WO1997001573A2

WO1997001573A2 - Early onset alzheimer's disease gene and gene products

Info

Publication number: WO1997001573A2
Application number: PCT/US1996/011002
Authority: WO
Inventors: Eric H. Karran
Original assignee: Smithkline Beecham Corporation; Smithkline Beecham Plc
Priority date: 1995-06-28
Filing date: 1996-06-26
Publication date: 1997-01-16
Also published as: AU6341696A

Description

Early Onset Alzheimer's Disease Gene and Gene Products

Field of the Invention

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides and polynucleotides of the present invention are the Early Onset Alzheimer's Disease (herein "EOAD") gene, gene products and mutants and fragments thereof. The invention also relates to inhibiting the action of such polypeptides. More particularly, this invention relates to EOAD genes and segments of EOAD genes useful as probes and amplification primers for the identification of a predisposition for or diagnosis of Alzheimer's Disease.

Background of the Invention

Alzheimer's disease (AD) is the fourth most common cause of death in the U.S. after heart disease, cancer and stroke. It presently afflicts more than four million people and this number is expected to double during the next forty years as the population ages. There is presently no cure for AD and treatments are largely palliative rather than treating the underlying causes of disease. A stated aim of the National Institute of Aging is to delay the age of onset by five years during the next five years and by ten years within the next ten years thus reducing significantly the number of people affected by AD.

Apart from advanced age and Down syndrome the only consistent risk factor for the development of AD identified in epidemiological surveys has been the presence of a positive family history of disease. The most striking evidence in support of genetic factors is the existence, amongst early onset cases of AD, of families in which the disease is inherited as a fully penetrant autosomal dominant disorder (Nee et al., Arch. Neurol 40:203-208). The existence of large families with an inherited form of AD has enabled a genetic linkage strategy to be used to localize the disease senes. The observation of AD neuropathology in aging Down syndrome (DS) patients led researchers to analyze chromosome 21 in families with an inherited form of AD. Genetic linkage between FAD and markers on the long arm of chromosome 21 was first reported in 1987 (St. George-Hyslop, et al., Nature, 347:194-197. (1990)). Since that time it has been demonstrated that early onset FAD is genetically heterogeneous and that many pedigrees do not show linkage to chromosome 21 markers (St. George-Hyslop, et al., Nature Genetics, 2:330-334 (1992) and Schellenberg, et al., Annals Of Neurology 57:223-227 (1992)). Analysis of only those families which show clear evidence of linkage to chromosome 21 revealed that the β-amyloid precursor protein (APP) gene was tightly linked to the disease locus in these families (Goate, et al., Nature 349:104-706 (1991)). The β- amyloid peptide, that is deposited in senile plaques in AD brains, is encoded by parts of exons 16 and 17 of the APP gene. Sequencing of these exons of the APP gene in chromosome 21 linked AD families led to the identification of a point mutation at position 2149, which causes an amino acid substitution of valine to isoleucine at codon 717 of APP770 in two families (Goate, et al., Nature 349:104-106 (1991)). Subsequent screening of affected individuals from many FAD cases has identified a further nine families with the same mutation (Yoshioka et al., Biochem. Biophys. Res. Commun. 778:1141-1146 (1991); Sorbi et al., Nature Genetics 4: 10 (1993); Naruse et al., Lancet 557:978-979.(1991); Karlinsky et al, Neurology 42:1445-1453 (1992); Fidani, et al., Mol. Genet. 7:165-168(1992)). Another three mutations have been identified in affected members of a single FAD pedigree in each case. Two of these mutations are also in codon 717 and result in valine to glycine and valine to phenylalanine substitutions (Chartier-Harlin, et al., Nature 555:844-846 (1991); Murrell, et al., Science 254:97-99(1991)). The occurrence of three different mutations at the same codon in affected AD individuals strongly supports the view that these mutations are pathogenic. The third mutation is a double point mutation in codons 670/671 which result in lysine/methionine being substituted by asparagine/leucine (Mullan, et al.. Nature Genetics 2:340-342 (1992)). Screening large numbers of individuals has identified several apparently silent mutations not associated with disease and several mutations that lead to different phenotypes (Hendriks, et al., Nature Genetics 1:218-221 (1992); Jones, et al. Nature Genetics, 7:306-309 (1992); Kamino, et al., Am. 1. Hum. Genet. 57:998-1014 (1992); Levy, et al., Science 245:1124-1126 (1990) and Zubenko, et al., Neuropath, and Exp. Neurol. 57:459-463 (1992)). The first of these mutations to be described was a glutamic acid to glutamine (APP693) substitution observed in individuals with hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (Levy, et al., Science 248:1124-1126 (1990)). Individuals with this disorder die from strokes in early middle age. Neuropathological examination of their brains shows β-amyloid deposited in the cerebral vessels and in the brain parenchyma as diffuse plaques. A family in which individuals develop either AD or HCHWA-D has also been described. These people have a mutation at codon 692 (Hendriks, et al., Nature Genetics 1:218-221 (1992)). It is presently not known what additional factors determine whether an individual in this family develops AD or HCHWA-D. Analysis of a large number of familial and sporadic cases of AD has indicated that the reported mutations in the APP gene are likely to cause the disease in only a small proportion of cases, at least in the Caucasian population (Tanzi, et al., Am. 1. Hum. Genet. 57:273-282 (1992)).

Some of the reported mutations are within the p-amyloid fragment (APP693 and APP692) whereas others appear to flank the β-amyloid sequence (APP717 and APP670/671) suggesting that different pathogenic mechanisms could be involved. Those mutations within the β-amyloid fragment might be expected to alter the physical properties of β-amyloid such that it is more likely to form fibrils whereas those mutations flanking the β-amyloid fragment could affect proteolytic processing of the precursor, one consequence of which might be increased β-amyloid production. Recent studies on the proteolytic processing of APP provide further support for this hypothesis. Several pathways of APP processing have so far been identified, at least one of which can lead to the production of potentially amyloidogenic fragments. The β-amyloid peptide deposited in AD and DS can be detected as a normal product of APP processing, in the medium from cells in tissue culture and in bodily fluids. Introduction of a construct containing the double mutation at APP670/671, which was reported in a Swedish pedigree, into human kidney 293 cells has been shown to lead to a 6-8 fold increase in the levels of soluble β-amyloid detectable in the medium whilst transfection of the APP717 mutations leads to a decrease the ratio of β-amyloid 1-40/β-amyloid 1-42 without altering the total amount of β-amyloid peptide produced. The predicted effect of both of these mutations would be. to increase the amount of β-amyloid deposited.

Despite the relatively small numbers of families with these mutations, they remain the first and only known cause of AD. It has been suggested that these observations support the hypothesis that β-amyloid deposition is central to the disease process and that in these cases, mutations in the APP gene lead to premature deposition of β-amyloid whilst in DS overexpression of APP due to an extra copy of the normal gene leads to the same pathology. Further evidence in support of this hypothesis has recently come from the introduction of an APPV717F transgene into mice. These mice develop an age related neurodegeneration and deposit large amounts of p-amyloid peptide in their brains. The hypothesis also predicts that other genes which lead to AD will be involved in the regulation or processing of APP.

Genetic linkage studies have identified a second locus causing early onset FAD (herein "early onset Alzheimers Disease" or "EOAD") on the long arm of chromosome 14 (Schellenberg, et al., Annals Of Neurology 57:223-227 (1992); Van Broeckhoven, et al., Nature Genetics 2:335-339 (1992); St. George-Hyslop, et al., Nature Genetics, 2:330-334 (1992)) linkage was first reported to D14S43 and localized to a region of about 23cM between D14S52 and D14SS3. The isolation of additional genetic markers has led to the candidate region being narrowed to a distance of 6.4cM between D14S289 and D14S61 (Cruts, et al., Human Molecular Genetics. A positional cloning strategy is presently being used in our tab and others to identify the defective gene. Although the majority of EOAD families studied show linkage to this locus, at least one more locus causing early onset FAD must exist because the Volga German families show recombination with the APP gene and the markers tightly linked to the FAD gene on chromosome 14 (Schellenberg, et al.. Science 25:668-671(1992). Apart from age of onset of disease no phenotypic or neuropathological markers have been identified that distinguish between the different causes of FAD. Identification of new chromosome 14-linked families for meiotic mapping and the successful application of linkage disequilibrium techniques (both of which could narrow down the region of interest considerably) could be key factors in the rapid identification of this gene since 6.4cM of DNA could contain hundreds of candidate genes.

When the AD3 locus was first localized to chromosome 14 two genes were known to map to this region of the chromosome: the heat shock protein, HSPA2 and the protooncogene cfos. Refinement in the mapping of HSPA2 and the identification of additional recombinants in AD families now place the HSPA2 gene outside the candidate region cfos remains within the candidate region. However, extensive sequencing of the coding region by several groups has failed to reveal any pathogenic mutations although several polymoφhisms have been identified and physical characterization of the early onset Alzheimer's disease AD3 locus on chromosome 14q24.3. Two expressed sequence tagged sites (ESTs) have also been mapped just outside the candidate region: D14S1O2E, which maps between D14S289 and D14S251 and D14S1O1E. which maps between D14S61 and D14S59. Two other genes and one pseudogene have been mapped within the candidate region. The known genes are transforming growth factor beta (tg -β), and the Kreb's cycle enzyme dihydrolipoamide succinyltransferase (DLST). Since tg -β is known to modulate APP expression it represents a plausible candidate gene. To date no mutations have been identified in this gene in patients from chromosome 14-linked FAD cases. A reduction in the activity of DLST has been reported in brains from AD cases and also in the fibroblasts from chromosome 14-linked AD cases. However, to date no mutations have been identified in this gene in patients from chromosome 14-linked FAD cases.

The recent explosion in the number of genetic markers available now means that the availability of family material is most often the limiting factor in the resolution of genetic studies. This is particularly true for rarer diseases. Two different approaches can be used to get around this problem: the identification of additional families with closer flanking recombinants or the use of linkage disequilibrium studies in groups of families with a common ancestor. Linkage disequilibrium can narrow down the candidate region from several megabases to a few hundred kilobases and was successfully used in the positional cloning of the Huntington's disease (HD). linkage studies in HD narrowed down the candidate region to approximately 2.2Mb. However, linkage disequilibrium and haplotype studies indicated that the most likely location of the HD gene was in a segment between D4S18O and D14S182. The HD gene is located within this region close to D4S 1 80.

Genetic analysis of familial cases of late onset AD, using both parametric (Lod score method) and non-parametric (affected pedigree member) methods, has implicated a region on the long arm of chromosome 19 in predisposition to this form of the disorder (Pericak- Vance, et al., Am 1. Hum. Genet 4:1034-1050 (1991)). In addition, association studies have been carried out using two genes mapping in this region: apolipoprotein CII (ApoCII) and apolipoprotein E (ApoE). In the first case the frequency of the "F" allele of ApoCII was significantly increased amongst affected family members in twenty-three pedigrees when compared with unrelated controls. Whilst in more recent studies many groups have reported an association between the ApoE-e4 allele and late onset AD (Corder, et al., Science 267:921-923 Saunders, et al., Neurology, 43: 1467-1472 (1993); Po ier, et al., 77ιe Lancet 542:697-699 (1993)). The latter studies observed this association in both familial and sporadic cases of AD (Saunders, et al., Neurology, 45:1467-1472 (1993). Several lines of evidence support the hypothesis that ApoE-ε4 is the risk factor rather than a second locus in strong linkage disequilibrium with ApoE-ε4. ApoE has long been known to be a component of senile plaques (Namba, et al., Brain Research. 54h 163-166 (1991) but more recent in vitro studies suggest that ApoE may bind to β-amyloid in an allele-dependent manner (Strittmatter, et al., Proc. Natl Acad. Sci USA 90:8098-8102 (1993). Perhaps the strongest evidence in favor of ApoE-ε4 is the reported dose dependent effect of ApoE-ε4 on risk for AD and age of onset of AD (Corder, et al., Science 267:921-923 (1993)). Recent data (Chartier- Harlin. et al.. Human Molecular Genetics 5:569-574 (1994): Corder, et al., Nature Genetics 7: 180-183 (1994)) suggest that ApoE-ε2 allele is protective against AD, again supporting the hypothesis that it is alleles at the ApoE gene rather than a sequence variant in linkage disequilibrium with ApoE that influences risk to AD. Although ApoE-ε4 is a significant risk factor for late onset AD, it does not account for all of the risk. There are families/individuals who have no ApoE-ε4 alleles and yet they still develop AD.

The pathogenic mechanism by which ApoE alleles influence risk for AD is presently unknown but several hypotheses have been proposed (Strittmatter, et al. Proc. Natl. Acad. Sci. USA 90:8098-8012). In vitro studies suggest that ApoE may bind to both β-amyloid and tau in an isoform dependent manner proposed (Strittmatter, et al. Proc. Natl. Acad. Sci. USA 90: 8098-8012). However, the β- amyloid result is confusing since different labs have conflicting results concerning the effects of different ApoE alleles. ApoE has also been shown to increase fibril formation from soluble p-amyloid in vitro. ApoE alleles when bound to P-VLDL have also been reported to have different abilities to stimulate neurite outgrowth in several culture systems. This effect can be blocked by antibodies to the LDL related protein (IRP) receptor and by the endogeneous inhibitor of the LRP receptor, a 39kd regulatory protein.

There is a clear need for treatments for this disease and the present invention relates to compounds and methods of treatment. Moreover, identification of such EOAD has been hampered by the unavailability of convenient diagnostic materials and methods. Thus, there is also a need for a rapid, sensitive, and specific test to aid in the diagnosis of EOAD. DNA-based diagnostic tests not only are sensitive and specific but also have the advantage of being rapid. Early detection and identification of EOAD facilitate prompt, appropriate treatment and care. The invention includes embodiments which are DNA sequences that are unique to the EOAD gene and comprise nucleic acid mutations are useful as diagnostic probes to detect the EOAD or a predisposition for EOAD.

This invention provides a unique set of DNA sequences useful for the detection of EOAD gene mutations, and particularly useful as primers and probes for the detection of EOAD or a predisposition for EOAD. Brief Description of the Invention

In one aspect, the present invention is directed to each of the DNA sequences and molecules (and corresponding RNA sequences) identified in Figures 1-6 [SEQ ID NO: 1-7] and to fragments or portions of such sequences which contain at least 15 bases, and preferably at least 50 bases, and to those sequences which are at least 95% and preferably at least 97% identical thereto, and to DNA (RNA) sequences encoding the same polypeptide as the sequences of Figures 1-6 [SEQ ID NO: 1-7] as well as fragments and portions thereof. The sequences identified in Figures 1-6 [SEQ ID NO: 1-7] are hereinafter sometimes referred to as ESTs (Expressed Sequence Tags) of the EOAD gene. Each such identified sequence is a sequenced portion of an overall cDNA sequence contained in a cDNA clone derived from human tissue.

In accordance with a further aspect, the present invention is directed to a DNA sequence identical to one contained in and isolated from ATCC Deposit No. 75916. The DNA sequence contained in the deposit is hybridizable under stringent conditions with a DNA sequence (EST) identified in Figures 1-5 [SEQ ID NO: 1-5]. In addition, the present invention relates to fragments or portions of the isolated DNA sequences (and corresponding RNA sequences) containing at least 15 bases, preferably at least 40 bases and more preferably at least 50 bases, as well as sequences which are at least 97% identical thereto, as well as DNA (RNA) sequences encoding the same polypeptide.

As used herein, a first DNA (RNA) sequences is at least 95% and preferably at least 97% identical to another DNA (RNA) sequence if there is at least 95% and preferably at least a 95% or 97% identity, respectively, between the bases of the first sequence and the bases of the other sequence, when properly aligned with each other, for example when aligned by BLAST or FAST A.

In yet another aspect, the present invention is directed to an isolated DNA (RNA) sequence or molecule comprising at least the coding region of a human gene (or a DNA sequence encoding the same polypeptide as such coding region), in particular an expressed human gene, which human gene comprises a DNA sequence selected from the group listed in Figures 1-6 [SEQ ID NO: 1-7] or one at least 95% and preferably at least 97% identical thereto, as well as fragments or portions of the coding region which encode a polypeptide having a similar function to the polypeptide encoded by the coding region. Thus, the isolated DNA (RNA) sequence can include only the coding region of the expressed gene (or fragment or portion thereof as hereinabove indicated) or can further include all or a portion of the non¬ coding DNA of the expressed human gene.

In yet another aspect, the present invention is directed to an isolated DNA sequence (RNA) containing at least the coding region of a human gene of a DNA (RNA) sequence encoding the same peptide as such coding region (in particular, an expressed human gene) which human gene (either in the coding or non-coding region and in general, in the coding region) contains a DNA sequence identical to a DNA sequence present in ATCC Deposit No. 75916, which DNA sequence in such ATCC Deposit No. 75916 is hybridizable under stringent conditions with a DNA sequence listed in Figures 1-6 [SEQ ID NO: 1-7]. The invention further relates to fragments or portions of such coding region which encode a polypeptide having a similar function to the polypeptide encoded by the coding region.

The present invention further relates to polypeptides encoded by such hereinabove noted DNA (RNA sequences, as well as the production and use of such polypeptides and fragments, derivatives and structural modifications thereof with the same function(s) and use(s) and to antibodies against such polypeptides.

The present invention also relates to vectors or plasmids which include such DNA (RNA) sequences, as well as the use of the DNA (RNA) sequences.

The material deposited as ATCC Deposit No. 75916 is a mixture of cDNA clones deposited as phages derived from a variety of human tissues. The tissues from which the clones were derived are listed in Figures 1-5 above the sequences. The deposited material includes the cDNA clones which were partially sequenced and listed in Figures 1-6 [SEQ ID NO: 1-7]. Thus, the DNA sequence of Figures 1- 6 [SEQ ID NO: 1-7] is only a ponion of the sequence included in the clone from which the sequence was derived. Thus, a clone which is isolatable from the ATCC Deposit by use of a sequence listed in Figures 1-6 [SEQ ID NO: 1-7] may include the entire coding region of a human gene of in other cases such clone may include a substantial portion of the coding region of a human gene. Although the sequence listing lists only a portion of the DNA sequence in a clone included in the ATCC Deposit, it is well within the ability of one skilled in the art to complete the sequence of the DNA included in a clone isolatable from the ATCC Deposit by use of a sequence (or portion thereof) listed in Figures 1-6 [SEQ ID NO: 1-7] by procedures hereinafter further described, and other apparent to those skilled in the art.

In addition, in the case where a clone isolatable from the ATCC Deposit by use of a DNA sequence (or portion thereof) listed in Figures 1-6 [SEQ ID NO: 1-7] does not include the full coding region of a human gene, it is well within the scope of those skilled in the art to obtain the full coding region by techniques described herein or others in the art.

The EST sequences disclosed herein are markers for and components of human EOAD genes actually transcribed in vivo. Techniques are disclosed for using these ESTs to obtain the full coding region of the corresponding EOAD gene and mutants thereof. The use of ESTs, complete coding sequences, or fragments thereof for marking chromosomes, for mapping locations of expressed genes on chromosomes, for individual or forensic identification, for mapping locations of disease-associated genes, for identification of tissue type, and for preparation of antisense sequences, probes, and constructs is discussed in detail below. Unlike the random genomic DNA sequence tagged sites (STSs) (Olson et al., Science, 245: 1434 (1989)), the EOAD ESTs point directly to expressed EOAD genes. However, they can be used to detect unexpressed genes as described elsewhere herein.

Various aspects of the present invention thus include each of the individual EOAD ESTs, corresponding partial and complete EOAD cDNA, mRNA, antisense strands, triple helix probes, PCR primers, coding regions, and constructs.

Expression vectors and polypeptide expression products, are also within the scope of the present invention, along with antibodies, especially monoclonal antibodies, to such expression products.

This invention relates to an isolated DNA having the sequence selected from the group of sequences given herein as SEQ ID NO:l. SEQ. ID. NO:2, SEQ. ID. NO:3. SEQ. ID. NO:4, SEQ. ID. NO:5, SEQ. ID. NO:6 and SEQ ID NO:7. This invention relates to an isolated polypeptide sequences having the sequence selected from the group of sequences given herein as Figures 1-5 [SEQ ID NO: 8-37].

This invention further relates to a gene comprising the sequence selected from the group of sequences depicted in Figures 1-6 [SEQ ID NO: 1-7].

In a further embodiment the invention relates to a nucleic acid probe capable of selectively hybridizing to human mutant EOAD gene nucleic acids, said probe comprising a sequence selected from the group of sequences given herein as SEQ ID NO:l SEQ. ID. NO:2, SEQ. ID. NO:3, SEQ. ID. NO:4, SEQ. ID. NO:5, SEQ. ID. NO:6, and SEQ. ID. NO:7.

In another aspect, the invention relates to an isolated DNA sequence comprising DNA having at least a 95% identity to a DNA sequence selected from Figures 1-6 [SEQ ID NO: 1-7].

In yet another aspect, the invention relates to an isolated sequence comprising RNA corresponding to any of the DNA sequences or fragments of Figures 1-6 [SEQ ID NO: 1-7].

In a further embodiment the invention relates to a method for identifying a mutant EOAD gene nucleic acid sequence comprising the steps of: (a) isolating nucleic acid from a sample suspected to contain said mutant EOAD gene nucleic acid sequence; (b) contacting said nucleic acid with oligonucleotide primers consisting of two single-stranded oligonucleotides between 10 and 30 nucleotides in length being fragments of said length derived from sequences selected from the group consisting of the sense strand of SEQ. ID NO: 1-7 as a 5' primer and the antisense strand of SEQ. ID NO: 1-7 as a 3' primer and whereby the 5' primer and the 3' primer are non-overlapping and are capable of driving amplification; (c) amplifying said nucleic acid to form an amplified product; (d) and detecting the amplified product wherein the presence of the amplified product indicates the presence of a mutant EOAD gene. It is preferred that in the method for identifying human EOAD the nucleic acid sequence be DNA or RNA. If the nucleic acid is RNA, then the amplification reaction preferably uses reverse transcriptase to form the cDNA to be amplified prior to the amplifying step. Moreover, the method using RNA as the starting nucleic acid to be amplified can be used to detect both mutant and wild-type expression levels using quantitative PCR, as well as using RT-PCR to detect mutant EOAD gene sequences. The skilled artisan will be readily able to derive 3' and 5' primers of appropriate length from the disclosed nucleic acid sequences using methods well known in the art and will be readily able to determine which primer set will drive the amplification reaction. Moreover, the sequences in SEQ. ID. NO: 1-7 are useful in hybridization reactions well known to skilled artisans in the relevant art.

In a further embodiment the invention relates to amplification primer pairs comprising the sequences between 10 and 30 nucleotides in length being fragments of said length derived from sequences selected from the group consisting of a sense strand of SEQ. ID NO: 1-7 and an antisense strand of SEQ. ID NO: 1-7 as a 3' primer and whereby the 5' primer and the 3' primer are non-overlapping and are capable of driving amplification.

In yet another embodiment the invention relates to a kit for the detection of human a mutant EOAD gene comprising a carrier adapted to contain in close confinement therein a first container containing a hybridization solution and a second container containing a probe comprising a sequence selected from the group of sequences given herein as SEQ ID NO: 1-7 and selectively hybridizing fragments thereof.

In another embodiment the invention relates to a method of detecting human EOAD gene nucleic acid in a nucleic acid sample comprising:(a) contacting an oligonucleotide probe to the nucleic acid sample under hybridization conditions wherein said probe comprises a sequence selected from the group of sequences given herein as SEQ ID NO: 1-7 and hybridizing fragments thereof; and (b) detecting whether or not said oligonucleotide probe hybridized with the nucleic acid in the sample indicating the nucleic acid in the sample contains a mutant human EOAD gene.

In accordance with one aspect of the present invention. there is provided a novel mature polypeptide which is the EOAD gene, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin. In accordance with one aspect of the present invention, there is provided a polynucleotide encoding the same mature polypeptide as a human gene whose coding region includes a nucleotide sequence selected from the group consisting of the nucleotide sequences of Figures 1-6 [SEQ ID NO: 1-7].

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, for the treatment of Alzheimer's Disease, particularly EOAD.

In accordance with yet a further aspect of the present invention, there is provided an antibody against such polypeptides.

In accordance with yet another aspect of the present invention, there are provided antagonist/inhibitors to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of Alzheimer's Disease, especially for the treatment of EOAD.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Brief Description of the Figures:

Figures 1-5 [SEQ ID NO: 1-5 and 8-37] illustrate various polypeptide and polynucleotide sequences useful in the practice of this invention. Nucleotides identified as SEQ ID NO: 1-5 are presented. In the invention the nucleotide indicated by the letter N in any of the figures can be selected from the group consisting of A, C. G and T. In the invention the lower case letter x in the polypeptide sequences represents any of the naturally occurring amino acids, being alanine, arginine, asparagine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids are shown in single letter code having the meaning known and used in the art.

Figure 6 illustrates various DNA sequences useful in the practice of this invention. Sequences identified as SEQ ID NO:6 and 7 are presented. In the invention the nucleotide indicated by the letter X in any of the figures can be selected from the group consisting of A, C, G and T.

Figure 7 illustrates an assembly map comprising (A) SEQ ID NO:7 and (B) an assembly map comprising SEQ ID NO:6.

Detailed Description ofthe Invention:

Nucleic acid-based methods for the detection of human diseases or a predisposition for human disease using clinical samples can be separated into two broad categories, differing primarily in the lower limit of detection of the target nucleic acid sequence. The first category employs conventional molecular techniques to detect target sequences directly from clinical samples. The second category, predicated on nucleic acid amplification technologies, rapidly enriches the target sequences prior to detection. The determination of which approach to employ depends on a number of factors such as cost, labor and the clinical need for rapid results. The nucleic acid molecules of this invention may be advantageously and simply employed in either of the two systems.

Conventional methods for nucleic acid detection rely on physico-chemical methods to foster visualization the molecules or rely on hybridization methodology employing nucleic acid probes which are labeled with analytically detectable reagents. Examples include: Southern blotting whereby endonuclease-digested DNA is immobilized on supports such as nitrocellulose filters then probed with analytically labeled nucleic acid to detect specific complementary sequences. Analytically detectable reagents for this purposes include radioactive isotopes (e.g., ¹⁴C and ³²P) and non-radioactive reagents such as chemiluminescent materials, DNA dot blots whereby DNA is extracted from a number of clinical isolates by any convenient means and transferred by numerous methods known in the art, including but not limited to vacuum filtration, to a support and probed as is the case of Southern blotting; and Colony dot blots whereby the colonies comprising human gDNA or cDNA derived from clinical isolates are cultured on agar plates, transferred to paper and lysed in situ prior to probing.

Amplification systems rely on the existence of primer nucleic acid molecules of about 10-30 nucleotides in length which flank the target region. The primer acts as initiation points for multiple cycles of DNA replication on the region defined by the flanking primers. The Polymerase Chain Reaction (PCR) employing the Taq DNA polymerase (Mullis et al., Meth. Enzymol. 755:335-350(1987)) is a classic example of an amplification system.

EOAD Polynucleotides and Polypeptides

The EOAD EST sequences of the present invention have been isolated from cDNA libraries using a rapid screening and sequencing technique. In general, the method comprises applying automated DNA sequencing technology to screen clones, advantageously randomly selected clones, from a cDNA library. Preferably, the library is initially "enriched" by removal of ribosomal sequences and other common sequences prior to clone selection. According to the disclosed method, EOAD ESTs are generated from partial EOAD DNA sequencing of the selected clones. The EOAD ESTs of the present invention were generated using low redundancy of sequencing, typically a single sequencing reaction. While single sequencing reactions may have an accuracy as low as 97%, this nevertheless provides sufficient fidelity for identification of the sequence and design of PCR primers, as well as for full length sequence because of the exceptional amount of laboratory work and resultant chemical/biological disclosure reported herein, including that done by automatically cycle sequencing.

The automated sequencing reported here was performed on Catalyst robots (Applied Biosystems, Inc., Foster, CA) and 37 Automated DNA Sequencers (Applied Biosystems. Inc.). The Catalyst robot is a sophisticated pipetting and temperature controlled robot that has been developed specifically for DNA sequencing reactions. The Catalyst combines pre-aliquoted templated and reaction mixtures consisting of deoxy- and dideoxynucleotides, the Taq thermostable DNA polymerase, fluorescently-labelled sequencing primers, and reaction buffer. Reaction mixtures and templates are combined in the wells of an aluminum 96-well thermocycling plate. Thirty consecutive cycles of linear amplification (e.g. one primer synthesis) steps are performed including denaturation, annealing of primer and template, and extension of DNA synthesis. A heated lid on the thermocycling plate prevents evaporation without the need for an oil overlay. The Applied Biosystems, Inc. (ABI) system currently used for EST sequencing involves use of four dye-labeled sequencing primers, one for each of the four terminator nucleotides. Each dye-primer is labeled with a different fluorescent dye, permitting the four individual reactions to be combined into one lane of the 373 DNA Sequencer for electrophoresis, detection, and base-calling. ABI supplies pre-mixed reaction mixes (PRIZM Ready Reaction Kit) containing all the necessary non-template reagents for sequencing. These reaction mixtures are stable for at least a year at -20°C.

EOAD ESTs comprise DNA sequences corresponding to a portion of nuclear encoded messenger RNA. The EOAD ESTs of the invention are of sufficient length to permit: (1) amplification of the specific sequences from a cDNA library, e.g., by polymerase chain reaction (PCR); (2) use of a synthetic polynucleotide corresponding to a partial or complete sequence of the EST as a hybridization probe of a cDNA library, generally having about 30-50 base pairs; or (3) unique designation of the pure cDNA clone from which the EST was derived (the EST clone) for use as a hybridization probe of a cDNA library. The length of a partial EOAD EST according to the present invention can be. for example, approximately 30, 400, 50, 75, 90, 100, or 150 bases. Preferably, EST-derived primer pairs and sequences amplify or detectably hybridize to a sequence from a genomic library.

Complete Coding Region DNA Sequences Recovered Using ESTs

The EOAD ESTs of the present invention generally represent relatively small coding regions or untranslated regions of human genes. Although these EST sequences do not generally code for a complete gene product, they are highly specific markers for the corresponding complete coding regions. The EOAD ESTs are of sufficient length that they will hybridize, under stringent conditions, only with DNA for the EOAD gene and mutants thereof. Suitably stringent conditions comprise conditions, for example, where at least 95%, preferably at least 97% or 98% identity (base pairing), is required for hybridization. This property permits use of the EOAD ESTs to isolate the entire coding region and even the entire sequence of the EOAD gene. Therefore, only routine laboratory work is necessary to parlay the unique EST sequence into the corresponding unique complete gene sequence.

Thus, each of the ESTs of the present invention "corresponds" to or is a part of a particular unique human gene. Knowledge of the EOAD EST sequence permits isolation and sequencing of the complete coding sequence of the corresponding gene. The complete coding sequence is present in a full-length cDNA clone as well as in the gene carried on genomic clones. Therefore, each EOAD EST also "corresponds" to or is a part of a complete genomic EAOD gene sequence, and may or may not be DNA which is included in a polypeptide coding region of the gene.

The first step in determining where an EOAD EST is located in the cDNA is to analyze the EST for the presence of coding sequence. The CRM program predicts the extent and orientation of the coding region of a sequence. Based on this information, one can infer the presence of start or stop codons within a sequence and whether the sequence is completely coding or completely noncoding. If start or stop codons are present, then the EST can cover both part of the 5'- untranslated or 3'- untranslated part of the mRNA (respectively) as well as part of the coding sequence. If no coding sequence is present, it is likely that the EST is derived from the 3'- untranslated sequence due to its longer length and the fact that most cDNA library construction methods are biased toward the 3' end of the mRNA.

An EOAD EST is a specific tag for a EOAD messenger RNA molecule. The complete sequence of that messenger RNA, in the form of cDNA, can be determined using the EST as a probe to identify a cDNA clone corresponding to a full-length transcript, followed by sequencing of that clone. The EST of the full-length cDNA clone can also be used as a probe to identify a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons and introns.

ESTs are used as probes to identify the cDNA clones from which an EST was derived. ESTs, or portions thereof, can be nick-translated or end-labeled with "p using polynucleotide kinase using labeling methods known to those with skill in the art (Basic Methods in Molecular Biology, L.G. Davis, M.D. Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986). A lambda library can be directly screened with the labeled ESTs of interest of the library can be converted en masse to pBluescript (Stratgene Cloning Systems, 11099 N. Torrey Pines Road, La Jolla, CA 92037) to facilitate bacterial colony screening. Regarding pBluescript, see Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), pg. 1.20. Both methods are will known in the art. Briefly, filter with bacterial colonies containing lambda plaques are denatured and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al., supra. The EOAD ESTs of the invention, cloned into lambda or Bluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing.

The EOAD ESTs can additionally be used to screen Northern blots of mRNA obtained from various tissues or cell cultures, including the tissue of origin of the EST clone as described in more detail elsewhere herein. Northern analysis will most often produce one to several positive bands. The bands can be selected for further study based on the predicted size of the EOAD mRNA.

Positive EOAD cDNA clones in phage lambda will be analyzed to determine the amount of additional sequence they contain using PCR with one primer from the EST and the other primer from the vector. Clones with a larger vector-insert PCR product than the original EST clone are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size on a Northern blot.

Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined. The preferred method is to use exonuclease III digestion (McCombie, et al., Methods, 5:33-40, 1991). A series of deletion clones is generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.

A similar screening and clone selection approach can be applied to obtaining cosmid or lambda clones from a genomic DNA library that contains the complete gene from which the EST was derived (Kirkness, et al., Genomics 70: 985-995 (1991). Although the process is much more laborious, these genomic clones can be sequenced in their entirety also. A shotgun approach is preferred to sequencing clones with inserts longer than 10 kb (genomic cosmid and lambda clones). In shotgun sequencing, the clone is randomly broken into many small pieces, each of which is partially sequenced. The sequence fragments are then aligned to produce the final contiguous sequence with high redundancy. An intermediate approach is to sequence just the promoter region and the intron-exon boundaries and to estimate the size of the introns by restriction endonuclease digestion (ibid.).

Using the EOAD sequence information provided herein, the polynucleotides of the present invention can be derived from natural sources or synthesized using known methods. The sequences falling within the scope of the present invention are not limited to the specific sequences described, but include human allelic and species variations thereof and portions thereof of at least 15-18 bases, preferably at least 75, 90, 100, 125, or 150 bases. (Sequences of at least 15-18 bases can be used, for example, as PCR primers or as DNA probes.) In addition, the invention includes the entire coding sequence associated with the specific polynucleotide sequence of bases described in the Sequence Listing, as well as portions of the entire coding sequence of at least 15-18 bases, preferably at least 25. 40, or 50 bases, and more preferably at least 75, 90, 100, 125, or 150 bases, and allelic and species variations thereof. Allelic variations can be routinely determined by comparison of one sequence with a sequence from another individual of the same species. Furthermore, to accommodate codon variability, the invention includes sequences coding for the same amino acid sequences as do the specific sequences disclosed herein. In other words, in a coding region, substitution of one codon for another which encodes the same amino acid is expressly contemplated. (Coding regions can be determined through routine sequences analysis.)

Any specific sequence disclosed herein can be readily screened for errors by resequencing each EST in both directions (i.e., sequence both strands of cDNA). Alternatively, error screening can be performed by sequencing corresponding polynucleotide of human origin isolated by using part or all of the EST in question as a probe or primer.

In a cDNA library there are many species of mRNA represented. Each cDNA clone can be interesting in its own right, but must be isolated form the library before further experimentation can be completed. In order to sequence any specific cDNA, it must be removed and separated (i.e. isolated and purified) from all the other sequences. This can be accomplished by many techniques known to those of skill in the art. These procedures normally involve identification of a bacterial colony containing the cDNA of interest an further amplification of that bacteria. Once a cDNA is separated from the mixed clones library, it can be used as a template for further procedures such as nucleotide sequencing.

Although claims to large number of ESTs and conesponding sequences are presented herein, the invention is not limited to these particular groupings of sequences. Thus, individual sequences are considered as applicants' discoveries or inventions, as are subgroups of sequences.

RFLP is a pattern based technique, which does note require the DNA sequence of the individual to be sequenced. The sequences of the present invention can be used to provide an alternative technique that determines the actual base-by- base EOAD DNA sequences of selected portions of an individual's genome. These sequences can be used to prepared PCR primers for amplifying and isolating such selected EOAD DNA. One can, for example, take an EST of the invention and prepare two PCR primers from the 5' and 3' ends of the EST. These are used to amplify and individual's DNA, conesponding to the EST. The amplified DNA is sequenced.

Recombinant Production Techniques and Purification

"Substantially equivalent," can refer both to nucleic acid and amino acid sequences, for example a mutant sequence, that vary from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, sequences having equivalent biological activity, and equivalent expression characteristics are considered substantially equivalent. For purposes of determining equivalence, truncation of the mature sequences should be disregarded.

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide comprising a polypeptide fragment selected from the group consisting of polypeptides depicted in Figures 1-5 [SEQ ID NO: 8-37] and encoded by the nucleotide sequences in Figure 6 or for the mature polypeptide encoded by the clone obtained as disclosed herein from the cDNA library deposited as ATCC Deposit No. 75916.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA (gDNA), and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figures 1-6 [SEQ ID NO: 1-7] to that of the clone obtained from the deposited library (ATCC Deposit No. 75916) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the wild-type DNA comprising the DNA sequence of Figures 1-6 [SEQ ID NO: 1-7] or the clone in the deposited library (ATCC Deposit No. 75916).

The polynucleotide comprising a sequence of Figure 1-6 [SEQ ID NO: 1-7] which encodes for the mature polypeptide or for the mature polypeptide encoded by the cDNA clone from the deposited library (ATCC Deposit No. 75916) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide comprising a deduced amino acid sequence of Figure 1-5 [SEQ ID NO: 8-37] or the polypeptide encoded by the cDNA clone of the deposited library (ATCC Deposit No. 75916). The variant of the polynucleotide may be a naturally occuning allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide comprising a polypeptide as shown in Figures 1-5 [SEQ ID NO: 8-37] or the same mature polypeptide encoded by the cDNA clone of the deposited library (ATCC Deposit No. 75916) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide comprising a polypeptide of Figures 1-5 [SEQ ID NO: 8-37] or the polypeptide encoded by the cDNA clone of the deposited library (ATCC Deposit No. 75916). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the gene comprising a polynucleotide coding sequence shown in Figures 1-6 [SEQ ID NO: 1-7] or of the coding sequence of the clone in the deposited library (ATCC Deposit No. 75916). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., CeU, 37:161 (1984)).

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove- described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a prefened embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded the gene comprising a polynucleotide of Figures 1-6 [SEQ ID NO: 1-7] or a clone from the deposited cDNA library.

The deposit refened to herein (ATCC Deposit No. 75916) will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. This deposit are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The terms "fragment," "derivative" and "analog" when referring to the a polypeptide or gene product comprising the sequence of the polypeptide of Figures 1-5 [SEQ ID NO: 8-37] or that encoded by the clone in the deposited cDNA library (ATCC Deposit No. 75916), means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide ofthe present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of comprising a sequence of Figures 1-5 [SEQ ID NO: 8-37] or that encoded by a clone in the deposited cDNA library (ATCC Deposit No. 75916) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occuning polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid. a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the EOAD genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces. Salmonella typhimurium: fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a prefened aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHlόa, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRJT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R, P_L and tφ. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins comprising the amino acid sequence in Figure 1-5 can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incoφorated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a -downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK). α-factor, acid phosphatase. or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation. disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication. mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS- 7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 25:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem., 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

The polypeptides of the present invention may be employed in combination with a suitable pharmaceutical canier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable canier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol. ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds. The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The amounts and dosage regimens of EOAD polypeptides of the invention administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician. Generally speaking, they are given, for example, in therapeutically effective doses of at least about 10 mg kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day and preferably the dosage is from about 10 mg/kg to about 1 mg kg body weight daily, taking into account the routes of administration, symptoms, etc.

The present invention is further directed to inhibiting Alzheimer's Disease, preferably EOAD, in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 247:456 (1988); and Dervan et al., Science, 257:1360 (1991)), thereby preventing transcription and the production of mutant EOAD gene products. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the MCP-4 (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton. FL (1988)).

Alternatively, the oligonucleotides described above can be delivered to cells by procedures in the art such that the antisense RNA or DNA may be expressed in vivo to inhibit production of mutant EOAD gene products in the manner described above. Accordingly, antisense constructs to the EOAD polypeptide can be used to treat EOAD.

The present invention is also directed to antagonist/inhibitors of the mature polypeptides comprising the polypeptide sequences of the present invention. The antagonist/inhibitors are those which inhibit or eliminate the function of the mature polypeptide.

Thus, for example, antagonists bind to a mature polypeptide of the present invention and inhibit or eliminate its function. The antagonist, for example, could be an antibody against the polypeptide which binds to the polypeptide or, in some cases, an oligonucleotide. An example of an inhibitor is a small molecule which binds to and occupies the catalytic or binding site of the mature polypeptide thereby making the catalytic or binding site inaccessible to substrate or ligand such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.

Alternatively, antagonists to the genes and polypeptides comprising a polypeptide of the present invention may be employed which bind to the receptors to which a polypeptide of the present invention normally binds. The antagonists may be closely related proteins such that they recognize and bind to the receptor sites of the natural protein, however, they are inactive forms of the polypeptide and thereby prevent the action of the EOAD polypeptide since receptor sites are occupied. In these ways, the antagonist/inhibitors may be used to treat Alzheimer's Disease, preferably EOAD.

The antagonist/inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight. EOAD Primers, Probes and Methods of Use

The present invention advantageously provides both probes and primers which detect a variety of mutant EOAD genes. Probes of the invention are useful as an initial screen for EOAD or a predisposition for EOAD, and provide a rapid alternative to traditional behavioral diagnosis of EOAD using observation and analysis of patient behavior which may lead to misdiagnosis through confusion with other dementias.

Nucleotide sequences are presented herein by single- and double-strand in the 5' to 3' direction, from left to right. The skilled artisan can use double- and single-stranded probes for hybridization analyses using methods of the invention as for other methods known in the art. One letter nucleotide symbols, A,C, G and T, used herein have their standard meaning in the art in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission and the Patent Office Rules. Nucleotide symbols N and X disclosed herein can stand for any of the nucleotides A, C, G or T. All of the finite variations of the sequences herein are embodiments of the invention and are useful in the methods of the invention. Herein "complement" refers to sequence which is "complementary" as that term is used in the art.

The term "amplification pair," as used herein, refers to a pair of oligonucleotide probes of the present invention selected to be suitable for use together in amplifying a selected EOAD gene nucleic acid sequence by a process such as polymerase chain reaction, ligase chain reaction, or strand displacement amplification, as explained in greater detail below.

Nucleic acid (i.e., DNA, gDNA, cDNA or RNA) samples for practicing the present invention may be obtained from any suitable source. Typically, the nucleic acid sample will be obtained in the form of a sample of a biological fluid or biological tissue suspected of containing a mutant EOAD gene and or from a patient suspected of having EOAD or a predisposition for EOAD. Suitable biological fluids include. but are not limited to, blood, lymph, saliva, urine and plasma. Suitable tissue samples include, but are not limited to. skin, neural, brain and soft tissue samples. Oligonucleotide primers and probes of the present invention may be derived from the sequences of the present invention, being fragments of such sequences and being of any suitable length, depending on the particular assay format employed. In general, the oligonucleotide primers are at least about 10 to about 30 nucleotides in length. For example, oligonucleotide primers used for detecting EOAD are preferably 15 to 20 nucleotides in length. The oligonucleotide probes may incoφorate the elements of a strand displacement amplification pairs of oligonucleotide probes are directed are preferably 50 to 150 nucleotides in length. Fro the sequences disclosed, the skilled artisan can readily determine what length fragments to use for the particular analysis employed considering, for example, the nucleic acid content of the fragment.

With respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein, hybridization may be canied out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 0.5x SSC and 0.1% SDS at a temperature of 20 or 30 degrees below the melting temperature of the probe, or even conditions represented by a wash stringency of 0. IxSSC and 0.1% SDS at a temperature of 10 degrees below the melting temperature of the DNA sequence to target DNA) in a standard hybridization assay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory). In general, nucleic acid sequences which hybridize to the DNA disclosed herein will have at least 65% sequence similarity, 70% sequence similarity and even 75% or greater sequence similarity with the sequence of DNA disclosed herein.

Probes of the invention can be utilized with naturally occuning sugar- phosphate backbones as well as modified backbones including phosphorothioates, dithionates. alkyl phosphonates and α-nucleotides. Modified sugar-phosphate backbones are generally illustrated by Miller and T'so, Ann. Reports Med. Chem., 25:295 (1988) and Moran et al., Nuc. Acids Res., 74:5019 (1987). Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). with DNA prefened. Use of the probes in detection methods include Northern blots (RNA detection), Southern blots (DNA detection), western blots (protein detection), and dot blots (DNA, RNA or protein),as discussed above. Other detection methods include kits containing probes on a dipstick setup and the like.

To detect hybrid molecules formed from using the probes of the invention, typically an analytically detectable marker is added to one of the probes. Probes can be labeled by several methods. Probes can be radiolabelled and detected by autoradiography. Such labels for autoradiography include ³H, ^{1 5}I, ³⁵S, ¹⁴C, and ³²P. Typically the choice of radioactive isotopes depends on research preferences involving ease of synthesis, stability, and half lives of the isotopes. Other detectable markers include ligands, fluorophores chemiluminescent agents, electrochemical via sensors, time-resolved fluorescence, enzymes, and antibodies. For example, an antibody can be labeled with a ligand. Other detectable markers for use with probes ofthe invention include biotin, radionucleotides, enzyme inhibitors, co-enzymes, luciferins, paramagnetic metals, spin labels, and monoclonal antibodies. The choice of label dictates the manner in which the label is bound to the probe.

Radioactive nucleotides can be incoφorated into probes of the invention by several means. Such means include nick translation of double-stranded probes, copying single-stranded M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase I of E. coli or other such DNA polymerase in the presence of radioactive dNTP, transcribing cDNA from RNA templates using reverse transcriptase in the presence of radioactive dNTP, transcribing RNA from vectors containing strong promoters such as SP6 promoters or T7 promoters using SP6 or T7 RNA polymerase in the presence of radioactive rNTP, tailing the 3' ends of probes with radioactive nucleotides using terminal transferase, and by phosphorylation of the 5' ends of probes using gamma ³²P ATP and polynucleotide kinase.

Amplification of a selected, or target, nucleic acid sequence may be canied out by any suitable means. See generally, D. Kwoh and T. Kwoh, Am. Biotechnol. Lab. 8: 14-25(1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification (See: D. Kwoh et al., Proc. Nat'l. Acad. Sci. USA 86: 1173-1177 (1989)), self-sustained sequence replication (See: J. Guatelli et al., Proc. Natl. Acad. Sci. USA 82:1874-1878 (1990)), and the Qβ replicase system (See: P. Lizardi et al., BioTechnology 6: 1197-1202 (1988)).

Polymerase chain reaction (PCR) is canied out in accordance with known techniques. See, e.g.,: U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosure of all U.S. Patent references cited herein are to be incoφorated herein by reference). In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present. These steps are cyclically repeated until the desired degree of amplification is obtained. Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g.. an oligonucleotide probe of the present invention), the probe carrying a detectable label, and then detecting the label in accordance with known techniques.

Ligase chain reaction (LCR) is canied out in accordance with known techniques. See, e.g.,: R. Weiss, Science 254:1292 (1991). In general, the reaction is canied out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. Each pair together completely overlaps the strand to which it conesponds. The reaction is carried out by, first denaturing (e.g., separating) the strands of sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes is ligated together, then separating the reaction product, and then cyclically repeating the process until the sequence has been amplified to the desired degree. Detection may then be canied out in like manner as described above with respect to PCR.

Strand displacement amplification (SDA) is also canied out in accordance with know techniques. See: G. Walker, et al., Proc. Nat'l. Acad. Sci. USA 89:392- 396 (1992); G. Walker et al., Nucleic Acids Res. 20:1691-1696(1992). SDA may be carried out with a single amplification primer or a pair of amplification primers, with exponential amplification being achieved with the latter. In general, SDA amplification primers comprise, in the 5' to 3' direction, a flanking sequence (the DNA sequence of which is noncritical), a restriction site for the restriction enzyme employed in the reaction, and an oligonucleotide sequence (e.g., oligonucleotide probe of the present invention) which hybridizes to the target sequence to be amplified and/or detected. The flanking sequence, which simply serves to facilitate binding of the restriction enzyme to the recognition site is preferably about 15 to 20 nucleotides in length; the restriction site is functional in the SDA reaction (i.e., phosphorothioate linkages incoφorated into the primer strand do not inhibit subsequent nicking - a condition which may be satisfied through use of a nonpalindromic recognition site); the oligonucleotide probe portion is preferably about 13 to 15 nucleotides in length. SDA is carried out with a single amplification primer as follows: a restriction fragment (preferably about 50 to 100 nucleotides in length and preferably of low GC content) containing the sequence to be detected is prepared by digesting a DNA sample with one or more restriction enzymes, the SDA amplification primer is added to a reaction mixture containing the restriction fragment so that a duplex between the restriction fragment and the amplification primer is formed with a 5' overhang at each end, a restriction enzyme which binds to the restriction site on the amplification probe (e.g., Hindi) is added to the reaction mixture, an exonuclease deficient DNA polymerase (e.g. an exonuclease deficient form of E. coli DNA polymerase I. See: V. Derbyshire, Science 240:199-201 ( 1988)) is added to the reaction mixture, and three dNTPs and one dNTP(αS], with the dNTP[αS] selected so that a phosphorothioate linkage is incoφorated into the primer strand at the restriction site for the particular restriction enzyme employed (e.g., dGTP, dCTP, dTTP, amd dATPfαS] when the restriction enzyme is HincII) are added to the reaction mixture. The DNA polymerase extends the 3' ends ofthe duplex with the dNTPs to form a downstream complement of the target strand, the restriction enzyme nicks the restriction site on the amplification primer, and the DNA polymerase extends the 3' end of the amplification primer at the nick to displace the previously formed downstream complement of the target strand. The process is inherently repetitive because the restriction enzyme continuously nicks new complementary strands as they are formed from the restriction site, and the DNA polmerase continuously forms new complementary strands from the nicked restriction site. SDA can be carried out with a pair of primers on a double stranded target DNA sequence, with the second primer binding to the 5' end of the complementary strand, so that two sets of repetitive reactions are occurring simultaneously, with the process proceeding exponentially because the products of one set of reactions serve as target for the amplification primer in the other set of reactions. In addition, the step of first digesting the DNA sample to form a restriction fragment can be eliminated by exploiting the strand displacing activity of the DNA polymerase and adding a pair of "bumper" primers which bind to the substrate at a flanking position 5' to the position at which each amplification primer binds. Each bumper primer extension product displaces the conesponding amplification primer extension product, and the two displaced, complementary, amplification primer extension products bind to one another to form a double- stranded DNA fragment which can the serve as a substrate for exponential SDA with that pair of SDA primers.

When SDA is employed, the oligonucleotide probes of the invention are preferably selected so that guanine plus cytosine content is low, preferably comprising less than 70% of the total nucleotide composition of the probe. Similarly, the target sequence should be of low GC content to avoid the formation of secondary structures.

A kit for detecting mutant EOAD gene nucleic acid in a nucleic acid sample contains at least one probe fragment derived from a sequence of the present invention, and hybridization solution for enabling hybridization between the probe or probes and the nucleic acid sample, with the probe either suspended in the solution or provided separately in lyophilized form. One example of a suitable hybridization solution is a solution comprised of 6x SSC (0.9M sodium chloride. 0.09M sodium citrate, pH 7.0), O.lM EDTA pH 8.0, 5x Denhardt's solution (0.1% (w/v) Ficoll Type 400, 0.1% (w/v) polyvinylpynolidone, 0.1% (w/v) bovine serum albumin), and 100 μg/ml sheared, denature salmon sperm DNA, commercially available from Bethesda Research Laboratories, Gaithersburg, Md. 20877 USA under Catalog No. 5565UA. See also T. Maniatis et al., Molecular Cloning: A Laboratory Manual, 387-388 (1982)(Cold Spring Harbor Laboratory). The components of the kit are packaged together in a common container (e.g., a container sealed with a frangible seal), the kit typically including an instruction sheet for carrying out a specific embodiment of the method of the present invention. Additional optional components of the kit, depending on the assay format to be employed, include a second probe for carrying out PCR as explained above (or, in the case of a kit for carrying out a detecting step (e.g., a probe of the invention labeled with a detectable marker and optionally an enzyme substrate when the detectable marker is an enzyme).

The polypeptides having the amino acid sequence depicted in Figures 1-5 [SEQ ID NO: 8-37], their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides conesponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:12), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.

A kit for detecting mutant EOAD protein in a protein sample contains at least one antibody against a polypeptide of the present invention, and protein binding solution for enabling binding between the antibody and the polypeptide sample, with the antibody either suspended in the solution or provided separately in lyophilized form

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a cunent need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymoφhisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in conelating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA. thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene conesponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific- cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensityor simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map data. Such data are found, for example, in V. McKusick. Mendelian Inheritance in Man (publicly available on line via computer). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. With cunent resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).

Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymoφhisms. Skilled artisans can readily obtain the EOAD gene from normal human individuals using the nucleotide and amino acid sequences of the invention. Mutants of the gene can be determined by comparison of the normal sequence to that gene sequence derived from individuals with EOAD using the nucleotide and amino acid sequences of the invention.

The present invention is further described by the following non-limiting examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

Examples

Example 1 Complete Sequence of an EOAD EST Clone Inserts

There are a number of methods known to those with skill in the art of molecular biology to obtain sequence information from the cDNAs conesponding to the EOAD EST sequences. Procedures for these methods are provided in Basic Methods in Molecular Biology (David et al. supra). One was to acquire more information about the cDNA from which an EST was derived is to sequence the remainder of the EOAD cDNA clone. Briefly, EOAD EST clones are digested with the restriction enzymes Sail and Kpnl or Pstl and BamHI (for deletions from the Forward primer and Reverse primer ends ofthe insert, respectively). The Kpnl and Pstl enzymes leave 3' sticky ends following digestion, which Exonuclease III is unable to bind. This results in unidirectional deletions into the cDNA insert leaving the vector sequence undisturbed. After addition of Exonuclease III to the Forward and Reverse deletion reactions, aliquots of the reaction are removed at defined time intervals and the reaction is stopped to prevent further deletion. S 1 nuclease and Klenow DNA polymerase are added to create blunt ended fragments suitable for ligation. Samples for each time point are purified by electrophoresis through an agarose gel and religated. Two to four representative clones from each time point in each direction are sequenced to give between 200 and 400 base pairs of sequence data. Careful selection of deletion conditions and time points allow a deletion series of approximately 100-200 base pairs difference in length at each consecutive time point. Sequence fragments are reassembled into a redundant contiguous sequence using the INHERIT software from Applied Biosystems, Inc. (Foster City, CA) In this way, the complete insert from the cDNA clones is sequenced on both strands to an average redundancy between three and four (each base is sequenced between three and four times, on average).

Example 2 EOAD Gene Expression from DNA Sequences Corresponding to EOAD ESTs

An EOAD gene sequence of the present invention coding part of a human EOAD gene product is introduced into an expression vector using conventional technology. (Techniques to transfer cloned sequences into expression vectors that direct protein translation in mammalian, yeast, insect or bacterial expression systems are well known in the art.) Commercially available vectors and expression systems are available from a variety of suppliers including Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego. California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon expressing organism, as explained by Hatfield, et al., U.S. Patent No. 5,082,767, incoφorated herein by this reference.

The following is provided as one exemplary method to generate polypeptide(s) from cloned EOAD cDNA sequence(s) which include the coding region for the peptide of interest and which EOAD cDNA sequences are obtained by use of an EST of the present invention, as hereinabove described. For EOAD cDNA sequences lacking a poly A sequences, this sequence can be added to the construct by, for example, splicing out the poly A sequence from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and incoφorating it into the mammalian expression vector pXTl (Stratagene). pXTl contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRS in the construct allow efficient stable transfection. The vector includes the Heφes Simplex thymidine kinase promoter and the selectable neomycin gene. The EOAD cDNA is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the cDNA and containing restriction endonuclease sequences for Pstl incoφorated into the 5' primer and Bglll at the 5' end of the conesponding cDNA 3' primer, taking care to ensure that the cDNA is positioned such that its followed with the poly A sequence. The purified fragment obtained from the resulting PCR reaction is digested with Pstl. blunt ended with an exonuclease, digested with Bglll, purified and ligated to pXTl. now containing a poly A sequence and digested Bglll.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand, Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Missouri). The protein is preferably released into the supernatant. However, it the protein has membrane binding domains, the protein may additionally be retained within the cell or expression may be restricted to the cell surface.

Since it may be necessary to purify and locate the transfected product, synthetic 15-mer peptides synthesized form the predicted cDNA sequences are injected into mice to generate antibody to the polypeptide encoded by the cDNA. A method to make antibody production possible, the EOAD cDNA sequence is additionally incoφorated into eukaryotic expression vectors and expressed as a chimeric with, for example, β-globin. Antibody to β-globin is used to purify the chimeric. Conesponding protease cleavage sites engineered between the β-globin gene and the cDNA are then used to separate the two polypeptide fragments form one another after translation. A useful expression vector for generating β-globin chimerics is pSG5 (Stratagene). This vector encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incoφorated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as David et al. and many of the methods are available form the technical assistance representatives from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from either construct using in vitro translation systems such as In vitro Express™ translation Kit. (Stratagene).

Example 3 Production of an Antibody to a Human EOAD Protein

Substantially pure protein or polypeptide is isolated from the transfected or transformed cells using methods known in the art or described herein. The protein can also be produced in a recombinant prokaryotic expression system, such as E. coli, or can be chemically synthesized. Concentration of protein in the final ϊ preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, Nature, 256:495 (1975) or modifications of the methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth o the culture is continued. Anti body producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol, 70:419 (1980), and modified methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal anti body production are described in Davis, L. et al. Basic Methods in Molecular Biology, Elsvier, New York. Section 21-2 (1986).

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANTS: SmithKline Beecham Corporation and SmithKline Beecham PLC

(ii) TITLE OF THE INVENTION: EARLY ONSET ALZHEIMER'S DISEASE

GENE AND GENE PRODUCTS

(iii) NUMBER OF SEQUENCES: 37

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation

(B) STREET: 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19406-2799

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 1.5

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Gimmi, Edward R (3) REGISTRATION NUMBER: 38,891

(C) REFERENCE/DOCKET NUMBER: P50352 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-270-4478

(B) TELEFAX: 610-270-5090

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 806 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AATTCGGCAN AGTCCAAGTC TTCCTGACCA CCTTGCACTA TTGGACTTTG GAAGGAGGTG 60

TTAAGCCGTN TCAGGTTCAG AAGGACTGGT GGAACGTGAT AACCTGAAAC CTTCCTCCAC 120

CCTATAGAAA ACGATTTTGA ACATACTTCA TCGCAGTGGA CTGTGTCCCT CGGTGCAGAA 180

GGATATCTTT TGCTAAAACT TGTATGAAGT AGCGTCACCT GACACAGGGA GCCACGTCTT 240

ACTACCAGAT TTGAGGGACG NGGTCAAGGA GATATGATAG GCCCGGAAGT TGCTGTGCCC 300

TGATGGTCTA AACTCCCTGC NCCAGTTCCT CTATACTATC CGGGCCTTCA ACGACACGGG 360

CATCAGCAGC TTGACGCGTG GTCACAGGAC GATTTCACTG ACACTGCGAA CTCTCAGGAC 420

GTAGTCGTCG AACTGCGCAC CAGTGTCCTG CTAAAGTGAC TGTGACGCTT GAGAGTCCTG 480

TACCGTTACC AAGAGGTTAG GTGAAGTGGT TTAAACCAAA CGGAACTCTT CATCTTAAAC 540

ATGGCAATGG TTCTCCAATC CACTTCACCA AATTTGGTTT GCCTTGAGAA GTAGAATTTG 600

TACAAGTTGA AATCAACCCA TAATTCNGTA TTAACTGAAT TCTGAACTTT TCAAGGGGTA 660

ATGTTCAACT TTAGTTGGGT ATTAAGNCAT AATTGACTTA AGACTTGAAA AGTTCCCCAT 720

CTGTGAGGAA GAGCAGCACC ACAGCAGAAT GGGGATTGGN GAAGACACTC CTTCTCGTCG 780

TGGTGTCGTC TTACCCCTAA CCNCTT 806

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 832 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GGCAGAGTGG NATGTTTCTT CTTTGACTAT AACAAAATCN GGGGAGGNCA AAGGTGCATT 60

CCGTCTCACC NTACAAAGAA GAAACTGATA TTGTTTTAGN CCCCTCCNGT TTCCACGTAA 120

TTCCTGTGTC CACATCTAAC AAAGTCAAGG TTCCCGGCNT GGACTTTNNG AAGCTTCCTT 180

AAGGACACAG GTGTAGATTG TTTCAGTTCC AAGGGCCGNA CCTGAAANNC TTCGAAGGAA 240

CCAAGTCTTC CTGACCACCT TGGCACTATT GGACTTTGGA AGGAGGTGCC TATAGAAAAC 300

GGTTCAGAAG GACTGGTGGA ACCGTGATAA CCTGAAACCT TCCTCCACGG ATATCTTTTG 360

GATTTTGAAA CATACTTCAT CGCAGTGGNA CTGTGTCCCT GCGGTGCAGA AACTACCAGA 420

CTAAAACTTT GTATGAAGTA GCGTCACCNT GACACAGGGA CGCCACGTCT TTGATGGTCT 480

TTTNGAGGGA CGAGGTCAAG GNGNTGATGA TAGGCCCGGN AAGTTGCTGT GNCCCATNCA 540

AAANCTCCCT GCTCCAGTTC CNCNACTACT ATCCGGGCCN TTCAACGACA CNGGGTANGT 600

GCAGTTTGAC GCGTGGTCAC AGGACGATTT CACTGGACAN GGNGGAACTC TCNGGGNTTA 660

CGTCAAACTG CGCACCAGTG TCCTGCTAAA GTGACCTGTN CCNCCTTGAG AGNCCCNAAT 720

CCGTTTACCC AGAGGTTTAG GTGGAAGTTG GTTTAAACCA AACGGAACTT TTNCATGGCA 780

AATGGGTCTC CAAATCCACC TTCAACCAAA TTTGGTTTGC CTTGAAAANG TA 832

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 778 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

GGCANAGACG TATCTGGNTT GAAACAC-CTC AGGAGAGAAA TGAAACGCTT TTGGNAGCTC 60

CCGTNTCTGC ATAGACCNAA CTTTGTCGAG TCCTCTCTTT ACTTTGCGAA AACCNTCGAG 120

TNNTTTACTC CTCAACAATG GTGTGGTTGG TGAATATGGC AGAAGGAGAC CCGGTAAGCr 180

ANNAAATGAG GAGTTGTTAC CACACCAACC ACTTATACCG TCTTCCTCTG GGCCATTCGA 240 CAAAGGAGAG TATCCAAAAA TTCCAAGTAT AATGCAGAAA GCACAGAAAG GGAGTCACAA 300

GTTTCCTCTC ATAGGTTTTT AAGGTTCATA TTACGTCTTT CGTGTCTTTC CCTCAGTGTT 360

GACACTGTTG CAGAGAATGA TGGATGGCGG GTTCAGTGAG GAATGGGTAG CCCAGAGGNA 420

CTGTGACAAC GTCTCTTACT ACCTACCGCC CAAGTCACTC CTTACCCATC GGGTCTCCNT 480

CAGTGCATCT TAGGGCCTCA TNCGCTC AC ACCTGAGTNC ACGAGCTGCT G ACCAGGAA 540

GTCACGTAGA ATCCCGGAGT ANGCGAGATG TGGACTCANG TGCTCGACGA CATGGTCCTT 600

CTTTNCCAGC CAGTATTCCT CGCTGGTTAA AGACCCCAGA GGAAAGGGNG AGTTAAAACT 660

GAAANGGTCG GTCATAAGGA GCGACCAATT TCTGGGGTCT CCTTTCCCNC TCAATTTTGA 720

TGGGNTTGGG GANGTTTNCC ATTTCTTACA CCCNAACCCC TNCAAANGGT AAAGAATG 778

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 996 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GGCACGAGGG TTGGTGAATA TGGCAGAAGG AGACCCGGAA GCTCAAAGGA GAGTATCCAA 60

CCGTGCTCCC AACCACTTAT ACCGTCTTCC TCTGGGCCTT CGAGTTTCCT CTCATAGGTT 120

AAATTCCAAG TATAATGCAG AAAGCACAGA AAGGGAGTCA CAAGACACTG TTGCAGAGAA 180

TTTAAGGTTC ATATTACGTC TTTCGTGTCT TTCCCTCAGT GTTCTGTGAC AACGTCTCTT 240

TGATGATGGC GGGTTCAGTG AGGAATGGGA AGCCCAGAGG GACAGTCATC TAGGGCCTCA 300

ACTACTACCG CCCAAGTCAC TCCTTACCCT TCGGGTCTCC CTGTCAGTAG ATCCCGGAGT 360

TCGCTCTACA CCTGAGTCAC GAGCTGCTGT CCAGGAACTT TCCAGCAGTA TCCTCGCTGG 420

AGCGAGATGT GGACTCAGTG CTCGACGACA GGTCCTTGAA AGGTCGTCAT AGGAGCGACC 480

TGAAGACCCA GAGGAAAGGG GAGTAAAACT TGGATTGGGA GATTTCATTT TCTACAGTGT 540

ACTTCTGGGT CTCCTTTCCC CTCATTTTGA ACCTAACCCT CTAAAGTAAA AGATGTCACA 600

TCTGGTTGGT AAAGCCTTCA GCAACAGCCA GTGGAGACTG GGAACACAAC CATAGCCTGT 660

AGACCAACCA TTTCGGAAGT CGTTGTCGGT CACCTCTGAC CCTTGTGTTG GTATCGGACA 720

TTTCGTAGCC ATATTAATTG GTTTGTGCCT TACATTATTT ACTCCTTGCC ATTTTCAAGG 780

AAAGCATCGG TATAATTAAC CAAACACGGA ATGTAATAAA TGAGGAACGG TAAAAGTTCC 840

AAAGCATTGC CAGTTNTTTC CAATTTTCCC ATCANCTTTT GGGGTTTTGT TTTCTTACTT 900

TTTCGTAACG GTCAANAAAG GTTAAAAGGG TAGTNGAAAA CCCCAAAACA AAAGAATGAA 960

TNGGCACCAG ATTATCTTAN CCGTGGTCTA ATAGAA 996 (2) INFORMATION FOR SEQ ID NO:5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 688 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

AACAAAGTCA AGATTCCCGG CTGGACTTTT GCAGCTTCCT TCCAAGTCTT CCTGACCACC 60

TTGTTTCAGT TCTAAGGGCC GACCTGAAAA CGTCGAAGGA AGGTTCAGAA GGACTGGTGG 120

TTGCACTATT GGACTTTGGA AGGAGGTGCC TATAGAAAAC GATTTTGAAC ATACTTCATC 180

AACGTGATAA CCTGAAACCT TCCTCCACGG ATATCTTTTG CTAAAACTTG TATGAAGTAG 240

GCAGTGGACT GTGTCCCTCG GTGCAGAAAC TACCAGATTT GAGGGACGAG GTCAAGGAGA 300

CGTCACCTGA CACAGGGAGC CACGTCTTTG ATGGTCTAAA CTCCCTGCTC CAGTTCCTCT 360

TATGATAGGC CCGGAAGTTG CTGTGCCCCA TCAGCAGCTT GACGCGTGGT CACAGGACGA 420

ATACTATCCG GGCCTTCAAC GACACGGGGT AGTCGTCGAA CTGCGCACCA GTGTCCTGCT 480

TTTCACTGAC ACTNCGAACT CTC GGNCTA CCGTTNACCA AGAGGTTAGG TGAAAGTGGG 540

AAAGTGACTG TGANGCTTGA GAGTCCNGAT GGCAANTGGT TCTCCAATCC ACTTTCACCC 600

TTTAANANCA AAACGGAACT CTTTCATCTT NAAACTACAA CGGTAAATTN TNGTTTTGCC 660

TTGAGAAAGT AGAANTTTGA TGTTGCCA 688

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1184 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GGCANAGACG TATCTGGNTT GAAACAGCTC AGGAGAGAAA TGAAACGCTT TTGGNAGCTC 60

CCGTNTCTGC ATAGACCNAA CTTTGTCGAG TCCTCTCTTT ACTTTGCGAA AACCNTCGAG 120

TNNTTTACTC CTCAACAATG GTGTGGTTGG TGAATATGGC AGAAGGAGAC CCGGTAAGCT 180

ANNAAATGAG GAGTTGTTAC CACACCAACC ACTTATACCG TCTTCCTCTG GGCCATTCGA 240

CAAAGGAGAG TATCCAAAAA TTCCAAGTAT AATGCAGAAA GCACAGAAAG GGAGTCACAA 300

GTTTCCTCTC ATAGGTTTTT AAGGTTCATA TTACGTCTTT CGTGTCTTTC CCTCAGTGTT 360

GACACTGTTG CAGAGAATGA TGGATGGCGG GTTCAGTGAG GAATGGGWAG CCCAGAGGGA 420

CTGTGACAAC GTCTCTTACT ACCTACCGCC CAAGTCACTC CTTACCCWTC GGGTCTCCCT 480

CAGTGCATCT TAGGGCCTCA TNCGCTCTAC ACCTGAGTNC ACGAGCTGCT GTACCAGGAA 540

GTCACGTAGA ATCCCGGAGT ANGCGAGATG TGGACTCANG TGCTCGACGA CATGGTCCTT 600

CTTTNCCAGC CAGTATTCCT CGCTGGTKAA AGACCCCAGA GGAAAGGGNG AGTTAAAACT 660

GAAANGGTCG GTCATAAGGA GCGACCAMTT TCTGGGGTCT CCTTTCCCNC TCAATTTTGA 720

TGGRNTTGGG GAGRTTTNCM WTTTCTTACA GTGTTCTGGT TGGTAAAGCC TTCAGCAACA 780

ACCYNAACCC CTCYAAANGK WAAAGAATGT CACAAGACCA ACCATTTCGG AAGTCGTTGT 840

GCCAGTGGAG ACTGGGAACA CAACCATAGC CTGTTTTCGT AGCCATATTA ATTGGTTTGT 900

CGGTCACCTC TGACCCTTGT GTTGGTATCG GACAAAAGCA TCGGTATAAT TAACCAAACA 960

GCCTTACATT ATTTACTCCT TGCCATTTTC AAGGAAAGCA TTGCCAGTTN TTTCCAATTT 1020

CGGAATGTAA TAAATGAGGA ACGGTAAAAG TTCCTTTCGT AACGGTCAAN AAAGGTTAAA 1080

TCCCATCANC TTTTGGGGTT TTGTTTTCTT ACTTTNGGCA CCAGATTATC TTAGGGTAGT 1140

NGAAAACCCC AAAACAAAAG AATGAAANCC GTGGTCTAAT AGAA 1184

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 884 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :

AACAAAGTCA AGATTCCCGG CTGGAMTTYK GCANAGCTTC CTTCCAAGTC TTCCTGACCA 60

TTGTTTCAGT TCTAAGGGCC GACCTKAARM CGTNTCGAAG GAAGGTTCAG AAGGACTGGT 120

CCTTGCACTA TTGGACTTTG GAAGGAGGTG CCTATAGAAA ACGATTTTGA ACATACTTCA 180

GGAACGTGAT AACCTGAAAC CTTCCTCCAC GGATATCTTT TGCTAAAACT TGTATGAAGT 240

TCGCAGTGGA CTGTGTCCCT CGGTGCAGAA ACTACCAGAT TTGAGGGACG NGGTCAAGGA 300 AGCGTCACCT GACACAGGGA GCCACGTCTT TGATGGTCTA AACTCCCTGC NCCAGTTCCT 360

GATATGATAG GCCCGGAAGT TGCTGTGCCC CATCAGCAGC TTGACGCGTG GTCACAGGAC 420

CTATACTATC CGGGCCTTCA ACGACACGGG GTAGTCGTCG AACTGCGCAC CAGTGTCCTG 480

GATTTCACTG ACACTNCGAA CTCTCAGGNC TACCGTTNAC CAAGAGGTTA GGTGAAAGTG 540

CTAAAGTGAC TGTGANGCTT GAGAGTCCNG ATGGCAANTG GTTCTCCAAT CCACTTTCAC 600

GGTTTAANAN CAAAACGGAA CTCTTTCATC TTNAAACTAC AACGGTTGAA ATCAACCCAT 660

CCAAATTNTN GTTTTGCCTT GAGAAAGTAG AANTTTGATG TTGCCAACTT TAGTTGGGTA 720

AATTCNGTAT TAACTGAATT CTGAACTTTT CAAGGGGTAC TGTGAGGAAG AGCAGCACCA 780

TTAAGNCATA ATTGACTTAA GACTTGAAAA GTTCCCCATG ACACTCCTTC TCGTCGTGGT 840

CAGCAGAATG GGGATTGGNG AAGTCGTCTT ACCCCTAACC NCTT 884

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asn Ser Ala Xaa Ser Lys Ser Ser Pro Pro Cys Thr Ile Gly Leu Trp

1 5 10 15

Lys Glu Val Pro lie Glu Asn Asp Phe Glu His Thr Ser Ser Gin Trp

20 25 30

Thr Val Ser Leu Gly Ala Glu Thr Thr Arg Phe Glu Gly Arg Gly Gin

35 40 45

Gly Asp Met Ile Gly Pro Glu Val Ala Val Pro His Gin Gin Leu Asp

50 55 60

Ala Trp Ser Gin Asp Asp Phe Thr Asp Thr Ala Asn Ser Gin Asp Tyr 65 70 75 80

Arg Tyr Gin Glu Val Arg Ser Giy Leu Asn Gin Thr Glu Leu Phe Ile

85 90 95

Leu Asn Tyr Lys Leu Lys Ser Thr His Asn Ser Val Leu Thr Glu Phe

100 105 110

Thr Phe Gin Gly Val Leu Gly Arg Ala Ala Pro Gin Gin Asn Gly Asp 115 120 125 Trp Xaa

130

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Ile Arg Xaa Ser Pro Ser Leu Pro Asp His Leu Ala Leu Leu Asp Phe

1 5 10 15

Gly Arg Arg Cys Leu Lys Thr lie Leu Asn Ile Leu His Arg Ser Gly

20 25 30

Leu Cys Pro Ser Val Gin Lys Leu Pro Asp Leu Arg Asp Xaa Val Lys

35 40 45

Glu Ile Ala Arg Lyε Leu Leu Cys Pro Ile Ser Ser Leu Thr Arg Gly

50 55 60

His Arg Thr Ile Ser Leu Thr Leu Arg Thr Leu Arg Thr Thr Val Thr 65 70 75 80

Lys Arg Leu Gly Glu Val Val Thr Lys Arg Asn Ser Ser Ser Thr Thr

85 90 95

Ser Asn Gin Pro Ile Ile Xaa Tyr Leu Asn Ser Glu Leu Phe Lys Gly

100 105 110

Tyr Cys Glu Glu Glu Gin His His Ser Arg Met Gly lie Gly Glu 115 120 125

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 131 amino acids (3) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Phe Gly Xaa Val Gin Val Phe Leu Thr Thr Leu His Tyr Trp Thr Leu

1 5 10 15

Glu Gly Gly Ala Tyr Arg Lys Arg Phe- Thr Tyr Phe Ile Ala Val Asp

20 25 30

Cys Val Pro Arg Cys Arg Asn Tyr Gin Ile Gly Thr Xaa Ser Arg Arg

35 40 45

Tyr Asp Arg Pro Gly Ser Cys Cys Ala Pro Ser Ala Ala Arg Val Val

50 55 60

Thr Gly Arg Phe His His Cys Glu Leu Ser Gly Leu Pro Leu Pro Arg 65 70 75 80

Gly Val Lys Trp Phe Lys Pro Asn Gly Thr Leu His Leu Lys Leu Gin

85 90 95

Val Glu Ile Asn Pro Phe Xaa Ile Asn Ile Leu Asn Phe Ser Arg Gly

100 105 110

Thr Val Arg Lys Ser Ser Thr Thr Ala Glu Trp Glx 115 120

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Phe Xaa Asn Pro His Ser Ala Val Val Leu Leu Phe Leu Thr Val Pro 1 5 10 15 Leu Glu Lys Phe Arg Ile Gin Leu Ile Xaa Asn Tyr Gly Leu lie Ser

20 25 30

Thr Cys Ser Leu Arg Arg Val Pro Phe Gly Leu Asn His Phe Thr Pro

35 40 45

Leu Gly Asn Gly Ser Pro Glu Ser Ser Gin Cys Gin Asn Arg Pro Val

50 55 60

Thr Thr Arg Gin Ala Ala Asp Gly Ala Gin Gin Leu Pro Gly Leu Ser 65 70 75 80

Tyr Leu Leu Asp Xaa Val Pro Gin Ile Trp Phe Leu His Arg Gly Thr

85 90 95

Gin Ser Thr Ala Met Lys Tyr Val Gin Asn Arg Phe Leu Ala Pro Pro

100 105 110

Ser Lys Val Gin Cys Lys Val Val Arg Lys Thr Trp Thr Xaa Pro Asn 115 120 125

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Xaa Gin Ser Pro Phe Cys Cys Gly Ala Ala Leu Pro His Ser Thr Pro

1 5 10 15

Lys Val Gin Asn Ser Val Asn Thr Glu Leu Trp Val Asp Phe Asn Leu

20 25 30

Phe Lys Met Lys Ser Ser Val Trp Phe Lys Pro Leu His Leu Thr Ser

35 40 45

Trp Arg Ser Glu Phe Ala Val Ser Val Lys Ser Ser Cys Asp His Ala

50 55 60

Ser Ser Cys Trp Gly Thr Ala Thr Ser Gly Pro lie lie Ser Pro Pro 65 70 75 80

Arg Pro Ser Asn Leu Val Val Ser Ala Pro Arg Asp Thr Val His Cys 85 90 95 Asp Glu Val Cys Ser Lys Ser Phe Ser Ile Gly Thr Ser Phe Gin Ser

100 105 110

Pro Ile Val Gin Gly Gly Gin Glu Asp Leu Asp Xaa Ala Glu

115 120 125

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ser Pro Ile Pro Ile Leu Leu Trp Cys Cys Ser Ser Ser Gin Tyr Pro

1 5 10 15

Leu Lys Ser Ser Glu Phe Ser Tyr Xaa Ile Met Gly Phe Gin Leu Val

20 25 30

Val Asp Glu Glu Phe Arg Leu Val Thr Thr Ser Pro Asn Leu Leu Val

35 40 45

Thr Val Val Leu Arg Val Arg Ser Val Ser Glu Ile Val Leu Pro Arg

50 55 60

Val Lys Leu Leu Met Gly His Ser Asn Phe Arg Ala Tyr His Ile Ser 65 70 75 80

Leu Thr Xaa Ser Leu Lys Ser Gly Ser Phe Cys Thr Glu Gly His Ser

85 90 95

Pro Leu Arg Ser Met Phe Lys lie Val Phe Tyr Arg His Leu Leu Pro

100 105 110

Lys Ser Asn Ser Ala Arg Trp Ser Gly Arg Leu Gly Leu Cys Arg Ile 115 120 125

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Gly Arg Val Xaa Cys Phe Phe Phe Asp Tyr Asn Lys Ile Xaa Gly Gly

1 5 10 15

Gin Arg Cys Ile Phe Leu Cys Pro His Leu Thr Lys Ser Arg Phe Pro

20 25 30

Ala Trp Thr Xaa Xaa Ser Phe Leu Pro Ser Leu Pro Asp His Leu Gly

35 40 45

Thr Ile Gly Leu Trp Lys Glu Val Pro Ile Glu Asn Asp Phe Glu Thr

50 55 60

Tyr Phe Ile Ala Val Xaa Leu Cys Pro Cys Gly Ala Glu Thr Thr Arg 65 70 75 80

Phe Xaa Gly Thr Arg Ser Arg Xaa Ala Arg Xaa Val Ala Val Xaa His

85 90 95

Xaa Ala Val Arg Val Val Thr Gly Arg Phe His Trp Thr Xaa Xaa Asn

100 105 110

Ser Xaa Gly Leu Pro Phe Thr Gin Arg Phe Arg Trp Lys Leu Val Thr

115 120 125

Lys Arg Asn Phe Xaa 130

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: pepcide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Ala Glu Trp Xaa Val Ser Ser Leu Thr Ile Thr Lys Ser Gly Glu Xaa

1 5 10 15

Lys Gly Ala Phe Ser Cys Val His Ile Gin Ser Gin Gly Ser Arg Xaa

20 25 30

Gly Leu Xaa Glu Ala Ser Phe Gin Val Phe Leu Thr Thr Leu Ala Leu

35 40 45

Leu Asp Phe Gly Arg Arg Cys Leu Lys Thr Ile Leu Lys His Thr Ser

50 55 60

Ser Gin Trp Xaa Cys Val Pro Ala Val Gin Lys Leu Pro Asp Xaa Glu 65 70 75 80

Gly Arg Gly Gin Gly Xaa Asp Asp Arg Pro Gly Lys Leu Leu Xaa Pro

85 90 95

Xaa Gin Gin Phe Asp Ala Trp Ser Gin Asp Asp Phe Thr Gly Xaa Gly

100 105 110

Gly Thr Leu Xaa Xaa Tyr Arg Leu Pro Arg Gly Leu Gly Gly Ser Trp

115 120 125

Phe Lys Pro Asn Gly Thr Phe Xaa 130 135

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Gin Ser Gly Met Phe Leu Leu Leu Gin Asn Xaa Gly Arg Xaa Lys Val

1 5 10 15

His Phe Pro Val Ser Thr Ser Asn Lys Val Lys Val Pro Gly Xaa Asp

20 25 30

Phe Xaa Lys Leu Pro Ser Lys Ser Ser Pro Pro Trp His Tyr Trp Thr 35 40 45 Leu Glu Gly Gly Ala Tyr Arg Lys Arg Phe Asn Ile Leu His Arg Ser

50 55 60

Gly Thr Val Ser Leu Arg Cys Arg Asn Tyr Gin Ile Xaa Arg Asp Glu 65 70 75 80

Val Lys Xaa Xaa Met Ile Gly Pro Xaa Ser Cys Cys Xaa Pro Xaa Ser

85 90 95

Ser Leu Thr Arg Gly His Arg Thr Ile Ser Leu Asp Xaa Xaa Glu Leu

100 105 110

Ser Gly Xaa Thr Val Tyr Pro Glu Val Val Glu Val Gly Leu Asn Gin

115 120 125

Thr Glu Leu Xaa His 130

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Xaa Lys Ser Ser Val Trp Phe Lys Pro Thr Ser Thr Thr Ser Gly Thr

1 5 10 15

Val Xaa Pro Glu Ser Ser Xaa Xaa Ser Ser Glu Ile Val Leu Pro Arg

20 25 30

Val Lys Leu Leu Xaa Gly Xaa Gin Gin Leu Xaa Gly Pro Ile Ile Xaa

35 40 45

Xaa Leu Thr Ser Ser Leu Xaa lie Trp Phe Leu His Arg Arg Asp Thr

50 55 60

Val Pro Leu Arg Ser Met Phe Gin Asn Arg Phe Leu Ala Pro Pro Ser 65 70 75 80

Lys Val Gin Cys Gin Gly Gly Gin Glu Asp Leu Glu Gly Ser Phe Xaa

85 90 95

Lys Ser Xaa Pro Gly Thr Leu Thr Leu Leu Asp Val Asp Thr Gly Lys 100 105 110 Cys Thr Phe Xaa Leu Pro Xaa Phe Cys Tyr Ser Gin Arg Arg Asn Xaa

115 120 125

Pro Leu Cys 130

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Met Xaa Lys Phe Arg Leu Val Thr Asn Phe His Leu Asn Leu Trp Val

1 5 10 15

Asn Gly Xaa Pro Xaa Glu Phe Xaa Xaa Val Gin Asn Arg Pro Val Thr

20 25 30

Thr Arg Gin Thr Ala Xaa Trp Xaa Thr Ala Thr Xaa Arg Ala Tyr His

35 40 45

Xaa Xaa Leu Asp Leu Val Pro Xaa Asn Leu Val Val Ser Ala Pro Gin

50 55 60

Gly His Ser Xaa Thr Ala Met Lys Tyr Val Ser Lys Ser Phe Ser Ile 65 70 75 80

Gly Thr Ser Phe Gin Ser Pro Ile Val Pro Arg Trp Ser Gly Arg Leu

85 90 95

Gly Arg Lys Leu Xaa Lys Val Xaa Ala Gly Asn Leu Asp Phe Val Arg

100 105 110

Cys Gly His Arg Lys Met His Leu Xaa Pro Pro Xaa Ile Leu Leu Ser

115 120 125

Lys Lys Lys His Xaa Thr Leu 130 135 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Xaa Lys Val Pro Phe Gly Leu Asn Gin Leu Pro Pro Lys Pro Leu Gly

1 5 10 15

Lys Arg Xaa Xaa Arg Val Pro Pro Cys Pro Val Lys Ser Ser Cys Asp

20 25 30

His Ala Ser Asn Cys Xaa Met Gly His Ser Asn Xaa Pro Gly Leu Ser

35 40 45

Ser Xaa Pro Pro Arg Pro Ser Lys Ser Gly Ser Phe Cys Thr Ala Gly

50 55 60

Thr Gin Xaa His Cys Asp Glu Val Cys Phe Lys Ile Val Phe Tyr Arg 65 70 75 80

His Leu Leu Pro Lys Ser Asn Ser Ala Lys Val Val Arg Lys Thr Trp

85 90 95

Lys Glu Ala Ser Xaa Ser Pro Xaa Arg Glu Pro Leu Cys Met Trp Thr

100 105 110

Gin Glu Asn Ala Pro Leu Xaa Ser Pro Asp Phe Val lie Val Lys Glu

115 120 125

Glu Thr Xaa His Ser Ala 130

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Gly Xaa Asp Val Ser Gly Leu Lys Gin Leu Arg Arg Glu Met Lys Arg

1 5 10 15

Phe Trp Xaa Leu Xaa Phe Thr Pro Gin Gin Trp Cys Gly Trp Ile Trp

20 25 30

Gin Lys Glu Thr Arg Ala Gin Arg Arg Val Ser Lys Asn Ser Lys Tyr

35 40 45

Asn Ala Glu Ser Thr Glu Arg Glu Ser Gin Asp Thr Val Ala Glu Asn

50 55 60

Asp Gly Trp Arg Val Gin Gly Met Gly Ser Pro Glu Xaa Gin Cys Ile 65 70 75 80

Leu Gly Pro His Xaa Leu Tyr Thr Val His Glu Leu Leu Tyr Gin Glu

85 90 95

Leu Xaa Gin Pro Val Phe Leu Ala Gly Arg Pro Gin Arg Lys Gly Xaa

100 105 110

Val Lys Thr Trp Xaa Trp Gly Xaa Xaa Pro Phe Leu 115 120

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Ala Xaa Thr Tyr Leu Xaa Asn Ser Ser Gly Glu Lys Asn Ala Phe Gly 1 5 10 15 Ser Ser Xaa Leu Leu Leu Asn Asn Gly Val Val Gly Glu Tyr Gly Arg

20 25 30

Arg Arg Pro Gly Lys Leu Lys Gly Glu Tyr Pro Lys Ile Pro Ser Ile

35 40 45

Met Gin Lys Ala Gin Lys Gly Ser His Lys Thr Leu Leu Gin Arg Met

50 55 60

Met Asp Gly Gly Phe Ser Glu Glu Trp Val Ala Gin Arg Xaa Ser Ala 65 70 75 80

Ser Gly Leu Xaa Arg Ser Thr Pro Glu Xaa Thr Ser Cys Cys Thr Arg

85 90 95

Asn Phe Xaa Ser Gin Tyr Ser Ser Leu Val Lys Asp Pro Arg Gly Lys

100 105 110

Gly Glu Leu Lys Leu Gly Xaa Gly Xaa Val Xaa His Phe Leu 115 120 125

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Xaa Arg Arg Ile Trp Xaa Glu Thr Ala Gin Glu Arg Asn Glu Thr Leu

1 5 10 15

Leu Xaa Ala Leu Xaa Tyr Ser Ser Thr Met Val Trp Leu Val Asn Met

20 25 30

Ala Glu Gly Asp Pro Val Ser Ser Lys Glu Ser Ile Gin Lys Phe Gin

35 40 45

Val Cys Arg Lys His Arg Lys Gly Val Thr Arg His Cys Cys Arg Glu

50 55 60

Trp Met Ala Gly Ser Val Arg Asn Gly Pro Arg Gly Thr Val His Leu 65 70 75 80

Arg Ala Ser Xaa Ala Leu His Leu Ser Xaa Arg Ala Ala Val Pro Gly 85 90 95 Thr Xaa Pro Ala Ser Ile Pro Arg Trp Leu Lys Thr Pro Glu Glu Arg

100 105 110

Xaa Ser Asn Leu Gly Leu Gly Xaa Phe Xaa Ile Ser Tyr 115 120 125

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Glu Met Xaa Asn Xaa Pro Xaa Pro Lys Phe Leu Xaa Leu Ser Ser Gly

1 5 10 15

Val Phe Asn Gin Arg Gly Ile Leu Ala Gly Lys Val Pro Gly Thr Ala

20 25 30

Ala Arg Xaa Leu Arg Cys Arg Ala Xaa Glu Ala Leu Arg Cys Thr Val

35 40 45

Pro Leu Gly Tyr Pro Phe Leu Thr Glu Pro Ala Ile His His Ser Leu

50 55 60

Gin Gin Cys Leu Val Thr Pro Phe Leu Cys Phe Leu His Tyr Thr Trp 65 70 75 80

Asn Phe Trp Ile Leu Ser Phe Glu Leu Thr Gly Ser Pro Ser Ala Ile

85 90 95

Phe Thr Asn His Thr Ile Val Glu Glu Xaa Arg Ala Xaa Lys Ser Val

100 105 110

Ser Phe Leu Ser Ala Val Ser Xaa Gin Ile Arg Leu Cys 115 120 125

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Val Arg Asn Gly Lys Xaa Pro Gin Xaa Gin Val Leu Thr Xaa Pro Phe

1 5 10 15

Leu Trp Gly Leu Pro Ala Arg Asn Thr Gly Trp Xaa Ser Ser Trp Tyr

20 25 30

Ser Ser Ser Xaa Thr Gin Val Ser Xaa Gly Pro Lys Met His Cys Xaa

35 40 45

Ser Gly Leu Pro Ile Pro His Thr Arg His Pro Ser Phe Ser Ala Thr

50 55 60

Val Ser Cys Asp Ser Leu Ser Val Leu Ser Ala Leu Tyr Leu Glu Phe 65 70 75 80

Leu Asp Thr Leu Leu Ala Tyr Arg Val Ser Phe Cys His Ile His Gin

85 90 95

Pro His His Cys Gly Val Xaa Xaa Ser Xaa Gin Lys Arg Phe Ile Ser

100 105 110

Leu Leu Ser Cys Phe Xaa Pro Asp Thr Ser Xaa 115 120

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Lys Lys Trp Xaa Thr Ser Pro Xaa Pro Ser Phe Asn Ser Pro Phe Pro

1 5 10 15

Leu Gly Ser Leu Thr Ser Glu Glu Tyr Trp Leu Xaa Lys Phe Leu Val

20 25 30

Gin Gin Leu Val Xaa Ser Gly Val Glu Arg Met Arg Pro Asp Ala Leu

35 40 45

Xaa Leu Trp Ala Thr His Ser Ser Leu Asn Pro Pro Ser Ile Ile Leu

50 55 60

Cys Asn Ser Val Leu Leu Pro Phe Cys Ala Phe Cys Ile Ile Leu Gly 65 70 75 80

Ile Phe Gly Tyr Ser Pro Leu Ser Leu Pro Gly Leu Leu Leu Pro Tyr

85 90 95

Ser Pro Thr Thr Pro Leu Leu Arg Ser Lys Xaa Glu Leu Pro Lys Ala

100 105 110

Phe His Phe Ser Pro Glu Leu Phe Gin Xaa Arg Tyr Val Xaa Ala 115 120 125

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Gly Thr Arg Val Gly Glu Tyr Gly Arg Arg Arg Pro Gly Ser Ser Lys

1 5 10 15

Glu Ser Ile Gin Lys Phe Gin Val Cys Arg Lys His Arg Lys Gly Val

20 25 30

Thr Arg His Cys Cys Arg Glu Trp Arg Val Gin Gly Met Gly Ser Pro

35 40 45

Glu Gly Gin Ser Ser Arg Ala Ser Ser Leu Tyr Thr Val Thr Ser Cys 50 55 60 Cys Pro Gly Thr Phe Gin Gin Tyr Pro Arg Trp Arg Pro Arg Gly Lys 65 70 75 80

Gly Ser Lys Thr Trp Ile Gly Arg Phe His Phe Leu Gin Cys Ser Gly

85 90 95

Trp Ser Leu Gin Gin Gin Pro Val Glu Thr Gly Asn Thr Thr Ile Ala

100 105 110

Cys Phe Arg Ser His Ile Asn Trp Phe Val Pro Tyr Ile Ile Tyr Ser

115 120 125

Leu Pro Phe Ser Arg Lys Ala Leu Pro Val Xaa Ser Asn Phe Pro Ile

130 135 140

Xaa Phe Trp Gly Phe Val Phe Leu Leu Xaa Ala Pro Asp Tyr Leu 145 150 155

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Ala Arg Gly Leu Val Asn Met Ala Glu Gly Asp Pro Glu Ala Gin Arg

1 5 10 15

Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr Glu Arg Glu

20 25 30

Ser Gin Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe Ser Glu Glu

35 40 45

Trp Glu Ala Gin Arg Asp Ser His Leu Gly Pro His Arg Ser Thr Pro

50 55 60

Glu Ser Arg Ala Ala Val Gin Glu Leu Ser Ser Ser Ile Leu Ala Gly 65 70 75 80

Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile

85 90 95

Phe Tyr Ser Val Leu Val Gly Lys Ala Phe Ser Asn Ser Gin Trp Arg 100 105 110 Leu Gly Thr Gin Pro Pro Val Phe Val Ala Ile Leu Ile Gly Leu Cys

115 120 125

Leu Thr Leu Phe Thr Pro Cys His Phe Gin Gly Lys His Cys Gin Xaa

130 135 140

Phe Pro Ile Phe Pro Ser Xaa Phe Gly Val Leu Phe Ser Tyr Phe Xaa 145 150 155 160

His Gin Ile Ile

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

His Glu Gly Trp Ile Trp Gin Lys Glu Thr Arg Lys Leu Lys Gly Glu

1 5 10 15

Tyr Pro Lys Ile Pro Ser Ile Met Gin Lys Ala Gin Lys Gly Ser His

20 25 30

Lys Thr Leu Leu Gin Arg Met Met Met Ala Gly Ser Val Arg Asn Gly

35 40 45

Lys Pro Arg Gly Thr Val Ile Gly Leu Ile Ala Leu His Leu Ser His

50 55 60

Glu Leu Leu Ser Arg Asn Phe Pro Ala Val Ser Ser Leu Val Lys Thr 65 70 75 80

Gin Arg Lys Gly Glu Asn Leu Asp Trp Glu Ile Ser Phe Ser Thr Val

85 90 95

Phe Trp Leu Val Lys Pro Ser Ala Thr Ala Ser Gly Asp Trp Glu His

100 105 110

Asn His Ser Leu Phe Ser Pro Tyr Leu Val Cys Ala Leu His Tyr Leu

115 120 125

Leu Leu Ala Ile Phe Lys Glu Ser Ile Ala Ser Xaa Phe Gin Phe Ser 130 135 140 His Xaa Leu Leu Gly Phe Cys Phe Leu Thr Xaa Gly Thr Arg Leu Ser 145 150 155 160

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Asp Asn Leu Val Pro Lys Val Arg Lys Gin Asn Pro Lys Xaa Trp Glu

1 5 10 15

Asn Trp Lys Xaa Leu Ala Met Leu Ser Leu Lys Met Ala Arg Ser Lys

20 25 30

Cys Lys Ala Gin Thr Asn Tyr Gly Tyr Glu Asn Arg Leu Trp Leu Cys

35 40 45

Ser Gin Ser Pro Leu Ala Val Ala Glu Gly Phe Thr Asn Gin Asn Thr

50 55 60

Val Glu Asn Glu Ile Ser Gin Ser Lys Phe Tyr Ser Pro Phe Leu Trp 65 70 75 80

Val Phe Thr Ser Glu Asp Thr Ala Gly Lys Phe Leu Asp Ser Ser Ser

85 90 95

Leu Arg Cys Arg Ala Met Arg Pro Met Thr Val Pro Leu Gly Phe Pro

100 105 110

Phe Leu Thr Glu Pro Ala Ile Ile Ile Leu Cys Asn Ser Val Leu Leu

115 120 125

Pro Phe Cys Ala Phe Cys Ile Ile Leu Gly Ile Phe Gly Tyr Ser Pro

130 135 140

Leu Ser Phe Arg Val Ser Phe Cys His Ile His Gin Pro Ser Cys 145 150 155 (2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Arg Ser Gly Ala Xaa Ser Lys Lys Thr Lys Pro Gin Lys Xaa Met Gly

1 5 10 15

Lys Leu Glu Xaa Thr Gly Asn Ala Phe Leu Glu Asn Gly Lys Glu Ile

20 25 30

Met Gly Thr Asn Gin Leu Ile Trp Leu Arg Lys Gin Ala Met Val Val

35 40 45

Phe Pro Val Ser Thr Gly Cys Cys Arg Leu Tyr Gin Pro Glu His Cys

50 55 60

Arg Lys Asn Leu Pro Ile Gin Val Leu Leu Pro Phe Pro Leu Gly Leu 65 70 75 80

His Gin Arg Gly Tyr Cys Trp Lys Val Pro Gly Gin Gin Leu Val Thr

85 90 95

Gin Val Ser Asp Glu Ala Leu Asp Asp Cys Pro Ser Gly Leu Pro Ile

100 105 110

Pro His Thr Arg His His His Ser Leu Gin Gin Cys Leu Val Thr Pro

115 120 125

Phe Leu Cys Phe Leu His Tyr Thr Trp Asn Phe Trp Ile Leu Ser Phe

130 135 140

Glu Leu Pro Gly Leu Leu Leu Pro Tyr Ser Pro Thr Leu Val 145 150 155

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Lys Ile lie Trp Cys Xaa Lys Glu Asn Lys Thr Pro Lys Xaa Asp Gly

1 5 10 15

Lys Ile Gly Xaa Asn Trp Gin Cys Phe Pro Lys Trp Gin Gly Val Asn

20 25 30

Asn Val Arg His Lys Pro Ile Asn Met Ala Thr Lys Thr Gly Tyr Gly

35 40 45

Cys Val Pro Ser Leu His Trp Leu Leu Leu Lys Ala Leu .Pro Thr Arg

50 55 60

Thr Leu Lys Met Lys Ser Pro Asn Pro Ser Phe Thr Pro Leu Ser Ser 65 70 75 80

Gly Ser Ser Pro Ala Arg Ile Leu Leu Glu Ser Ser Trp Thr Ala Ala

85 90 95

Arg Asp Ser Gly Val Glu Arg Gly Pro Arg Leu Ser Leu Trp Ala Ser

100 105 110

His Ser Ser Leu Asn Pro Pro Ser Ser Phe Ser Ala Thr Val Ser Cys

115 120 125

Asp Ser Leu Ser Val Leu Ser Ala Leu Tyr Leu Glu Phe Leu Asp Thr

130 135 140

Leu Leu Ala Ser Gly Ser Pro Ser Ala Ile Phe Thr Asn Pro Arg Ala 145 150 155 160

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 114 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Asn Lys Val Lys Ile Pro Gly Trp Thr Phe Ala Ala Ser Phe Gin Val

1 5 10 15

Phe Leu Thr Thr Leu His Tyr Trp Thr Leu Glu Gly Gly Ala Tyr Arg

20 25 30

Lys Arg Phe Thr Tyr Phe Ile Ala Val Asp Cys Val Pro Arg Cys Arg

35 40 45

Asn Tyr Gin Ile Gly Thr Arg Ser Arg Arg Tyr Asp Arg Pro Gly Ser

50 55 60

Cys Cys Ala Pro Ser Ala Ala Arg Val Val Thr Gly Arg Phe His His 65 70 75 80

Xaa Glu Leu Ser Gly Leu Pro Xaa Thr Lys Arg Leu Gly Glu Ser Gly

85 90 95

Phe Xaa Xaa Lys Thr Glu Leu Phe His Leu Xaa Thr Thr Thr 100 105 110

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 114 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Thr Lys Ser Arg Phe Pro Ala Gly Leu Leu Gin Leu Pro Ser Lys Ser

1 5 10 15

Ser Pro Pro Cys Thr Ile Gly Leu Trp Lys Glu Val Pro Ile Glu Asn

20 25 30

Asp Phe Glu His Thr Ser Ser Gin Trp Thr Val Ser Leu Gly Ala Glu

35 40 45

Thr Thr Arg Phe Glu Gly Arg Gly Gin Gly Asp Met Ile Gly Pro Glu

50 55 60

Val Ala Val Pro His Gin Gin Leu Asp Ala Trp Ser Gin Asp Asp Phe 65 70 75 80 Thr Asp Thr Xaa Asn Ser Gin Xaa Tyr Arg Xaa Pro Arg Gly Val Lys

85 90 95

Val Gly Leu Xaa Xaa Lys Arg Asn Ser Phe Ile Xaa Lys Leu Gin Arg 100 105 110

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 114 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Gin Ser Gin Asp Ser Arg Leu Asp Phe Cys Ser Phe Leu Pro Ser Leu

1 5 10 15

Pro Asp His Leu Ala Leu Leu Asp Phe Gly Arg Arg Cys Leu Lys Thr

20 25 30

Ile Leu Asn Ile Leu His Arg Ser Gly Leu Cys Pro Ser Val Gin Lys

35 40 45

Leu Pro Asp Leu Arg Asp Glu Val Lys Glu Ile Ala Arg Lys Leu Leu

50 55 60

Cys Pro Ile Ser Ser Leu Thr Arg Gly His Arg Thr Ile Ser Leu Thr 65 70 75 80

Leu Arg Thr Leu Arg Xaa Thr Val Xaa Gin Glu Val Arg Lys Trp Val

85 90 95

Xaa Gin Asn Gly Thr Leu Ser Ser Xaa Asn Tyr Asn Gly 100 105

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 114 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Pro Leu Phe Xaa Asp Glu Arg Val Pro Phe Xaa Xaa Thr His Phe His

1 5 10 15

Leu Thr Ser Trp Xaa Thr Val Xaa Leu Arg Val Arg Ser Val Ser Glu

20 25 30

Ile Val Leu Pro Arg Val Lys Leu Leu Met Gly His Ser Asn Phe Arg

35 40 45

Ala Tyr His Ile Ser Leu Thr Ser Ser Leu Lys Ser Gly Ser Phe Cys

50 55 60

Thr Glu Gly His Ser Pro Leu Arg Ser Met Phe Lys Ile Val Phe Tyr 65 70 75 80

Arg His Leu Leu Pro Lys Ser Asn Ser Ala Arg Trp Ser Gly Arg Leu

85 90 95

Gly Arg Lys Leu Gin Lys Ser Ser Arg Glu Ser Leu Cys 100 105

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 114 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Thr Val Val Val Xaa Arg Lys Ser Ser Val Leu Xaa Leu Asn Pro Leu

1 5 10 15

Ser Pro Asn Leu Leu Val Asn Gly Xaa Pro Glu Ser Ser Xaa Cys Gin 20 25 30 Asn Arg Pro Val Thr Thr Arg Gin Ala Ala Asp Gly Ala Gin Gin Leu

35 40 45

Pro Gly Leu Ser Tyr Leu Leu Asp Leu Val Pro Gin Ile Trp Phe Leu

50 55 60

His Arg Gly Thr Gin Ser Thr Ala Met Lys Tyr Val Gin Asn Arg Phe 65 70 75 80

Leu Ala Pro Pro Ser Lys Val Gin Cys Lys Val Val Arg Lys Thr Trp

85 90 95

Lys Glu Ala Ala Lys Val Gin Pro Gly lie Leu Thr Leu 100 105

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 109 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Arg Cys Ser Xaa Lys Met Lys Glu Phe Arg Phe Xaa Xaa Lys Pro Thr

1 5 10 15

Phe Thr Pro Leu Gly Xaa Arg Xaa Glu Phe Xaa Val Ser Val Lys Ser

20 25 30

Ser Cys Asp His Ala Ser Ser Cys Trp Gly Thr Ala Thr Ser Gly Pro

35 40 45

Ile Ile Ser Pro Pro Arg Pro Ser Asn Leu Val Val Ser Ala Pro Arg

50 55 60

Asp Thr Val His Cys Asp Glu Val Cys Ser Lys Ser Phe Ser Ile Gly 65 70 75 80

Thr Ser Phe Gin Ser Pro lie Val Gin Gly Gly Gin Glu Asp Leu Glu

85 90 95

Gly Ser Cys Lys Ser Pro Ala Gly Asn Leu Asp Phe Val 100 105

Claims

What is claimed is:

1. An isolated polynucleotide comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence selected from the group consisting of SEQ ID NO:6 and 7 and variants and fragments thereof.

2. An isolated polypeptide encoded by a polynucleotide comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence selected from the group consisting of SEQ ID NO:6 and 7 and variants and fragments thereof.

3. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 8-37 and variants and fragments thereof.

4. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an isolated polynucleotide comprising a polynucleotide sequence at least 95% identical to a polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

5. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an antibody against an isolated polypeptide encoded by a polynucleotide comprising a polynucleotide at least 95% identical to the polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

6. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an antibody against an polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of Figures SEQ ID NO: 8-37 and variants and fragments thereof.

7. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample an isolated polynucleotide comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

8. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample with an isolated polypeptide encoded by a polynucleotide comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

9. A method of diagnosing Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample an antibody against an polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 8-37 and variants and fragments thereof.

10. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an isolated polynucleotide comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

11. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an antibody against an isolated polypeptide encoded by a polynucleotide comprising a polynucleotide at least 95% identical to a polynucleotide sequence sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

12. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and contacting said sample with an antibody against an polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 8-37 and variants and fragments thereof.

13. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample an isolated polynucleotide comprising a polynucleotide sequence at least

95% identical to a polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

14. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample an isolated polypeptide encoded by a polynucleotide comprising a polynucleotide sequence at least 95% identical to a polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

15. A method of detecting a predisposition to Alzheimer's Disease in a patient comprising: obtaining a bodily sample from an individual, and detecting in said sample a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 8-37 and variants and fragments thereof.

16. A kit comprising an antibody against an polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 8-37 and variants and fragments thereof.

17. A kit comprising an isolated an antibody against a polypeptide encoded by a polynucleotide comprising a polynucleotide sequence at least 95% identical to a polynucleotide sequence selected from the group consisting of polynucleotide sequences of SEQ ID NO: 1-7 and variants and fragments thereof.

18. An antagonist of a polypeptide encoded by a polynucleotide comprising a polynucleotide at least 95% identical to the polynucleotide sequence of

Figure 6 and variants and fragments thereof.

19. An agonist of a polypeptide encoded by a polynucleotide comprising a polynucleotide sequence at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6 and 7 and variants and fragments thereof.

- 80 -

SUBST1TUTE SHEET (RULE 25)

20. A method of treating Alzheimer's Disease comprising: contacting an individual having said disease with an antagonist of a polypeptide encoded by a polynucleotide comprising a polynucleotide at least 95% identical to the polynucleotide sequence selected from the group consisting of SEQ ID NO:6 and 7 and variants and fragments thereof.

21. A method of treating Alzheimer's Disease comprising: contacting an individual having said disease with an agonist of a polypeptide encoded by a polynucleotide comprising a polynucleotide at least 95% identical to the polynucleotide sequence selected from the group consisting of SEQ ID NO:6 and 7 and variants and fragments thereof.

- 81