose synthase. Lysozyme C and α-lactalbumin appear to be evolutionarily related (Q
Asba and Kumar, Crit. Rev. Biochem. Mol. Biol. 32 : 255-306 (1997)).

Taken together, these data indicate that the protein of SEQ ID NO: 87, or a portion thereof,
In particular, it is thought that the domain corresponding to the lysozyme C precursor described above plays a role in the metabolism of glycoprotein and / or peptidoglycan, possibly as a glycosyl hydrolase. Thus, this protein or a portion thereof may be useful in diagnosing and / or treating some dysfunctions, including but not limited to cancer and amyloidosis. It may also be useful for modulating the defense response to infectious agents such as bacteria.

(Protein of SEQ ID NO: 86 (Company No. 116-054-3-0-G12-FLC)) The protein of SEQ ID NO: 86, which is encoded by the cDNA of SEQ ID NO: 36 found in the liver, is obtained from bovine, mouse, MLRQ subunit of human species NADH-quinone oxidoreductase (complex I) (Genbank accession number X64897, U59509 and EMBL accession number U94586, respectively)
Shows homology to In addition, the 83 amino acid long protein of SEQ ID NO: 86 is close to the size of the known MLRQ subunit. Complex I is part of the mitochondrial electron transport chain and is involved in NADH dehydrogenation and transport of electrons to coenzyme Q. It is also thought to play a role in regulating apoptosis and necrosis. Mitochondrial cytopathy due to complex I deficiency
y) often occurs, causing the brain (mental retardation, convulsions, movement disorders), the heart (
Energy demanding tissues such as cardiomyopathy, conduction disorders), kidneys (Fanconi syndrome), skeletal muscles (exercise intolerance, weakness, hypotension) and / or eyes (ophthalmoplegia, ptosis, cataracts and retinopathy) Is affected. For a review on Complex I, see Smeit
See ink et al., Hum. Mol. Gent., 7 : 1573-1579 (1998).

Taken together, these data suggest that the protein of SEQ ID NO: 86 is an MLRQ-like protein of NADH-quinone oxidoreductase. Therefore, this protein or a part thereof, brain disorders (mental retardation, convulsions, movement disorders), heart disorders (cardiomyopathy, conduction disorders), renal disorders (Fanconi syndrome), skeletal muscle disorders (exercise intolerance, muscle weakness, May be useful in the diagnosis and / or treatment of some dysfunctions, including but not limited to (hypotension) and / or eye disorders (ophthalmoplegia, ptosis, cataracts and retinopathy) .

(Protein of SEQ ID NO: 91 (Company No. 117-005-4-0-E5-FLC)) The protein of SEQ ID NO: 91, which is encoded by the cDNA of SEQ ID NO: 41 found in the liver, is located on the inner mitochondrial membrane. 1 shows homology to the domains of the family of mitochondrial substrate carrier proteins found. These carrier proteins are evolutionarily related and are composed of three tandem repeats of a domain of about 100 residues, each domain containing two transmembrane domains. The 308 amino acid long protein of SEQ ID NO: 91 is close in size to one of the mitochondrial carrier proteins and shows three times the PROSITE signature characteristic of this protein family (positions 19-28, 115-124 And positions 237-246, see Table V). In addition, the protein of SEQ ID NO: 91 contains six potential transmembrane segments of 20 amino acids, and the TopPred II
Software (Claros and von Heijne, CABIOS applic.Notes, 10 : 685-
686 (1994)), four of the six (positions 1-21, 54-74, 135-155 and 217-237) were predicted with high confidence and two (96-116 Rank and 191-211 rank)
Is predicted with low confidence.

Taken together, these data suggest that the protein of SEQ ID NO: 91 or a portion thereof may play some role in energy transfer, possibly as a carrier protein for mitochondrial substrates. Thus, this protein or a portion thereof may be useful in diagnosing and / or treating some dysfunctions, including but not limited to mitochondrial cytopathy and obesity.

In particular, the protein of SEQ ID NO: 91, encoded by the cDNA of SEQ ID NO: 41, exhibits homology to apolipoprotein A-IV related proteins. Lipoproteins, such as HDL and LDL, contain the characteristic apolipoproteins necessary to target them to specific tissues and activate the enzymes required for transport of the lipid fraction of lipoproteins (including cholesterol). Apolipoprotein A-IV-related protein (AA4RP), a member of the apolipoprotein family, is 52% similar (29% identical) to apolipoprotein A-IV (ApoA-IV) and therefore may have similar functions. Many. ApoA-IV has been found to be associated with chylomicron and HDL fractions of blood. Its specific function is currently unknown, but is expressed in the liver and intestine and is regulated by a high-fat diet (up-regulation) and leptin (down-regulation). ApoA-IV levels have been correlated with glycemic control in young patients with type I diabetes (IDDM). Overexpression of the protein protects ApoE knockout mice from atherosclerosis. Finally, ApoAIV contributes to inter-individual variability in blood cholesterol response to changes in dietary fat / cholesterol intake.

Since AA4RP circulates with the blood, therapeutic actions can be facilitated by administering a synthetic peptide analog that mimics the activity or function of the synthetic peptide as a competitive antagonist (overtly inactive) directly into the blood. Can be applied. Because this protein is involved in the transport of fat and cholesterol in the body, and changes blood cholesterol in response to dietary changes, targeting this protein could result in cholesterol reduction and anti-atherosclerosis. It is useful in treatment and helps control diabetes and obesity.

(Protein of SEQ ID NO: 74 (Company No. 105-016-3-0-E3-FLC)) The 325 amino acid long protein of SEQ ID NO: 74 encoded by the cDNA of SEQ ID NO: 24 And control of differentiation and possibly direct interactions,
That is, E2F-1 (Galiana et al., Proc. Natl.Acad.Sci. USA 92: 1560-1564 (1
(955), Dupont et al., J. Neurosci. Res. 51: 257-267 (1998)), and are thought to play an important role in cell survival in the absence of cell cycle regulators. , 332 amino acid long mouse neural proliferative differentiation and regulatory 1 protein or NPDC
-1 (Genbank accession number X67209) shows homology to full length.

Taken together, these data suggest that the protein of SEQ ID NO: 74, or a portion thereof, may play a role in cell growth and differentiation. Thus, the protein or a portion thereof may be useful in diagnosing and / or treating some dysfunctions, including but not limited to cancer and neurodegenerative disorders.

(Protein of SEQ ID NO: 111 (Internal Reference No. 108-013-5-O-H9-FL)) The protein of SEQ ID NO: 111 encoded by the extended cDNA of SEQ ID NO: 61 is a eukaryote (
4 shows homology to a family of lysophospholipases conserved in yeast, rabbits, rodents and humans). Also, some members of this family (Rat: Genban
k accession number U97146, rabbit: Genbank accession number U97147), some exhibit calcium-independent phospholipase A2 activity (Portilla et al, J. Am. Soc.
: 1178-1186 (1998)). All members of this family are also found in the proteins of the invention (positions 54-58) Carboxylesterase active site consensus GXSX
It has a G motif. In addition, this protein is a software called TopPred II (
As predicted by Claros and von Heijne, CABIOS applic. Notes, 10: 685-686 (1994)), it may be a membrane protein containing one transmembrane domain.

Taken together, these data suggest that the protein of SEQ ID NO: 111 may play a role in fatty acid metabolism, possibly as a phospholipase. Thus, this protein or a portion thereof may be useful in the diagnosis and / or treatment of several dysfunctions, including but not limited to cancer, neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, diabetes There is. It may also help modulate the inflammatory response to infectious agents and / or suppress graft rejection.

(Protein of SEQ ID NO: 101 (In-house accession number: 108-005-5-O-F9-FL)) The protein of SEQ ID NO: 71 encoded by the extended cDNA of SEQ ID NO: 51 was obtained from Drosophila
Shows homology to rhythmically expressed gene 2 protein (Genbank accession number U65492). Expression of mRNA encoding a matching protein depends on day-night cycle, feeding conditions and expression per gene that is essential for the function of the endogenous circadian pacemaker (Van Gelder et al., Curr. Biol. ., 5: 1424-1436 (1995)
).

Taken together, these data suggest that the protein of SEQ ID NO: 101 may play a role in circadian control. Thus, this protein or a portion thereof may be useful in the diagnosis and / or diagnosis of several dysfunctions, including but not limited to insomnia, depression, stress and other disorders of the circadian rhythm.
Or may be useful for treatment. Such proteins may also be useful in modulating the physiological response to night work and jet lag.

C) A protein homologous to the domain of a protein having a known function (protein of SEQ ID NO: 94 (in-house identification number 121-004-3-0-F6-FLC)) cDNA of SEQ ID NO: 44 found in the brain The encoded protein of SEQ ID NO: 94 is
Found in both human (EMBL accession number 075786) and mouse species (EMBL accession number 088741)
Shows homology to ganglioside-induced differentiation associated protein 1. Gangliosides, possibly as modulators of membrane properties, are involved in neuronal development,
It is thought to be involved in differentiation, survival and lesions (Brigande and Seyfried, An
n. NY Acad. Sci. 845 : 215-218 (1998); Schengrund and Mummert, Ann. N.
Y. Acad. Sci. 845 : 278-284 (1998)).

Taken together, these data suggest that the protein of SEQ ID NO: 94, or a portion thereof, may play a role in central nervous system development and differentiation. Thus, the protein or portions thereof may be useful in the diagnosis and treatment of several dysfunctions, including but not limited to cancer and neurological disorders.

(Protein of SEQ ID NO: 89 (Internal Reference No. 117-005-2-0-E10-FLC)) The protein of SEQ ID NO: 89 encoded by the cDNA of SEQ ID NO: 39 is apolipoprotein of human, mouse and chicken species. Low homology to domains of protein A-IV (Genbank accession numbers M13654, M13966, EMBL accession number O93601, respectively). These apolipoproteins are thought to play a role in chylomicron and VLDL secretion and catabolism, and may be involved in reverse cholesterol transport. The size of the protein having a length of 366 amino acids in SEQ ID NO: 89 is close to the size of the apolipoprotein A-IV described above.

The protein of SEQ ID NO: 89, encoded by the cDNA of SEQ ID NO: 39, exhibits homology to carnitine carrier-related proteins. Carnitine carrier-related protein (CCRP) is likely to have a similar function because it is 45% similar (30% identical) to acyl-carnitine / carnitine carriers. Acyl-carnitine / carnitine carriers are mitochondrial carrier proteins required for the transport of fatty acids to mitochondria, and energy is generated when fatty acids are oxidized in mitochondria. CCRP is also similar in structure to the uncoupling protein (UCP-1), another mitochondrial transporter protein involved in weight regulation and temperature homeostasis. UCP protein activity is relatively well conserved in the predicted CCR protein, compared to 4/9 with the acyl-carnitine / carnitine carrier itself (6/9 are identical, 9/9 Analogous) regulated by nucleotides via a 9 amino acid protein domain. Thus, the function of CCRP can be easily activated or suppressed via small molecule nucleotide analogs.

The acyl-carnitine / carnitine carrier is required to transport fatty acids to the mitochondria before they are oxidized and turned into energy, but genetically mutating this gene will cause weight disturbances. Absent. In other words, other proteins should be used to transport fatty acids, and CCRP is likely to be this transporter.

The rate of lipids burned by mitochondria depends on the rate of delivery of fatty acids to mitochondria by these transporters. When the activity of CCRP is regulated by the nucleotide binding domain of CCRP or other interventions to increase mitochondrial availability or activity, the ability of tissues to burn fat also increases. Since elevated plasma fatty acid levels are associated with the cause of type II diabetes (NIDDM), such interventions can be designed to increase the rate of lipid removal from the blood. Other therapeutic effects targeted at CCRP include the ability to burn more fat by the liver and muscle than fat accumulation by fat tissue, and consequently reduce weight.

Taken together, these data suggest that the protein of SEQ ID NO: 89 may play a role in lipid metabolism. Thus, this protein or a portion thereof includes hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disorders such as coronary artery disease, neurodegenerative disorders such as Alzheimer's disease or dementia, and obesity. Without limitation, it can be useful in diagnosing and treating some dysfunctions.

(Protein of SEQ ID NO: 95 (Company No. 122-005-2-0-F11-FLC)) The protein of SEQ ID NO: 95 encoded by the cDNA of SEQ ID NO: 45 is a domain of the reductase family, It exhibits homology to rat, bovine and human species NADH-cytochrome b5 reductase (Genbank accession numbers J03867, M83104, Y09501 respectively). Homology includes NADH, a member of the flavin enzyme family, whose members are involved in photosynthesis, nitrogen and sulfur assimilation, fatty acid oxidation, methemoglobin reduction, as well as the metabolism of many pesticides, drugs and carcinogens. Contains the flavin-adenine dinucleotide-binding domain of the cytochrome b5 reductase protein.

Taken together, these data suggest that the protein of SEQ ID NO: 95 may play some role in the cell redox reaction, possibly as a flavin enzyme reductase. Thus, this protein or a portion thereof may be used in the diagnosis and treatment of several dysfunctions, including but not limited to cancer, methemoglobinemia, hyperlipidemia, obesity and vascular dysfunction. Could help. It may also help regulate the metabolism of pesticides, pharmaceuticals and carcinogens.

(Protein of SEQ ID NO: 106 (Internal Reference No. 108-011-5-O-B12-FL)) The protein of SEQ ID NO: 106 encoded by the extended cDNA of SEQ ID NO: 56 was obtained from human and mouse species (Genbank No.W04185 and W04184) both interleukin-17
Shows homology to the predicted extracellular domain of the receptor and part of the transmembrane domain. These IL-17R proteins are thought to belong to a new family of receptors for cytokines that induce T cell proliferation, I-CAM expression, and preferential maturation of hematopoietic progenitors to neutrophils (Yao et al. al., Cytokine., 9: 794-8001 (1997)). It is also thought to play a role in inducing inflammation and to induce nitric oxide. The protein of the present invention is obtained using software called TopPred II (Claros and von Heijne, CABI).
OS applic.Notes, 10: 685-686 (1994)), has a 21 amino acid transmembrane domain consistent with the 21 amino acid putative transmembrane domain of the human interleukin-17 receptor (position 172). ~ 192).

Taken together, these data suggest that the protein of SEQ ID NO: 106 may play a role in regulating immune and / or inflammatory responses. Thus, this protein or a portion thereof may be useful in the diagnosis and treatment of several dysfunctions, including but not limited to cancer, immunological dysfunction, septic shock and impotence is there. The protein may also help modulate the immune and / or inflammatory response to the infectious response and / or suppress graft rejection.

(Protein of SEQ ID NO: 114 (Internal Reference No. 108-014-5-O-D12-FL)) The protein of SEQ ID NO: 114 encoded by the extended cDNA of SEQ ID NO: 64 includes Drosoph
There is a cysteine-rich C3H2C3 region that is also found in the G1 protein of ila melanogaster (Swissprot accession number Q06003). This cysteine-rich region resembles a zinc finger of the RING type, a domain that binds two atoms of zinc, and is probably involved in mediating protein-protein interactions.

[0480] Taken together, these data indicate that the protein of SEQ ID NO: 114
It may play a role in protein interactions.

Utilizing the sequence of nucleic acids SEQ ID NOS: 24-73 or fragments thereof,
Fusion proteins in which 123 polypeptide sequences or fragments thereof are fused to a heterologous polypeptide can also be constructed. For example, a fragment of the polypeptide of SEQ ID NO: 74-123 contained in the fusion protein comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75 contiguous of the polypeptide of SEQ ID NO: 74-123. , 100 or
It can include 150 amino acids, or can be of any length suitable for the intended purpose of the fusion protein. The nucleic acids of SEQ ID NOs: 24-73 are cloned in frame with the nucleic acid encoding the heterologous polypeptide to produce the nucleic acid encoding the desired fusion protein. The nucleic acid encoding the desired fusion protein is operably linked to a promoter contained in a suitable vector, such as any of the vectors described above, and introduced into a host capable of expressing the fusion protein.

An antibody against the polypeptide of SEQ ID NO: 74-123 or a fragment thereof is used for immunoaffinity chromatography to isolate the polypeptide of SEQ ID NO: 74-123 or a fragment thereof, or A fusion protein comprising the polypeptide or a fragment thereof can be isolated.

Example 58 Immunoaffinity Chromatography The antibody prepared as described above is coupled to a support. Preferably, the antibodies are monoclonal antibodies, but polyclonal antibodies can also be used. The support was Sepharose CL-4B (Pharmacia Piscat, Piscataway, NJ).
away, NJ), Sepharose CL-2B (Pharmacia Piscataway, NJ), Affi-gel 10 (Biorad
, Richmond, CA) or glass beads, or any other suitable one commonly used in immunoaffinity chromatography.

The antibody can be coupled to the support using a suitable coupling reagent commonly used in immunoaffinity chromatography, including cyanogen bromide. After the antibody has been coupled to the support, the support is contacted with a sample containing the target polypeptide for which isolation, purification or enrichment is desired. The target polypeptide may be a polypeptide of SEQ ID NOs: 74-123, a fragment thereof, or a fusion protein comprising a polypeptide of SEQ ID NOs: 74-123 or a fragment thereof.

Preferably, the sample is contacted with the support for a sufficient time under suitable conditions that allow at least 50% of the target polypeptide to specifically bind to the antibody coupled to the support. .

Thereafter, the support is washed with an appropriate washing solution to remove the polypeptide non-specifically adhered to the support. The washing solution was PBS, Tris-lithium chloride buffer (0.1 M lysine base and 0.5 M lithium chloride, pH 8.0), and Tris-hydrochloride buffer (0.05 M Tri
s-hydrochloride, pH 8.0) or Tris / Triton / NaCl buffer (50 mM Tris.cl, p
H8.0 or 9.0, 0.1% Triton X-100 and 0.5 M NaCl), or any other commonly used in immunoaffinity chromatography.

After washing, the specifically bound target polypeptide is eluted from the support using a high or low pH eluate commonly used for immunoaffinity chromatography. In particular, the eluate may contain an effluent such as triethanolamine, diethylamine, calcium chloride, sodium thiocyanate, potassium bromide, acetic acid or glycine. In some embodiments, the eluate is Triton X-100
Alternatively, it may contain a detergent such as octyl-β-D-glycoside.

As described above, the cDNA of the present invention or a fragment thereof can be used for various purposes. Using a polynucleotide (constitutively, or,
As a marker for tissues where the corresponding protein is preferentially expressed (at a particular stage of tissue differentiation or development or in disease states); as a molecular weight marker in Southern gels; identifying chromosomes and mapping relevant gene locations As a chromosomal marker or tag (if labeled) to compare the patient's endogenous DNA sequence to identify potential genetic diseases; hybridize the relevant novel DNA sequence;
Thereby as a probe to find such sequences; as a source to derive PCR primers for genetic fingerprinting; binding to "gene chips" or other supports, including studying expression patterns To select and produce oligomers for use; to produce anti-protein antibodies using DNA immobilization techniques; to analyze, characterize, or treat antigens as antibodies that raise anti-DNA antibodies or elicit other immune responses Can be expressed. If the polynucleotide encodes a protein that binds or potentially binds another protein (such as in a receptor-ligand interaction), the polynucleotide may be interacted with by an interaction trap assay (Gyuris et al., Cell 75: 791- 803 (1993
) Can be used to identify polynucleotides encoding other proteins to which binding occurs, or to identify inhibitors to binding interactions.

[0489] Proteins or polypeptides obtained according to the present invention can also be utilized in assays to quantitatively determine the level of protein (or its receptor) contained in biological fluids. (Including labeling reagents); (constitutively, or at a particular stage of tissue differentiation or development, or in a disease state
Determining biological activity, including activity on multiple protein panels for high-throughput screening, as a marker for tissues where the corresponding protein is preferentially expressed, generating antibodies or other immune responses. It is possible to elicit and, of course, to isolate the relevant receptor or ligand. If a protein binds or potentially binds to another protein (such as in a receptor-ligand interaction), the protein can be used to identify other proteins where binding occurs, or It is possible to identify inhibitors. Utilizing proteins involved in these binding interactions, it is also possible to screen for peptides or small molecule inhibitors or agonists of binding interactions.

All of these research and investigation utilities can be developed into reagent grade or kit formats for commercial sale as research and investigation products.

The methods for utilizing the above-mentioned applications are well known to those skilled in the art. References disclosing such methods include "Molecular Cloning; A Laboratory Manual",
2d ed., Cole Spring Harbor Laboratory Press, Sambrook, J., EF Fritsch
and T. Maniatis eds., 1989 and "Methods in Enzymology; Guide to Molecul
ar Cloning Techniques ", Academic Press, Berger, SL and AR Kimmel eds
., 1987, but is not limited thereto.

[0492] The polynucleotides and proteins of the present invention can also be utilized as nutrients or supplements. Such uses include, but are not limited to, use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source, use as a carbohydrate source. In such a case, the protein or polynucleotide of the present invention can be added to the diet of the particular organism or can be added to another solid, such as in the form of a powder, pill, solution, suspension or capsule. It can be administered as a formulation or liquid formulation. In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium for culturing such a microorganism or to the surface of the medium.

Although the present invention has been described in terms of certain preferred embodiments, other embodiments apparent to those skilled in the art in light of the disclosure herein are also within the scope of the invention. Therefore, it is to be understood that the scope of the invention is to be defined only by the appended claims.

[0494]

[Table 1]

[0495]

[Table 2]

[0496]

[Table 3]

[0497]

[Table 4]

[0498]

[Table 5]

[0499]

[Table 6]

[0500]

[Table 7]

[0501]

[Table 8]

[0502]

[Table 9]

[0503]

[Table 10]

(Free text of the Sequence Listing) Von Heijne Matrix Score Oligonucleotide used as primer Predicted by MatInspector Name Complement

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a table listing all parameters that can be used in each step of cDNA analysis.

FIG. 2. Using the signal peptide identification method described herein, 4% of all human proteins contained in SwissProt to determine the frequency of false positives and false negatives.
FIG. 3 is a view showing a result of analyzing three amino terminal amino acids.

FIG. 3 shows a RT-PCR based method for isolating cDNA containing sequences flanking the 5 ′ EST used to obtain cDNA.

FIG. 4 is a diagram schematically illustrating isolated promoters and a method for assembling these promoters into corresponding 5 ′ tags.

FIG. 5 is a diagram showing transcription factor binding sites present in each of these promoters.

FIG. 6 is a block diagram illustrating an example of a computer system.

FIG. 7 illustrates one embodiment of a process 200 for comparing a new nucleotide or protein sequence to a sequence database to determine the level of homology between the new sequence and a sequence stored in the database. It is a flowchart shown.

FIG. 8 is a flowchart illustrating one embodiment of a process 250 for determining whether two sequences are homologous in a computer.

FIG. 9 is a flowchart illustrating one embodiment of an identifier process 300 for detecting the presence of features in an array.

FIG. 10 is a view showing the alignment of the protein of SEQ ID NO: 76 encoded by the cDNA of SEQ ID NO: 26 with the parotid HPSP protein (SEQ ID NO: 124)

FIG. 11 shows an alignment between the protein of SEQ ID NO: 93 encoded by the cDNA of SEQ ID NO: 43 and the human transmembrane protein (SEQ ID NO: 125). Conserved cysteines are shown in bold. The conserved region around the second cysteine is underlined. Potential active sites QxVxG are shown in italics.

FIG. 12 shows an alignment of the protein of SEQ ID NO: 75 encoded by the cDNA of SEQ ID NO: 25 with a putative human sialyltransferase (SEQ ID NO: 126), with 89.4% of 301 amino acid overlap Identical residues are indicated. The sialyl motif is shown in bold. The sialyl motif L is shown in italics. Potential transmembrane segments are underlined.

FIG. 13 shows an alignment between the protein of SEQ ID NO: 104, which is encoded by the extended cDNA of SEQ ID NO: 54, and the mouse recombination activation gene 1-inducible protein (SEQ ID NO: 177).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/21 1/19 C12P 21/02 C 1/21 G01N 37/00 102 5/10 33/53 M C12P 21/02 C12N 15/00 ZNAA G01N 37/00 102 F // G01N 33/53 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, T , TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Catherine Cruissel 93100 Montreuil-Souis-Bois Ave. De Predida Wilson, 110 F term (reference) 4B024 AA01 AA11 CA04 FA10 HA03 HA14 HA17 4B029 AA08 AA23 CC13 FA12 4B064 AG01 CA19 CC24 DA03 4B065 AB01 BA02 CA24 CA44 CA46 4H045 AA10 AA30 CA40 DA75 EA50 FA74

Claims (30)

[Claims] 1. A purified or isolated nucleic acid comprising one of SEQ ID NOs: 24-73 or a sequence complementary thereto. 2. A sequence comprising at least 12 contiguous sequences of one of SEQ ID NOs: 24-73.
A purified or isolated nucleic acid comprising one of a base or a sequence complementary thereto. 3. Purification or isolation comprising the complete coding sequence of one of SEQ ID NOs: 24-73, wherein the complete coding sequence comprises a sequence encoding a signal peptide and a sequence encoding a mature protein. Nucleic acid. 4. A purified or isolated nucleic acid comprising the nucleotide of one of SEQ ID NOs: 24-73, encoding a mature protein. 5. One of SEQ ID NOs: 24-73 encoding a signal peptide.
A purified or isolated nucleic acid comprising two nucleotides. 6. A purified or isolated nucleic acid encoding a polypeptide having one of the sequences of SEQ ID NOs: 74-123. 7. A purified or isolated nucleic acid encoding a polypeptide having the sequence of the mature protein contained in one of the sequences SEQ ID NOs: 74-123. 8. A purified or isolated nucleic acid encoding a polypeptide having the sequence of a signal peptide contained in one of the sequences SEQ ID NOs: 74-123. 9. A purified or isolated protein comprising the sequence of one of SEQ ID NOs: 74-123. 10. Contiguous at least one of the sequences SEQ ID NO: 74 to 123
A purified or isolated polypeptide comprising 10 amino acids. 11. An isolated or purified polypeptide comprising a signal peptide of one of the polypeptides of SEQ ID NOs: 74-123. 12. An isolated or purified polypeptide comprising the mature protein of one of the polypeptides of SEQ ID NOs: 74-123. 13. A method for producing a protein comprising one of the sequences of SEQ ID NOs: 74 to 123, comprising: obtaining a cDNA comprising one of the sequences of SEQ ID NOs: 24 to 73; Inserting the cDNA into an expression vector so as to be operably linked to a promoter; and introducing the expression vector into a host cell, thereby producing a protein encoded by the cDNA in the host cell. The method comprising: 14. The method of claim 13, further comprising the step of isolating said protein. 15. A protein obtained by the method according to claim 14. 16. A host cell comprising the recombinant nucleic acid according to claim 1. 17. A purified or isolated antibody capable of binding specifically to a protein having one of SEQ ID NOs: 74 to 123. 18. An array of polynucleotides of at least 15 nucleotides in length, wherein the improvement is at least one of the sequences of SEQ ID NOs: 24-73 or complementary to the sequence of SEQ ID NOs: 24-73. The array comprising one of the sequences, or a fragment of at least 15 contiguous nucleotides thereof, in the array. 19. At least 15 bases capable of hybridizing under stringent conditions with a sequence of one of SEQ ID NOs: 24-73 or a sequence complementary to one of the sequences of SEQ ID NOs: 24-73. A purified or isolated nucleic acid that is made up. 20. Consecutive at least one of the sequences of SEQ ID NOs: 74 to 123
A purified or isolated antibody capable of binding a polypeptide containing 10 amino acids. 21. A computer-readable medium storing a sequence selected from the group consisting of the cDNA codes of SEQ ID NOS: 24 to 73 and the polypeptide codes of SEQ ID NOS: 74 to 123. 22. A computer system comprising a processor and a data storage device, wherein the data storage device is a sequence selected from the group consisting of a cDNA code of SEQ ID NOs: 24-73 and a polypeptide code of SEQ ID NOs: 74-123. The computer system, wherein the computer system is stored. 23. The computer system according to claim 22, further comprising an array comparer and a data storage device storing a reference sequence. 24. The computer system of claim 23, wherein said sequence comparer includes a computer program indicating a polymorphism. 25. The computer system of claim 22, further comprising an identifier for identifying a feature of the array. 26. A method for comparing a first sequence selected from the group consisting of the cDNA codes of SEQ ID NOs: 24 to 73 and the polypeptide codes of SEQ ID NOs: 74 to 123 with a reference sequence, comprising comparing the sequences. Reading the first sequence and the reference sequence using a computer program to perform the method, and determining a difference between the first sequence and the reference sequence using the computer program. 27. The step of determining a difference between a first sequence and a reference sequence,
27. The method of claim 26, comprising identifying a polymorphism. 28. A method for identifying a feature of a sequence selected from the group consisting of a cDNA code of SEQ ID NOs: 24-73 and a polypeptide code of SEQ ID NOs: 74-123, comprising: Using the computer program to identify features of the sequence;
The method comprising: 29. A purified or isolated nucleic acid comprising a contiguous span of at least 12 nucleotides of one of SEQ ID NOs: 24-73 or one of the sequences complementary thereto, wherein The nucleic acid, wherein the span comprises at least one of the nucleotide positions of the polynucleotide set forth in Table III. 30. A purified or isolated nucleic acid comprising a continuous span of at least 12 nucleotides of one of the polynucleotides shown in Table III or one of its complementary sequences.