AU3708501A - Partial intron sequence of von hippel-lindau (VHL) disease gene and its use in diagnosis of disease - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Invention Title: PARTIAL INTRON SEQUENCE OF VON HIPPEL-LINDAU (VHL) DISEASE GENE AND ITS USE IN DIAGNOSIS OF DISEASE The following statement is a full description of this invention, including the best method of performing it known to me/us: TITLE OF THE INVENTION PARTIAL INTRON SEQUENCE OF VON HIPPEL-LINDAU (VHL) DISEASE GENE AND ITS USE IN DIAGNOSIS OF DISEASE FIELD OF THE INVENTION The invention is in the field of tumor suppressor genes. More specifically, the invention relates.to the Von Hippel-Lindau (VHL) disease gene and its corresponding cDNA and to methods for detecting carriers of the VHL disease gene using probes derived from the DNA sequences of the present invention.

BACKGROUND OF THE INVENTION Von Hippel-Lindau (VHL) disease is a familial cancer syndrome. This disease is an autosomal dominant disorder and patients who are heterozygous for mutations in the VHL disease gene are predisposed to a variety of cancers, the most frequent being hemangioblastomas of the central nervous system and retina, renal cell carcinoma (RCC) and pheochromocytoma. The multisystem character of the illness, combined with the fact multiple tumors may form in each target organ, produces considerable morbidity and mortality as evidenced by the reduction in life expectancy of affected individuals to 49 years (McKusick, V.A., Mendelian Inheritance in Man (1983) Johns Hopkins University Press, Baltimore and London, p 534-535). Although the prevalence of VHL disease is only 1 in 36,000, because of its late onset most individuals have children before they realize they have inherited VHL disease. For many years, the only method of presymptomatic or prenatal diagnosis of the disease has been periodic examination of the eye, brain, and abdomen in all asymptomatic members of VHL families.

Unfortunately, examination of all target organs is required to ensure detection of disease that may be limited to a single organ. In addition to the obvious inconvenience and the cost of these examinations, they have the additional 2 0 drawback that they may not yield definitive diagnostic information. Therefore, in order to develop a method which allows the unequivocal diagnosis of VHL disease in individuals at risk, researchers have focused intensive efforts on identifying and isolating the VHL disease gene.

Results of this research have shown that the VHL disease gene is a member of the family of tumor suppressor genes (Tory, K. et al. J. Natl. Canc. Inst. (1989) 81:1097- 1101; Maher, E.R. et al. J. Med. Genet. (1990) 27:311-314) and that it behaves in accordance with Knudson's theory of 0 human carcinogenesis (Knudson, Proc. Natl. Acad Sci. USA (1971) 68:816-823). In addition, the identification of DNA k markers tightly linked to the VHL disease gene has allowed localization of the VHL disease gene to human chromosome 3p25-p26. (Hosoe, S. et al. Genomics (1990) 8:634-640; 5 Maher, E.R. et al. Genomics (1990) 8:957-960; Glenn, G.M. et al. Hum. Genet. (1990) 87: 207-210, Latif, F. et al. Am J.

Hum. Genet. (1992) 51 (suppl.) A63; Tory, K. et al. Genomics (1992) 13:275-286; Richards, F.M. et al. J. Med. Genet.

(1993) 30:104-107); Seizinger, B.R. et al. Nature (1988) 332:268-269; Seizinger, B.R. et al. Proc. Natl. Acad. Sci.

USA (1991) 88:2864-2868 and Vance J.M. et al. Am J. Hum.

Genet. (1993) 51:203-209)). Recently, Glenn et al. (Glenn, G.M. et al. JAMA (1992) 1226-1231) have used DNA markers flanking the VHL disease gene as probes to detect linkage to the VHL disease gene via restriction fragment polymorphism analysis of DNA isolated from individuals who are members of families at risk for VHL disease. Although this DNA polymorphism method results in enhanced accuracy of identification of carriers of VHL disease gene, the method is inherently flawed in that DNA polymorphism analysis does not detect the VHL disease gene itself. More recently, a gene located in the VHL region has been cloned (Latif, F. et al. Cancer Res. (1993) 53:861-867). However, this gene was found to detect no mutation in VHL patients and thus, there are currently no available- methods which can identify 3 0 carriers of the VHL disease gene with 100% accuracy.

However, the recent identification and isolation of the VHL disease gene (Latif et al., Science,(1993) 260:1317-1320) and its corresponding cDNA should allow the development of diagnostic methods which provide unequivocal detection of carriers of the VHL disease gene.

SUMMARY OF THE INVENTION The present invention relates to the von Hippel- Lindau (VHL) disease gene and its corresponding cDNA.

0 The invention further relates to methods for detecting carriers of the VHL gene. The first method comprises analyzing DNA of a subject for mutations of the VHL disease gene associated with VHL disease or other diseases, including, but not limited to, sporadic renal 15 cancer, lung cancer, uterine cancer, breast cancer, testicular cancer, ovarian cancer, adrenal tumors, brain tumors, lung tumors or other cancers.

The second method comprises analyzing RNA of a subject for mutations or alterations in the VHL-specific 20 *mRNA associated with VHL disease or other diseases, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer and ovarian cancer.

The third method comprises analyzing protein of a subject for alterations in VHL protein expression associated with VHL disease or other diseases, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer and ovarian cancer.

The invention also encompasses recombinant VHL proteins derived from the VHL cDNA and antibodies directed against said VHL proteins or peptides derived therefrom.

The invention further relates to a method for treating a carrier of the VHL gene in which an expression vector containing a nucleic acid sequence representing the wild-type VHL gene is administered to the carrier.

4 0 The invention also provides a diagnostic kit for detecting carriers of the VHL gene. The kit comprises purified and isolated nucleic acid sequences useful as PCR primers in analyzing DNA or RNA for mutations of the VHL gene associated with VHL disease and diseases related thereto, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer and ovarian cancer.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word S"comprises" has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Figure 1 (Panel A) shows a genetic and physical map of the chromosome 3p region encompassing the VHL gene.

Genetic and physical distances between selected markers are shown in centiMorgans and kilobases, respectively. The location of selected cross-overs is indicated by crosses.

Figure 1 (Panel B) shows the 160 kb cosmid and phage contig covering the VHL region. An enlarged restriction map of cos3, cos 1 l, and phage pl91 detailing the position of g7 cDNA isolated by screening a Xgtll teratocarcinoma cDNA library with a conserved 7kb fragment from the centromeric end of cosll. The beginning of the smallest constitutional deletion is indicated by an asterisk and line. Restriction sites: B, Bam Hl; E, Eco Rl; N, Not I; Nr, Nru I; M, Mlu I.

4a Figures 2A and 2B: Figures 2A and 2B set forth a Northern blot analysis of the expression of the VHL gene represented by g7 cDNA in various human tissues. Figure 2A shows a low resolution blot containing 2 Ag poly A mRNA. The tissues are indicated above the lanes. Figure 2B shows a high resolution blot containing 1 pg of poly A mRNA from: lane 1, fetal brain; lane 2, adult brain; lane 3, fetal kidney; lane 4, adult kidney; lane 5, cerebellum; lane 6, adult adrenal; and lane 7, prostate. The sizes of the transcripts were determined by the position of the 28S and 18S rRNA bands.

o 5 Figures 3A, 3B, 3C, 3D and 3E: Figures 3A, 3B, 3C, 3D and 3E show detection by Southern blotting analysis of rearrangement mutations in constitutional DNA of VHL affected patients using g7 cDNA as probe. Figure 3A shows DNA from lymphoblastoid cell lines of 7 unrelated VHL patients was digested with EcoRI and analyzed by standard blotting procedures. The normal invariant band is about to 22 kb, the sizes of the aberrant bands probably resulting from intragenic deletions range from 4 to 25 kb. The patients code numbers are indicated above the lanes. Figure 3B shows DNAs from lymphoblastoid cell lines of pedigree members from a new mutation family (coded digested with Dral, HindIII, and PstI. The pedigree with the position of the affected (dotted circles) and predicted (hatched circle) members is shown (Figure 3C). Males are represented by 1. 5 squares and females by circles. Figures 3D and 3E show genetic transmission of the mutant allele (the aberrant band) in a regular VHL family (coded The DNAs were .digested with EcoRI and analyzed by Southern blotting (Figure 3D); the pedigree is shown (Figure 3E).

Figure 4: Figure 4 shows a Southern blot analysis of genomic DNA of VHL patients (only the initials of each patients name are given). The DNAs were digested with EcoRI and probed using different regions of g7 cDNA. Panel A: Total g7 cDNA 25 probe; Panel B: 5' end probe, nucleotides 3-146; Panel C: 3' end probe nucleotides 1277-1600.

Figures 5A and 5B: Figures 5A and 5B show the results of polymerase chain reaction-single stranded conformation analysis (PCR-SSCP) of the genomic DNA of VHL patients with the 8 bp insertion mutation (Table Portions of the DNA sequencing gels are shown that display normal (Figure and 714insTTGTCCGT mutation sequences (Figure 5B). The DNA sequence is of the antisense strand; therefore, the inserted bases are 5'-ACGGACAA-3'. Adjacent to the sequencing ladder 6 are shown the positions of the insertion, and the nature of the insertion, as predicted from the sequence.

Figure 6: Figure 6 shows the results of a "zoo" blot illustrating evolutionary conservation of the putative

VHL

gene. The g7 cDNA shows cross species homology to DNA from mammals, birds, fly, and sea urchin. Lanes: 1, human (Homo sapiens); 2, chimpanzee (Pan troqlodytes); 3, macaque (Macaca fascicularis); 4, cow (Bovis domesticus); 5, rat (Rattus norviqicus); 6, mouse (Mus musculus); 7, chicken (Gallus domesticus); 8, frog (Xenopus laevis); fly (Drosophila melanoqaster) 10, sea urchin (Stronqylocentrotus purpuratus); and 11, yeast (Saccharomvces ceriviseae).

15 Figures 7A, 7B and 7C: Figures 7A-7C show the RNase H mapping of the VHL mRNA. Figure 7A sets forth a Northern analysis of the RNase H digest of the VHL mRNA: 1-undigested RNA: 2-RNase H digest with oligomer 1: 3-RNase H digest with oligomer 2. Probe-extended exon 1 (bases 1-553; Latif, et al., 1993b). Figure 7B sets forth the same plot probed with exon 3 VHL group 7 cDNA (bases 740-1810). RNA markers: 0.24-9.5 kb RNA ladder (Gibco-BRL) human 28S (5000 nt) and 18S (2000 nt) rRNAs: Figure 7C shows the alignment of the VHL group cDNA and VHL mRNA according to RHase H mapping; 25 Oligomers 1 and 2 are represented by black boxes, exon 1 sequences are shown as hatched bars, exon 2 black bars, exon 3 open bars. Putative reading frame and scale (in kb) are shown below.

Figures 8A, 8B and 8C: Figures 8A-8C show the identification of the transcription initiation sites.

Figure 8A sets forth the templates and probes used for RNase protection assays. Genomic DNA is represented by solid line, pBluescript II SK vector is represented by an open bar, RNA probes are represented by dashed lines (with the 7 end nucleotides numbered from VHL mRNA transcription start site Probe numbers are shown in the right column. T3 and T7 promoters and their orientation are indicated.

Filled bars represent protected fragments. Figure 8B sets forth an RNase protection assay using probes 1, 2, 3 and poly(A)" RNA from the 293 cell line. i, 2 probe 1 hybridized to 293 RNA 3 probe 1 and yeast tRNA pg): 4-probe 2 and yeast tRNA; 5.6 probe 2 and 293 RNA.

7-probe 3 and yeast tRNA; 8.9 probe 3 and 293 RNA.

'Century markers' (Ambion): 500: 400: 300: 200: 100 nt C- RNase protection using probe 5 and 293 poly(A)-RNA 1 hybridization of the probe 5 and yeast tRNA: probe 5 and 293 RNA. Markers: protected fragments obtained after hybridization of the control sense RNA (probe 4) and probes 5: 6: 7 or 8 (194:182, 170 and 147nt, respectively) Figure 9: Figure 9 represents the identification of the VHL promoter region. Luciferase activity (right column) was compared to those for full length construct (residues -468/- 195) which represents 100% activity in 293 cells (mean value). Restriction map of the 5' flanking genomic region is shown at the top of the Figure. The positions of ~transcription initiation and first methionine AUG condon are indicated.

25 Figures 10A and 10B: Figures 10A and 10B depict

VHL

minigene expression in UMRC 6 cells. Figure 10A describes expression constructs used for stable transfection of the UMRC 6 cell line. VHL sequences were shown as black bars, vector sequences as open bars and solid lines. Predicted transcripts from VHL transgene represented by dashed line (size is indicated). Figure 10B describes Northern analysis of the expression of the VHL transgenes. Total RNA was isolated from four pools each containing 40 to 50 colonies transfected with different expression constructs: pRc- HAVHL; original UMRC 6 cells; pRcp VHL3U; (4) 8 pRcpVHL; pRcpVHLm. Arrows indicate endogenous expression, double arrows exogenous. Note: Previously, the size of the VHL mRNA on Northern blots was calculated as 6 to 6.5kb (Latif, et al., 1993b). In this study, the size of the VHL mRNA was defined more precisely as 4.4 to (depending on conditions of electrophoresis) 0.24 to RNA ladder (BRL) and 28S/18S human ribosomal RNA was used as a reference.

Figures 11A and 11B: Figures 11A and 11B show an analysis of the UMRC 6 clone 4 transfected with pRcpVHLm. Figure 11A shows a Southern blot: 1.2 HindIII digest: 3, 4 HindIII/EcoRI digest: 1, 3 original UMRC 6 cell line: 2, 4 UMRC 6 transfected with pRcpVHLm. A single arrow indicates signals for endogenes, double arrow for exogenes.

Figure 11B shows a Northern blot: 1 original UMRC 6 cells: 2 UMRC 6 clone 4.

Figure 12: Figure 12 sets forth the sequence of the VHL promoter and surrounding genomic region. This sequence has been deposited in the GenBank database (accession no.

U19763). The-minimal VHL promoter is underlined. Putative SP1 and AP2 binding sites and upstream terminationpolyadenylation site are shown in frame. Horizontal arrows show the start of transcription. Restriction sites for some 25 GC-specific rare cutters are indicated. Position of the end of the group 7 cDNA is shown as vertical arrow. The putative upstream splice acceptor site is double underlined.

The first AUG codon in VHL mRNA is shown in a black box.

Figure 13: Figure 13 sets forth the nucleic acid sequences of the partial intron sequences of the VHL disease gene.

The upper case letters depict the exon sequences and the lower case letters depict the intron sequences.

9 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the VHL disease gene, its corresponding cDNA and primers corresponding to the VHL wild-type gene sequence. Recently, the region of human chromosome 3 containing the VHL disease gene has been cloned by genomic walking with yeast artificial chromosomes (YACS) and the cloned DNA recovered with cosmids from a chromosome 3 specific library. The phage 191 which contains the VHL disease gene was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on May 13, 1993 and has been granted ATCC deposit number 69311. This VHL gene represents the wild-type VHL gene where wild-type means the gene not causing VHL disease or other disease associated with the VHL gene.

The present invention is also directed to a cDNA corresponding to the VHL gene. This cDNA sequence, designated g7, is set forth below as SEQ ID NO: 1 and was deposited with the American Type Culture Collection on May 13, 1993, and has been granted ATCC deposit number 69312.

This cDNA also has GenBank accession No. L15409.

a aooo CCTCGCCTCC GTTACAACAG GCGCACAGCC TCCGGCCGGC 25 GAGCGCGCGC

GAAGACTACG

CGCCCCGCGT CCGACCCGCG GATCGCGGAG GGAATGCCCC TAGGCGCGGA GGAGGCAGGC GGGGAGGAGT CGGGCGCCGA GGAACTGGGC GCCGAGGAGG TGCGCTCGGT GAACTCGCGC AGTCCGCGCG TCGTGCTGCC GCCCTACCCA ACGCTGCCGC GAGGTCACCT TTGGCTCTTC GTTAACCAAA CTGAATTATT

CCTACGGTGC

TATTTCCGCG

GAGGTCGACT

GATCCCGCGG

GGAGGGCGGA

GTCGAAGAGT

GGAGTCCGGC

AGATGGAGG C

GAGCCCTCCC

CGTATGGCTC

CTGGCACGGG

AGAGATGCAG

TGTGCCATCT

TGGAGGATCC TTCTGCGCAC AGCGCGTTCC ATCCTCTACC CGGGAGCGCG CACGCAGCTC CGTCCGGCCC GGGTGGTCTG GAACTGGGAC GAGGCCGAGG ACGGCCCTGA AGAAGACGGC CCGGAAGAGT CCGGCCCGGA CGGGCGGCCG CGGCCCGTGC AGGTCATCTT CTGCAATCGC AACTTCGACG GCGAGCCGCA CCGCCGCATC CACAGCTACC GGACACACGA TGGGCTTCTG CTCAATGTTG ACGGACAGCC 100 150 200 250 300 350 400 450 500 550 600 650 10 TATTTTTGCC AATATCACAC TGCCAGTGTA TACTCTGAAA

GAGCGATGCC

TCCAGGTTGT CCGGAGCCTA

GTCAAGCCTG

ATCGTCAGGT CGCTCTACGA

AGATCTGGAA

AGAATTACAG

GAGACTGGAC

GACCACCCAA

ATGTGCAGAA

TGCACATCAA. CGGATGGGAG AGACCTGGAG

CGGCTGACAC

ATTGAAGATT

TCTGTTGAAA

TACTGATGAG

TCTTGATCTA

AAGTGTCTCA

TTCTCAGAGT

S

S**S

S

AACTGACTTC

TAATTTAATG

CATTTTTGCT

ATAAGTAATT

TTGGCATCTG

CGGTTGGTGA

AGGCAGGGAC

TGAGCCTTCA

15

TAGAAAGTGC

GGGACCTTAA

TGTAGGAAGC

CTCTTGTTGA

CTACAGTTGT

ACGAGAAAAT

TGCTAAATGT

ATTGATTTGG

ACTAGGCATT

CCTGCCCATT

TCCTAGTAAG

CAGTGGGAAT

CTTTTAATGG

CTTGTCTGCC

AAGTCTTTCT

GTCAGGGTTT

TTAGAGGTTC

AATGTGTACA

AGGAGCGCAT

CTTACACTGT

GATACAGGAC

AAA ATAGGCA

GTGATGTTTA

AGAGAAGTAT

TCAGGACAGC

TGCAGCATAT

ATGTATAATA

TCCTGCTTTG

CCTCTTTGAG

CTCAGAGGAA

TGCCTCTATT

GTGAACAAAT

TTCATCTCAG

TGGTTCCTTC

CCATTGCTTA

GGGGCAAACA

TTATCAGGAG

TTGTATGTAA

CGTTTAATTT

CATCCATTCT

GGAAGACTGA

ACCCCAGTGC

CAAACCAGGG

TTTGTTGGGG

GTCTTAAAGG

TCGTGCACTT

TCTAAACTAG

TTGCCATCCG

TTAATGGACA

GTAAATATGT

TTGTTTTGTT

CTTTTGATGG

CTTAGTTTCA

AAAGAAAGTT

TCACAAAATG

AAGGTGGTGG

GGAGGTTTAT

TAAGAAGGCA

ACATCCGTAG

GGCATCCGTG

CTGCACATCA

GACACTTTGT

GGTGGGAGAG

GAATCATTTT

TCTCGGTCCA

GATTGACATT

CACAGAAAAT

AATAAGTTTT

GACATTCCTG

TTGTTTTTTT

700 750 800 850 .900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 ATTTTTTATA ATTTTCTAAG AGTGCTGTTT

TATTACTGTT

GATAATAGCA

TTTTTGTAAC

CTGCATGTTT

GATTATAGTA

GAGTATTTCT GTTCCTrTTTT GTTTTTTTGT TGTTGTTGTT GGGATGGAGG

GAATTC

S

1800 The abbreviations used f or the nucleotides are 25 those standardly used in the art.

The deduced amino acid sequence of the g7 cDNA is shown as SEQ ID NO: 2 below and starts at nucleotide 1 of SEQ ID NO:1 and extends 851 nucleotides.

Pro Arg Leu Arg Tyr Asn Ser Leu Arg Cys Trp Arg Ile Leu Leu 10 Arg Thr Arg Thr Ala Ser Gly Arg Leu Phe Pro Arg Ala Arg Ser 25 Ile Leu Tyr Arg Ala Arg Ala Lys Thr Thr Glu Val Asp Ser Gly 40 Ala Arg Thr Gin Leu Arg Pro Ala Ser Asp Pro Arg Ile Pro Arg 5560 11 0 Arg Pro Ala Glu Val Glu Ala Glu Ala Glu Ser Val Ser Pro 10 Pro Gln His Ser His Asp Leu Asn 15 Val Tyr Val Lys Tyr Glu Arg Leu Ala Arg Asn Trp Glu Tyr Glu Ser Glu Glu Asn Ser Arg Val Pro Tyr Tyr Arg Gly Leu Val Asp Thr Leu Pro Glu Asp Leu Thr Gin Val Asp Gly Gly 110 Met 125 Arg 140 Val 155 Pro 170 Gly 185 Leu 200 Gly 215 Lys 230 Asn 245 Glu 260 Glu 275 Val Glu Pro Pro Glu Glu Leu Thr His Val Gin Glu Tyr Asp Arg Trp Ala Glu Glu Ala Pro Pro Leu Leu Asn Pro Arg Arg His Ile Ile Glu Glu Glu Gly Ser Val Pro Trp Gin Ile Cys Arg Pro Ala Ala Glu Gly Met Pro 70 Val Gly Ala Glu Glu 85 Asp Gly Gly Glu Glu 100 Ser Gly Pro Glu Glu 115 Arg Pro Arg Pro Val 130 Gin Val Ile Phe Cys 145 Trp Leu Asn Phe Asp 160 Pro Gly Thr Gly Arg 175 Leu Phe Arg Asp Ala 190 Thr Glu Leu Phe Val 205 Phe Ala Asn Ile Thr 220 Leu Gin Val Val Arg 235 Leu Asp Ile Val Arg 250 Asn Val Gin Lys Asp 265 His Gin Arg Met Gly 280 Arg Ala Ser Leu Leu Asn Gly Arg Gly Pro Leu Ser Ser Leu Asp Arg Gly Gly 105 Gly 120 Arg 135 Arg 150 Glu 165 Ile 180 Thr 195 Ser 210 Pro 225 Leu 240 Leu 255 Glu 270 9* The present invention is also directed to intron 5505 0 0 sequences of the wild-type VHL disease gene. These intron sequences are set forth below as SEQ. ID. NO: 3, SEQ. ID.

NO: 4, and SEQ. ID. NO: 5. The lower case letters represent 25 the intron sequences, and the upper case letters represent the surrounding exon sequences.

-12 SEQ. ID. NO: 3 TACCCAACG CTGCCGCCTG GCACGGGCCG CCGCATCCAC

AGCTACCGAG

gtacgggCCC ggcgcttagg cccgacccag caggacgata gcacggtcta agcccctcta ccgccccggg gtccattcag acggggaa-ct aggccccttg aggcaggaca catccagggt -3' SEO. ID. NO: 4 51 -ctcctgacct ctatgatccg cctgcctcgg cctccaaagt gctgggatta caggtgtggg ccaccgtgcc cagccaccgg tGTGGCTCtt taacaacctt tgcttgtccc gatagGTCAC CTTTGGCTCT TCAGAGATGC

AGGGACACAC

GATGGGCTTC TGGTTAACCA AACTGAATTA TTTGTGCCAT

CTCTCAATGT

TGACGGACAG CCTATTTTTG CCAATATCAC ACTGCCAGgt actgacgttt tactttttaa aaagataagg ttgttgtggt aagtacagga tagaccactt .:gaaaaattaa gcccagttct caatttttgc c'tgatgtcag gcacggtatc *caatcttttt gtatcctatt ctctaccata aataaaatgg aagtgatgat ttt 31 .:9 S* 4@**15 SEO. ID. NO: ctacagaagg catgaacacc atgaagtgtc cataggggcc acagcataca a.....cactgccaca tacatgcact cacttttttt ctttaaccta aaagtgaaga S tccatcagta gtacaggtag ttgttggcaa aagcctcttg *ttcgttcctt gtactgagac cctagtctgc cactgaggat ttggtttttg ccc 3' 06...

13 Variations are contemplated in the cDNA sequence shown in SEQ. ID. NO: 1 which will result in a DNA sequence that is capable of directing production of analogs of the VHL protein shown in SEQ. ID. NO: 2. It should be noted that the DNA sequences set forth herein represent preferred embodiments of the present invention.

Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant VHL protein or its analogs. As such, DNA sequences which are functionally equivalent to the sequences set forth herein or which are functionally equivalent to sequences that would direct production of analogs of the VHL protein produced pursuant to the amino 15 acid sequence set forth above, are intended to be *e encompassed within the present invention.

*S

H:\janelKeep\Speci\25931-97.doc 5/04/01 14 0 The term analog includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the function of the VHL protein as described herein. Examples of conservative substitutions include the substitution of nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting protein or polypeptide displays the requisite functional activity.

20 "Chemical derivative" refers to a VHL protein or polypeptide having one or more residues chemically derivatized -by reaction of a functional side group.

Examples of such derivatized molecules include, but are not limited to, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For 15 example: 4-hydroxyproline may be substituted for proline; may be substituted for lysine; 3methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. A VHL protein or polypeptide of the present invention also includes any protein or polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.

The present invention also relates to methods for detecting carriers of the VHL gene.

It is understood by one skilled in the art that the methods for detection disclosed in the present invention can be used prenatally to screen a fetus or presymptomatically to screen a subject at risk through his/her family history. In addition, these methods can be used to determine the involvement of the VHL gene in other human malignancies such as sporadic renal cancer, uterine cancer, breast cancer, testicular cancer, bladder cancer, 20 pancreatic cancer, ovarian cancer and lung cancer.

Specifically, the methods of the present invention may be used- to detect familial types of renal cell carcinoma. Examples of familial types of renal cell carcinoma include, but are not limited to, hereditary, nonpappillary renal cell carcinoma; VHL disease; and hereditary papillary RCC.

Additionally, the methods of the present invention may be used to detect sporadic, noninherited malignancies, such as, for example, renal cell carcinoma.

In one embodiment of the invention, the method for detecting carriers of the VHL gene comprises analyzing the DNA of a subject for mutations of the VHL gene associated with VHL disease, or diseases related thereto.

For purposes of the present invention, subject means a mammal and mutation means inversion, translocation, 16 0 insertion, deletion or point mutation of the VHL gene.

For analysis of the DNA, a biological specimen is obtained from the subject. Examples of biological specimens that may be analyzed by the methods of the present invention include, but are not limited to, tissue biopsies, whole blood, serum, urine, feces, cerebrospinal fluid or other samples normally tested in the diagnosis of disease.

Preferred biological specimens are whole blood or urine.

Although it is not always required, it is preferable to at least partially purify DNA from the biological specimen prior to analysis. For example, after disruption of cells in the specimen, nucleic acid can be extracted from contaminating cell debris and other protein substances by extraction of the sample with phenol. In phenol extraction, the aqueous sample is mixed with an 15 approximately equal volume of redistilled phenol and centrifuged to separate the two phases. The aqueous phase containing the nucleic acid is removed and precipitated with S. ethanol to yield nucleic acid free of phenol.

Alternatively, DNA can be purified from the biological sample according to Sidransky, D. et al. (Science (1992) 256:102-105; Science (1991) 252:706) or by the method of Glenn, et al. (Glenn, G.M. et al. JAMA (1992) 267:1226- 1231). The DNA to be analyzed can be either single- or double-stranded.

Methods for analyzing the DNA for mutations in the VHL gene include Southern blotting after digestion with the appropriate restriction enzymes (restriction fragment length polymorphism, RFLP) (Botstein, D. Amer. J. Hum. Genet.

(1980) 69:201-205), denaturing gradient electrophoresis technique (Myers, Nature (1985) 313:495-498), oligonucleotide hybridization (Conner, R. et al., EMBO J.

(1984) 3:13321-1326), RNase digestion of a duplex between a probe RNA and the target DNA (Winter, E. et al., Proc. Natl.

Acad. Sci. U.S.A. (1985) 82:7575-7579), polymerase chain reaction (PCR) (Saiki, P.K. et al., Science (1988) 239:487- 17 491; U.S. Patents 4,683,195 and 4,683,202), ligase chain reaction (LCR) (European Patent Application Nos. 0,320,308 and 0,439,182), and PCR-single stranded conformation analysis (PCR-SSCP) (Orita, M. et al., Genomics (1989) 5:874-879; Dean, M. et al. Cell (1990) 61:863-871). In one preferred embodiment, DNA is analyzed by Southern analysis.

The DNA to be analyzed via Southern analysis is digested with one or more restriction enzymes. The restriction enzymes to be used in the present invention are those enzymes for whom the presence or absence of their recognition site is linked to a disease, including, but not limited to, VHL disease and sporadic renal carcinoma.

Preferred restriction enzymes include EcoRI, HindIII, PstI, Dral, BamHI, BglI, BglII, and PvuII. Following restriction digestion, resultant DNA fragments are separated by gel 15 electrophoresis and the fragments are detected by hybridization with a labelled nucleic acid probe (Southern, E.M. J. Mol. Biol. (1975) 98:503-517).

The nucleic acid sequence used as a probe in Southern analysis can be labeled in single-stranded or double-stranded form. Labelling of the nucleic acid sequence can be carried out by techniques known to one skilled in the art. Such labelling techniques can include radiolabels and enzymes (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In addition, there are known non-radioactive techniques for signal amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al. (1973) Proc. Natl. Acad. Sci., 70:2238-2242; Heck, R.F. 1968)

S.

Am. Chem. oc., 90:5518-5523), methods which allow detection by chemiluminescence (Barton, S.K. et al. (1992) J. Am.

Chem. Soc., 114:8736-8740) and methods utilizing biotinylated nucleic acid probes (Johnson, T. K. et al.

(1983) Anal. Biochem., 133:126-131; Erickson, P.F. et al.

(1982) J. of Immunology Methods, 51:241-249; Matthaei,

F.S.

18 et al. (1986) Anal. Biochem., 157:123-128) and methods which allow detection by fluorescence using commercially available products. The size of the probe can range from about 200 nucleotides to about several kilobases. A preferred probe size is about 500 to about 2000 nucleotides. Each of the nucleic acid sequences used as a probe in Southern analysis is substantially homologous to the corresponding portion of the cDNA sequence shown in SEQ ID NO: 1. By "substantially homologous" is meant a level of homology between the nucleic acid sequence used as a probe and the corresponding sequences shown in SEQ. ID. NO: 1 and SEQ. ID. NOS: Preferably, the level of homology is in excess of 70%, most preferably in excess of 80%, with a particularly preferred nucleic acid sequence being in excess of 90% homologous with the sequences shown in SEQ. ID. NO: 1 and SEQ. ID. NOS: 15 Once the separated DNA fragments are hybridized to the labelled nucleic acid probes, the restriction digest pattern can be visualized by autoradiography and examined for the presence or absence of a restriction fragment length polymorphism (RFLP) associated with VHL disease, or diseases related thereto.

In a second preferred embodiment, the DNA is o* analyzed for mutations in the VHL gene by PCR-SSCP (Orita et al., (1989), Dean et al., (1990)). In this method, each of the pairs of primers selected for use in PCR are designed 25 to hybridize with sequences in the VHL gene which are an appropriate distance apart (at least about 50 nucleotides) in the gene to permit amplification and subsequent detection of mutations in the amplification product. Primer pairs which can specifically hybridize to such VHL gene sequences can be derived from the VHL gene sequence.

In a preferred embodiment, the primers are derived from the cDNA sequences shown in SEQ. ID. NO: 1 and SEQ. ID.

NOS: 3-5. Each primer of a pair is a single-stranded oligonucleotide of about 15 to about 50 bases in length which is complementary to a sequence at the 3' end of one of 19 the strands of a double-stranded target sequence. Each pair comprises two such primers, one of which is complementary to the 3' end and the other of which is complementary to the other 5' end of the target sequence. The target sequence is generally about 100 to about 300 base pairs long but can be as large as 500-600 base pairs. Optimization of the amplification reaction to obtain sufficiently specific hybridization to the VHL gene is well within the skill in the art and is preferably achieved by adjusting the annealing temperature.

The present invention also provides purified and isolated pairs of primers for use in analysis of DNA for mutations in the VHL disease gene. The nucleic acid sequences of the primers are set forth below as SEQ. ID.

NOS: 7-12.

SEQ. ID. NO: 7 ATAGTGGAAA TACAGTAACG AGTTGGCCTA GCCTCGC SEQ. ID. NO: 8 CCCAGCTGGG TCGGGCCTAA GCGCCGGGCC CGT SEQ. ID. NO: 9 GTGGCTCTTT AACAACCTTT GCTTGTCCCG ATA SEQ. ID. NO: CAAGTGGTCT ATCCTGTACT TACCACAACA CCT SEQ. ID. NO: 11 TGTATACTCT GAAAGAGCGA TGCCTCCAGG T SEQ. ID. NO: 12 TACCATCAAA AGCTGAGATG AAACAGTGTA AGT where SEQ ID NO: 7 and SEQ ID NO: 8 represent one pair of primers; SEQ ID NO: 9 and SEQ ID NO: 10 represent a second pair of primers and SEQ ID NO: 11 and SEQ ID NO: 12 represent a third pair of primers.

Additional primers provided by the present invention for use in analysis of DNA for mutations in the VHL disease gene include the following primers, set forth as SEQ. ID. NOS: 13-22: SEQ. ID. NO: 13 20 AGTGGAAATA CAGTAACGAG

TTGGCCT

SEQ. ID. NO: 14 GAAATACAGT AACGAGTTGG

CCTAGC

SEQ. ID. NO: GTCCCAGTTC TCCGCCCTCC GGGGCAT SEQ. ID. NO: 16 TGGGTCGGGC CTAAGCGCCG GGCCCGT SEQ. ID. NO: 17 CTTTAACAAC CTTTGCTTGT CCCGATA SEQ. ID. NO: 18 GTGGCTCTTT AACAACCTTG C SEQ. ID.-NO: 19 GTCTATCCTG TACTTACCAC AACACCT *SEQ. ID. NO: CCTGTACTTA CCACAACACC

TTAT

SEQ. ID. NO: 21 *CTGAGACCCT AGTCTGCCAC TGAGGAT SEQ. ID.- NO: 22 *TTCCTTGTAC TGAGACCCTA GT SEQ. ID. NO: 23 GGAAATACAGT AACGAGTTGG CCT SEO. ID. NO: 24 GGAAATACAG TAACGAGTTG GCCTAGC SEQ. ID. NO: ACGGGCCCGG CGCTTAGGCC CGACCCA SEQ. ID.-NO: 26 ACGGGCCCGG CGCTTAGGCC CGACCCAGCA GG SEQ. _ID.NO:- 27 GTGGCTCTTT AACAACCTTT GCTTGTCCCG

ATA

SEQ. ID NO: 28 CTTTAACAAC CTTTGC EQ. ID N 02 9 GATAAGGTTG TTGTGGTAAG TACAGGA SlEQ ID NO: AGGTTGTTGT GGTAAGTACA GGATAGC SEQ. ID- NO: 31 21 CTCCTTGTAC TGAGACCCTA

GT

SEO. ID. NO: 32 GTGAGACCCT AGTCTGCCAC

TGAGGAT

Examples of primers useful in the present invention which may be used to hybridize-to mutant forms of.

the VHL gene include, but are not limited to, primers that possess the following mutated sequences: GAGGTCAC (SEQ. ID. NO. 33) A mutation from the nucleotide sequence

GATAGGTCAC

to GAGGTCAC in the VHL gene results in the loss of the exon 2 splice acceptor and the loss of expression of exon 2.

GATTGGTCAC (SEQ. ID. NO. 34) A mutation from the nucleotide sequence GATAGGTCAC .to GATTGGTCAC in the VHL gene results in the loss of the exon 2 splice acceptor.

A mutation from G to A at nucleotide 676 of SEQ. ID. NO: 1 and an eight nucleotide deletion of GTACTGAC.

A VHL gene possessing these mutations results in the loss of the exon 2 splice donor.

The primers of this invention can.be synthesized using any of the known methods of oligonucleotide synthesis the phosphodiester method of Agarwal et al. 1972.

Agnew. Chem. Int. Ed. Engl. 11:451, the phosphotriester method of Hsiung et al. 1979. Nucleic Acids Res. 6:1371, or the automated diethylphosphoramidite method of Beuacage et al. 1981. Tetrahedron Letters 22:1859-1862), or they can be isolated fragments of naturally occurring or cloned DNA. In addition, those skilled in the art would be aware that oligonucleotides can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially custom ordered and prepared. In one embodiment, the primers can be derivatized to include a detectable label suitable for detecting and/or identifying the primer extension products biotin, avidin, or radiolabeled dNTP's), or with a substance which aids in the isolation of the products of amplification biotin or avidin). In a preferred 22 embodiment, SEQ. ID. NO: 7 through SEQ. ID. NO: 34 are synthetic oligonucleotides.

In an alternative embodiment, primer pairs can be selected to hybridize to mutant forms of the VHL gene. The selected primer pairs will hybridize sufficiently specifically to the mutated gene sequences such that nonspecific hybridization to VHL gene sequences will not prevent identification of the amplification product of the mutant gene sequence. Primer pairs which hybridize to mutations in the VHL gene sequence can be used to amplify specific mutant gene sequences present in the DNA of a biological sample.

The amplification products of PCR can be detected either directly or indirectly. In the PCR-SSCP method direct detection of the amplification products is carried 1 5 out via labelling of primer pairs. Labels suitable for Slabelling the primers of the present invention are known to one skilled in the art and include radioactive labels, biotin, avidin, enzymes and fluorescent molecules. The derived labels can be incorporated into the primers prior to performing the amplification reaction. A preferred labelling procedure utilizes radiolabeled ATP and T4 polynucleotide kinase (Sambrook, J. et al. (1989) in .o "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY). Alternatively, the desired label can be incorporated into the primer extension products during the amplification reaction in the form of one or more labelled dNTPs. In the present invention, the labelled amplified PCR products can be analyzed for mutations of the VHL gene associated with VHL disease gene, or diseases related thereto, via separating the PCR products by denaturing polyacrylamide gel electrophoresis or via direct sequencing of the PCR-products.

In yet another embodiment, unlabelled amplification products can be analyzed for mutations in the VHL gene via hybridization with nucleic acid probes 23 radioactively labelled or labelled with biotin, in Southern blots or dot blots. Nucleic acid probes useful in the embodiment are those described previously for Southern analysis.

In a second embodiment, the method for detecting carriers of the VHL gene comprises analyzing the RNA of a subject for mutations or alterations in VHL-specific mRNA associated with VHL disease and diseases related thereto, including, but not limited to, sporadic renal cancer, uterine cancer, breast cancer, testicular cancer, bladder cancer, pancreatic cancer, ovarian cancer and lung cancer.

For the analysis of RNA by this method,

RNA

I* derived from blood or a tumor biopsy sample is obtained from said subject where said tumors include, but are not limited to, tumors of the eye, brain, liver, kidney, pancreas, and 15 pheochromocytomas.

The RNA to be analyzed can be isolated from blood or tumor biopsy samples as whole cell RNA or as poly(A) RNA. Whole cell RNA can be isolated by methods known to those skilled in the art. Such methods include extraction of RNA by differential precipitation (Birnbiom, H.C. (1988) SNucleic Acids Res., 16:1487-1497), extraction of RNA by organic solvents (Chomczynski, P. et al. (1987) Anal.

Biochem., 162:156-159) and extraction of RNA with strong denaturants (Chirgwin, J.M. et al. (1979) Biochemistry, 18:5294-5299) Poly(A) RNA can be selected from whole cell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. et al. (1972) Proc. Natl. Acad. Sci., 69:1408-1412).

A

preferred method of isolating RNA is extraction of whole cell RNA by acid-phenol (Chomczynski et al. 1987).

The methods for analyzing the RNA for alterations in the pattern or level of VHL specific mRNA expression linked to VHL disease and diseases related thereto, include Northern blotting (Alwine, J.C. et al. (1977) Proc. Natl.

Acad. Sci., 74:5350-5354), dot and slot hybridization (Kafatos, F.C. et al. (1979) Nucleic Acids Res., 7:1541- 24 0 1522), filter hybridization (Hollander, M.C. et al. (1990) Biotechniques; 9:174-179), RNase protection (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY) and reversetranscription polymerase chain reaction (RT-PCR) (Watson, J.D. et al. (1992) in "Recombinant DNA" Second Edition, W.H.

Freeman and Company, New York). One preferred method is Northern blotting.

The nucleic acid sequence used as a probe for detecting VHL-specific mRNA expression is substantially homologous to SEQ. ID. NO: 1. By "substantially homologous" is meant a level of homology between the nucleic acid *..sequence and the cDNA sequence of SEQ. ID. NO: 1.

Preferably, the level of homology is in excess of 70%, more preferably in excess on 80%, with a particularly preferred 15 nucleic acid sequence being in excess of 90% homologous with the cDNA sequence shown in SEQ. ID. NO: 1.

A most preferred method is reverse transcriptionpolymerase chain reaction (RT-PCR) where the primers used to amplify the cDNA produced via reverse transcription of RNA 20 are derived from the cDNA sequence shown in SEQ. ID. NO: 1.

These primers can be labelled as described earlier and the RT-PCR products can be analyzed for mutations of the VHL gene associated with VHL disease, or diseases related O* thereto, via denaturing polyacrylamide gel electrophoresis of the RT-PCR products or via direct sequencing of the RT- PCR products.

In a third embodiment, the method for detecting carriers of the VHL gene comprises analyzing the DNA of a subject for mutations or alterations in VHL-specific DNA associated with VHL disease, or diseases related thereto, such as sporadic renal cancer, uterine cancer, breast cancer, testicular cancer, bladder cancer, pancreatic cancer, ovarian cancer and lung cancer.

The present invention also encompasses recombinant proteins derived from the cDNA shown in SEQ. ID. NO: 1 and 0 antibodies directed to said proteins (called VHL proteins) Recombinant VHL proteins can be produced by recombinant DNA methodology known to one skilled in the art. For example, a nucleic acid sequence capable of encoding a protein comprising all or part of the amino acid sequence shown in SEQ. ID. NO: 2 can be cloned into a vector capable of being transferred into, and replicated in, a host organism. A suitable nucleic acid sequence for the purpose of this invention are the sequences shown in SEQ. ID. NO: 1 and SEQ.

ID. NOS: 3-5. Suitable expression vectors include, but are not limited to, vaccinia virus vectors, baculovirus vectors, and E. coli pTRCHIS (Invitrogen Co. San Diego) The recombinant expression vector produced by inserting a nucleic acid sequence capable of directing synthesis of VHL protein in a suitable expression vector can be transfected *e 15 into E. coli or into suitable eukaryotic cell systems by methods known to one skilled in the-art.

Cells containing the expressed recombinant VHL e* protein, cell lysate from cells transfected with a eo recombinant expression vector or a culture supernatant containing the expressed VHL protein can be used as an immunogen to elicit production of anti-VHL antibodies in a mammal. Alternatively, one can generate synthetic peptides for use as immunogens from the amino acid sequence shown in SEQ. ID. NO: 2.

Preferred synthetic peptide sequences for use as immunogens are shown below: SEQ ID NO. Glu Glu Tyr Gly Pro Glu Glu Asp Gly Gly Glu Glu Ser Gly SEQ ID NO. 36: Gly Thr Gly Arg Arg Ile His Ser Tyr Arg Gly His Leu While it is possible for the immunogen to be 26 0 administered to the mammal in pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. Suitable mammals for immunization include mice, rabbits and the like. The anti-VHL antibody of the present invention is typically produced by immunizing a mammal with an immunologically effective amount of synthetic peptide of this invention.

The preparation of polyclonal or monoclonal antibodies against such a peptide is well known in the art (Standt, et al. (1988) J. Exp. Med. 157:687-704). The anti-VHL peptide antibody molecules induced by immunization of a mammal with the recombinant VHL protein are -then collected from the mammal and those immunospecific for the VHL protein are isolated to the extent desired by well known techniques such as, for example, immunochromatography.

15 In a third embodiment, the method for detecting carriers of the VHL gene comprises: analyzing the protein of a subject for alterations in VHL protein expression.

For analysis of protein by this method, protein is 20 obtained from biological specimens such as tumor biopsy samples and urine and the like. The protein can be obtained as a crude lysate or it can be further purified by methods known to one skilled in the art (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor press, Plainview, NY).

Crude protein lysate can be analyzed for VHL.

protein by immunoassays using anti-VHL antibody.

Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard techniques known in the art for ELISA are described in Method in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al., Methods of Immunology, W.A. Benjamin, Inc., 1964, both of which are 27 incorporated herein by reference. Such assays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art. (Oellerich, M. 1984. J. Clin Chem. Clin. BioChem. 22:895-904).

Detection of the VHL protein anti-VHL antibody complex formed can be accomplished by reaction of the complex with a secondary antibody such as labelled antirabbit antibody. The label may be an enzyme which is detected by incubating the complex in the presence of a suitable fluorimetric or colorimetric reagent. Other detectable labels may also be used, such as radiolabels, or colloidal gold, and the like. The labelled VHL proteinanti-VHL antibody complex is then visualized by autoradiography.

The present invention also relates to a method for 15 treating a carrier of the VHL gene in which an expression vector containing a nucleic acid sequence representing the VHL gene is administered to the carrier. Nucleic acid sequences representing the VHL gene are SEQ. ID. NO: 1 and SEQ. ID. NOS: 3-7. Such nucleic acid sequences may be inserted into a suitable expression vector by methods known to those skilled in the art (Example Expression vectors suitable for producing high efficiency gene transfer in vivo include retroviral, adenoviral and vaccinia viral vectors.

Expression vectors containing a nucleic acid sequence representing the VHL gene can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or orally. A preferred route of administration is intravenously.

The invention also provides a diagnostic kit for detecting carriers of the VHL gene. This diagnostic kit comprises purified and isolated nucleic acid sequences according to SEQ ID. NO: 7 through SEQ ID NO: 34, said sequences useful as PCR primers in analyzing DNA for the presence of mutations of the VHL gene linked to VHL disease, or diseases related thereto.

28 The invention also provides a diagnostic kit for detecting regulatory defects of the VHL gene. This diagnostic kit comprises purified and isolated nucleic acid sequences according to SEQ. ID. NO: 7 through SEQ. ID. NO: 34, said sequences useful as PCR primers in analyzing DNA for mutations of the VHL gene linked to VHL disease and diseases related thereto, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer, ovarian cancer, adrenal tumors, brain tumors, lung tumors or other cancers.

The nucleic acid sequences of the present invention according to SEQ. ID. NO: 7 through SEQ. ID. NO: 34 are useful in the detection of hereditary and sporadic kidney cancers by the detection of abnormalities of the VHL gene in biological samples using the primers of the present 15 invention.

The present invention further provides a method of preventing or treating regulatory defects linked to VHL disease. Specifically, the present invention provides a method of treating or preventing cancer in a subject by 20 contacting the cancer with an amount of the VHL gene of the S. present invention effective to treat the cancer. This method comprises administration of the VHL gene in an amount effective to prevent or treat regulatory defects associated with VHL disease and diseases related thereto, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer and ovarian cancer.

In one embodiment of the invention, the VHL gene sequence or -analog thereof is administered in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers such as sterile solution, tablets, coated tablets and capsules. Such carriers may typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stensic acid, talc, vegetable fats 29 or olis, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives and other ingredients.

Types of cancer that may be treated using the VHL sequences or proteins of the present invention include, but are not limited to, VHL disease and diseases related thereto, including, but not limited to, sporadic renal cancer, lung cancer, uterine cancer, breast cancer, testicular cancer, ovarian cancer, adrenal tumors, brain tumors, lung tumors or other cancers.

Specific carcinomas which may be treated using the VHL sequences or proteins of the present invention include, Sbut are not limited to, renal cell carcinoma, pheochromocytoma, retinal angioma, hemangioblastoma, .o pancreatic cysts, pancreatic tumors and epididymal cystadenoma.

Any articles or patents referenced herein are incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.

MATERIALS

The subjects analyzed in the following examples were kindred identified by ophthalmologists, urologists, medical geneticists and neurosurgeons in the United States, Europe, and Canada. The members of the families resided in Louisiana, Tennessee, Mississippi, Virginia, Pennsylvania, New York, Michigan, Quebec, Nova Scotia, United Kingdom, and the Netherlands. Medical records of each family member known to be affected were reviewed. Asymptomatic family members and family members in whom there was uncertainty about the diagnosis were examined after informed consent for occult evidence of the illness at the Clinical Center of the National Institutes of Health. The examination consisted of a history and physical examination of the scrotum. An asymptomatic member of a VHL family was considered to be 30 affected if one or more of the following disease manifestations were detected: retinal angioma(s), spinal or cerebellar hemangioblastoma(s), pheochromocytoma(s), multiple pancreatic cysts, and multiple bilateral renal cysts accompanied by renal cell carcinoma. Disease diagnosis was made without knowledge of restriction fragment length polymorphism (RFLP) status.

Restriction enzymes were from Bethesda Research Laboratory (BRL) (Bethesda, MD), New England Biolabs (Beverly, MA) and Boehringer Mannheim (Indianapolis, IN) and were used as recommended by the manufacturers. 6-nPdCTP (-3000iu/mmol) was from Amersham (Arlington Heights, IL) The various human tissue polyadenylated RNAs used in Northern blotting were purchased from Clontech (Palo Alto, CA) as was the adult kidney double-stranded complementary 15 DNA sample. PCR and RT-PCR bits were from Perkin Elmer/Cetus (Norwalk, CT) deoxynucleotide triphosphates and flourescently labelled dideoxynucleotides were from Applied S. Biosystems, Inc. (Foster City, CA). Nylon membranes were purchased from MSI, Inc. (Westlore, MA).

METHODS

Southern and Northern blottings, filter hybridization and probe labelling were carried out using random priming and were otherwise performed by standard protocols (Sambrook, J. et al. (1989)). DNA inserts were purified following the GeneClean (Bio 101) (BioRad, Richmond, CA) protocol and used for subcloning or labelling.

Oligonucleotides used as primers in PCR or RT-PCR or for sequencing were synthesized on the Applied Biosystems, Inc.

Model 392 DNA/RNA synthesizer, according to the manufacturers recommendations. Pulse field goal electrophoresis was carried out using CHEF-DRII or CHEF mapper XA systems as described by the manufacturer (BioRad) under conditions optimal for obtaining the desired resolution.

31 0 PCR The PCR was performed in a 50 ul reaction volume in a mixture containing luM of each primer, 250uM of each deoxynucleotide triphosphate, 5ul of 10X PCR buffer (500MM KC1; 120MM Tris-HCl, pH 8.0; 1.5MM MgC1 2 and 0.1% gelatin) and 1.25 units of AmpTaq (Cetus) DNA polymerase, in a first generation automated thermal cycler (Perkin- Elmer/Cetus). The PCR conditions consisted of 40 cycles of denaturation for one minute at 94 0 C, annealing for one minute at specified temperatures (55-65 0 C) and extension for 4 minutes at 72 0 C followed by 7 minutes of final extension of 72°C.

RNA Preparation and Northern Blotting Total cellular RNA was isolated by extraction of lymphoblastoid cell lines of affected VHL patients or kidney tissues in guanidine thiocyanate followed by centrifugation through a 15 5.7 M CsCl cushion according to standard protocols (Sambrook, J. et al. (1989)) RNA samples were separated by electrophoresis in 1% agarose gels containing 2.2M formaldehyde, transferred to nylon membranes and hybridized to g7 cDNA probe (Sambrook, J. et al. (1989)).

20 RT-PCR About 5 ug of total cellular RNA was isolated by extraction of lymphoblastoid cell lines or kidney tissues of VHL patients or 2.5 ng of normal adult kidney double-stranded complementary DNA samples were analyzed for expression using RT-PCR kit from Perkin- Elmer/Cetus. The primers were derived from the g7 cDNA sequence shown in SEQ. ID. NO: 1 and the reactions were run using various annealing temperatures. The reaction products were analyzed by gel electrophoresis and Southern blotting (Sambrook, J. et al (1989)).

Cell Culture The 293 cells (Graham, et al. 1977) and UMRC 6 cells (Grossman, et al. 1995) were grown in DMEM medium supplemented with 10% fetal bovine serium (Life Technologies Inc., NY) penicillin (25 000 U/I) and streptomycin (25,000 jg- 1 with 8% CO 2 Isolation of RNA for Identification of Promoter 32 0 Region Total RNA from cell cultures was isolated using Ultraspec II RNA isolation system (Biotex, TX) Poly(A) RNA was purified twice on oligo-dT Cellulose (Stratagene, CA).

RNAse H mapping Ten micrograms of total RNA and 200 ng of VHL-specific antisense oligomer were annealed and RNA was digested with RNAse H essentially as described by Berger (1987). The following oligonucleotides were used; for VHL exon 1 (SEQ. ID. NO. 37): ACG ACG CGC GGA CTG CGA TTG CAG AAG AT-3': for exon 3 (SEQ. ID. NO. 38): GAC CTG ACG ATG TCC AGT CTC-3'. After ethanol precipation, RNA was separated in 0.75% agarose-formaldehyde gels (Lehrah, et al., 1977) transferred to nylon membrane and hybridized to the probe.

Mapping of the Transcription Start Site Transcription start mapping was performed using Ribonuclease 15 Protection Assay Kit (RPA II, Ambion, TX) according to manufacturer instructions. Protected fragments were separated in a standard urea sequencing gel (6% polyacrylamide). The gel was vacuum dried and exposed to Xray film (Kodak X-OMAT AR) Sequencing ladder was made using control template, primer and reagents from Sequenase Version 2.0 DNA sequencing kit (United States Biochemical,

OH).

RNA markers, probes and control sense VHL RNA were obtained by in vitro transcription using RNA Maxiscript T3/T7 kit (Ambion, TX) and three groups of templates. The first group (Figure 8A, probes 1, 2, 3 and 4) derived from plasmid pBluescript II S/K (Stratagene, CA) carrying an inserted 892bp EcoRI-NotI genomic fragment, containing the part of VHL exon 1 and 5' flanking genomic region 647/+245). For generation of probes no. 1, no. 2, no. 3 and no. 4 some parts of the insert were deleted and derivative plasmids were linearized as shown in Figure 8A. The second group of templates was generated by PCR using the primers CGC CTC CGT TAC AAC A-3' (SEQ. ID. NO. 39) and TCC TAA TAC GAC TCA CTA TAG GGA GGC GCC CGA CTC CTC CC-3' 33 (SEQ. ID. NO. 40). This PCR fragment contained part of the genomic EcoRI-NotI sequence (residues 166/+173) and the promoter of T7 RNA polymerase to make antisense VHL probe.

To generate several marker probes, the template was cleaved around presumptive transcription start sites with EagI, BssHII, Alul or BamHI (Figure 8A, probes 5, 6, 7 and 8).

These probes were hybridized to probe no. 4 (control sense RNA) and the protected fragments were used as markers on Figure 8C. The third set of templates (RNA Century Marker Template Set) was purchased from Ambion All templates were blunt ended with Klenow fragment.

Luciferase Plasmid Construction Presumptive promoter region was amplified by PCR using upstream (sense) primer 5'-CTA TCT AGA GGC CAA GGC AGG AGG ATC-3' (SEQ. ID.

NO. 41) and two downstream (antisense) primers: 5'-CAT TCT S. 15 AGA TTC CCT CCG CGA TCC AGA-3' (SEQ. ID. NO. 42) and TCT AGA CTC TTC CGG GCC GGA CTC-3' (SEQ. ID. NO. 43). The two PCR fragments contained residues 180-716 and 180-842 of the genomic EcoRI-NotI fragment (respectively residues -468- 69 and -468+195 on Figure 12) and XbaI linkers.

PCR

20 fragments were digested with XbaI and cloned in both orientations into the NheI site of the pGL-2 enhancer vector (Promega, WI). Series of 3' and 5' deletion constructs were generated using appropriate unique restrictases within the insert and in pGL-2 polylinker (MluI for 5' deletions and BglII for 3' deletions). The plasmids carrying SV 40 early promoter (in pGL-2 control: Promega) and thymidine kinase promoter (in pTK, Gill, et al., 1994) were used as positive controls.

Transfection and Assays of Luciferase Activity 293 and UMRC 6 cells were transfected using the lipofectin protocol as described elsewhere (Chang and Brenner, 1988).

For each 35mm plate 2Ag of the luciferase reporter plasmid, l1g of pCMV3 (Clontech, CA) and 1041 of Lipofectin (Gibco- BRL) were added. Luciferase and 0-galactosidase assays were performed 40h after transfection using luciferase and 3- 34 galactosidase assay kits (Promega). The luciferase assay was performed using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, CA).

Construction of the VEL Minigenes Expression construct (pRc-HAVHL), which contained VHL reading frame subcloned into pRc CMV vector (Invitrogen, CA), was kindly provided by Dr. William G. Kaelin Jr. (Division of Neoplastic Disease Mechanisms, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA). Group 7 VHL cDNA in pBluescript II KS was described elsewhere (Latif, et al., 1993), 1.4kb NotI fragment from group 7 construct (exons 3, 2 and 3' part of exon 1) was inserted in correct orientation into NotI site of plasmid pNE (pBluescript II SK carrying VHL 5' flanking 892bp EcoRI-NotI genomic fragment, including o.o 5' part of exon The final plasmid (pVHL) was used to 15 generate three expression constructs in which VHL minigene was driven by its own promoter as follows: pRcpVHL: after digestion of pRc-HAVHL with NruI-BstEII, CMV promoter/enhancer and part of the VHL reading frame were removed and substituted by VHL promoter and exon 1 from pVHL 20 (EcoRV-BstEII digest); pRcpVHLm: plasmid pRcpVHL was linearized with BstEII, filled-in with Klenow fragment and religated: pRcpVHL3U: BstEII-XbaI fragment in pRcpVHL was substituted by BstEII-XbaI fragment from pVHL, which contained additional 0.9kb from 3' untranslated region.

Stable Transfection of the UMRC6 Cells Eighty percent confluent UMRC 6 cells were transfected with 25 zg DNA and 40 Al of lipofectin in 5 ml OPTI-MEM medium (Life Technologies Inc., NY) per 100 mm plate for 12 h and grown in DMEM medium. In a day, 400 g ml-' of active geneticin was added and resistant colonies were grown for 2 to 3 weeks. Selective media was changed every 3 days.

EXAMPLES

The Examples herein are meant to exemplify the various aspects of carrying out the invention and are not 35 intended to limit the scope of the invention in any way.

Example 1 Isolation of the VHL Disease Gene The isolation of the VHL disease gene resulted from the use of positional cloning strategies (Latif et al., Cancer Res. (1993) 63:861-867; Trofatter et al., Cell (1993) 72:791-800 and The Huntington's Disease Collaborative Research Group; Cell (1993) 72:971-983) previously used in isolating disease genes and is described in Latif, et al., Science, (1993) 260:1317-1320. Genetic and physical map of the chromosome 3p region encompassing the VHL gene is shown in Figure 1. The VHL locus was positioned on the map (Fig.

1 Panel A) by multipoint linkage analysis and meiotic mapping (Tory et al., 1989); the location of selected cross-overs is indicated by crosses.

.YAC Library Screening and Analysis of YACs. Copies of the aWU and CEPH YAC libraries were obtained from Dr. Craig Chinault (Baylor Institute of Human Genetics, Houston, 20 Texas) and Dr. Daniel Cohen, respectively (centre d' Etude du Polymorphisme Humain, Paris). The WU and CEPH libraries are total human genomic DNA libraries constructed in the PYAC4 vector (Burke, D.T. et al. Science (1987) 236:806-812; Anand, R. et al. Nucleic Acids Res. (1990) 18: 1951-1956) These libraries were screened by sib selection using PCRbased techniques (Greene, E.D. et al., Proc. Natl. Acad Sci.

(1990) 87:1213-1217) with primers for the D3S601, D3S587 and D3S18 loci in the VHL region (Figure The sequences of the primers used to positively identify YACs Y52A10, YA101D4, Y132F2 and Y70D2 are shown below as SEQ. ID. NO. 44 thru SEQ. ID. NO. 49: Locus/ Location Designation Sequence D3S18/3p26 ML-1 CACAAGTGAT GCCTTGTAGC

TG

SEQ. ID. NO. 44 36 D3S18/3p2 6 ML-2 CAGTAGTGTC CTGTATTTAG

TG

SEQ. ID. NO. D3S601/3p25.3 ML-7 GTTGGCTATG GGTAGAATTG

G

SEQ. ID. NO. 46 D3S601/3p2 5 3 ML-8 CAGGGTAGCC TTGATCTAAG

T

SEQ. ID. NO. 47 D3S587/3p2 5 2 ML-10 GGAGGTCCTG AGAATATGTG

TCC

SEQ. ID. NO. 48 D3S587/3p 25 2 ML-11 TGTTCAGGCA CACAGTAGAT

G

SEQ. ID. NO. 49 Screening Chromosome 3 Cosmid Library and Cosmid Contig Assembly. The chromosome 3 cosmid library was constructed as described in Lerman, et al. (Lerman, M.I. et al. Hum.

Genet. (1991) 86:567-577). This library was screened by colony hybridization (Sambrook, J. et al. (1989)) using the YAC DNA inserts as probes as described in Baxendale, et al.

(Baxendale, S. et al. Nucl. Acids Res. (1991) 19:6651) After labeling with 2 nP-dCTP, the probes were preassociated with a 1000X excess of sheared human DNA. Cosmid contigs 20 were constructed by finding overlapping bands on Southern blots of EcoRI-digested cosmids using whole cosmids as probes. Gaps in the cosmid contigs were closed by chromosome walking using insert-end fragment probes, which were identified by restriction mapping and hybridization to restricted genomic DNA. These insert-end fragment probes were used for each walk step. Figure 1 shows the 160 kb cosmid and phage contig covering the VHL region. The phage T42 was isolated by screening a total genomic phage library with YAC DNA inserts as described above. The phage p191, which contains the VHL gene, was isolated by screening a three-hit pi phage genomic library (Genome System, Inc. St.

Louis, MO) with primers chosen from within an exon of the g7 cDNA sequence shown in SEQ ID NO. 1. The phage p191 was deposited with the ATCC on May 13, 1993.

37 Example 2 Isolation of a cDNA Corresponding to VHL Disease Gene Screening cDNA Libraries. A Xgtll teratocarcinoma library (gift of Dr. Maxine Singer, National Cancer Institute) was S screened by plaque hybridization (Sambrook, J. et al.

(1989)) to 100 filter-immobilized cDNA phage clones at a density of 4 x 104 pfu/150-mm filter. Figure 1 (Panel

B)

shows the position of the g7 cDNA isolated by screening the Agtll teratocarcinoma cDNA library with a conserved Fkb fragment at the centromeric end of cosll used as a probe in the screening. The orientation of the g7 cDNA was established by sequencing and restriction mapping to the contig.

The beginning of the smallest constitutional deletion is indicated by an asterisk and line. Restriction sites:

B,

15 Ba m HI; E, Eco RI; N, Not I; Nr, Nru I; M, Mlu I.

•cDNA Sequence and Sequence Analysis. The g7 cDNA clone was sub-cloned into the Bluescript KS plasmid (Stratagene, S• La Jolla, CA). Double-stranded plasmid DNA was used in sequencing reactions performed with Tag Dye Deoxy terminator cycle sequencing kits (Applied Biosystems, Inc.) All sequences were obtained by running the reactions in an ABI 373A automatic sequencing system (Applied Biosystems, Inc.) Initial sequencing was performed with T3 and T7 primers, and 25 walking" primers were then constructed to continue Ssequencing. The cDNA clone was sequenced multiple times in one orientation or both orientations. Database searching, sequence editing, sequence assembly, and sequence analysis were carried out with the University of Wisconsin Genetics Computer Group sequence analysis software package, version 7.0 (Devereaux, J. et al. Nucl. Acids Rev. (1984) 12:387- 395). The sequence of the g7 cDNA is shown in SEQ ID No. i.

This cDNA was deposited with the ATCC on May 13, 1993. The cDNA sequence revealed an open reading frame (ORF) of 284 amino acids indicating that the rest represents part of the 3' untranslated region of the mRNA. This ORF showed a high 38 0 probability score 95%) for being a protein coding sequence Fickett, Nucl. Acids Rev. (1982) 10:5303).

Neither the nucleotide nor the predicted amino acid sequences showed any significant homology to genes or proteins in the databases.

Example 3 Detection of q7-Specific mRNA Expression in Target Tissues RNA Preparation and Northern Blotting Analysis. To identify the VHL gene, the g7 loci was evaluated by analyzing its 0 expression in target tissues.

The expression pattern of the g7 gene was examined by Northern (RNA) blotting. Figure 2A shows a low resolution blot where each lane contains poly A' mRNA (2 pg) from: lane 1, fetal brain; lane 2, adult brain; lane 3, 15 fetal kidney; lane 4, adult kidney; lane 5, adult cerebellum; lane 6, adult adrenal; and lane 7, adult prostate while Figure 2B shows a high resolution blot of 1 ug of poly A+ mRNA from tissues as indicated in Figure 2A.

The sizes of the transcripts were determined from the 20 position of the 28S and 18S rRNA bands of total RNA run on the same gel..- Transcripts were observed in all' human tissues tested, including brain and kidney, tissues frequently affected in VHL disease. The transcripts were of two distinct sizes, 6 and 6.5 kb, and were expressed in a tissue-specific and developmentally selective manner, i.e.

only 6 kb or the 6.5 kb species was expressed in fetal brain and fetal kidney, while both were expressed in adult tissues. The two transcripts may represent alternatively spliced forms of g7 mRNA.

Example 4 Detection of Mutations of the VBL Disease Gene Associated With VHL Disease and Related Diseases RT-PCR Studies of Gene Expression. In order to detect DNA of affected patients in mutations in constitutional DNA of affected patients in 39 pedigrees and in new mutation patients, an extensive search for mutations small intragenic and nonoverlapping deletions or insertions) which were of the loss -offunction type was conducted in constitutional DNA derived from 221 unrelated VHL patients. Southern blot analysis of genomic DNA isolated from the blood (Sambrook, J. et al.

(1989)) of seven patients and then digested with EcoRI is shown in Figure 3A. This blot was probed using the g7 cDNA as probe. This probe has been shown to detect a single invariant 20-22 kb EcoRI fragment in normal DNA, as determined by previous tests on more than 100 unrelated

DNA

samples provided by Centre d'Etude du Polymorphisme Humain (CEPH). A high incidence (r 12%) of aberrant bands was observed with the bands ranging in size from 4 to 25 kb (Figure 3A), and these VHL patients were thus classified as new mutations.

0:0 In order to determine that the single aberrant bands originating from the 20-22 kb invariant fragment were deletions or insertions within this fragment or deletions removing the flanking EcoRI sites, Southern blot analysis was conducted with several other restriction enzyme digests besides EcoRI (BamHI, BglI, BglII, Dral, EcoRV, HindIII, PstI, and PvuII). The results of the Southern analysis with a few of these enzymes is shown in Figure 3B. These results demonstrated that the mutations were transmitted with the 25 disease (Figure 3C). Figure 3D shows the results of Southern blotting analysis of DNA isolated form a regular VHL family (coded and digested with EcoRI. The results clearly demonstrate transmission of the mutant allele (the aberrant band) in this VHL family (Figures 3D and 3E).

Example Detection and Mapping of Deletions of the VHL Disease Gene To prove the presence of deletions and to map them precisely, subfragments representing regions of the g7 cDNA generated by PCR were used as probes in Southern blotting 40 0 analysis of genomic DNA isolated from blood of VHL patients and digested with EcoRI. (Figure 4, where the probes used in each panel are: Panel A, total g7 cDNA; Panel B, nucleotides 3-146 of g7 cDNA; and Panel C, nucleotides 1277- 1600 of g7 cDNA). The results unequivocally demonstrated that 18 of the rearrangements were deletions as only part of the cDNA failed to detect the novel band in each patient (Figure 4).

These deletions could then be classified into three groups, as shown in Table 1.

S

S

5030 41

S.

Oe 0 660 0

SS

0 6S *000

C

@0 0 OS @0 0 S 0 0000C@ 0 TABLE 1 Deletion analysis of VHL patients with aberrant bands at the VHL locus (detected by g7 cDNA).

Paient Aberrant Apparent Code Probe :cDNA residue Band Deletion Size (kb) 3-146 169-391 291-501 585-940 921-1231 1277-1600 3567 ND ND ND ND ND ND 14 3607 ND ND ND ND ND ND 12 3639 ND ND ND ND ND ND 14 3648 ND ND ND ND ND ND 13 3654 ND ND ND ND ND ND 14 JD ND ND ND ND ND ND 17 PEM ND ND ND ND ND ND 15 MS ND ND ND ND ND ND 15 KA ND ND ND ND ND ND 15 3547 D D D ND ND ND 23-25 15-18 JM D D D ND ND ND 23-25 15-18 GD D D, D ND ND ND 23-25 15-18 3512 ND ND. ND ND D- D 10 11 3516 ND ND ND ND- D D 10 11 3557 ND ND ND ND D D 10 11 3574 ND ND ND ND D D 10 11 VIA ND ND ND ND D D 10 11 IC ND ND ND ND D D 10 11 NE ND ND ND ND D D 10 11 EP ND ND ND ND D D 10 11 MO ND ND ND ND D D 10 11 3569 ND ND ND D D D 12 9 3667 ND ND ND D D D 10 11 3761 ND ND ND D D D 4 17 3819 ND ND ND D D D 12 9 ND Not deleted D Deleted 00 0:0 0 io -42 The finding of three overlapping deletions within the same cDNA provides strong evidence for the identification of the g7 cDNA as the VHL gene.

Example 6 Detection of Intragenic Deletions or Insertions by PCR-SSCP and RT-PCR To find intragenic deletions or insertions, genomic DNA isolated from VHL patient lymphoblastoid cell lines (Lymphoblastoid cells were immortalized by transformation with Epstein Barr Virus according to standard protocols (Nilison, K. et al., Adv. Cancer Res. (1982) 37:319-380)) was analyzed for alterations by PCR-singlestrand-conformational polymorphism (PCR-SSCP) analysis using primers shown in SEQ. ID. NO. 7 thru SEQ. ID. NO. 12 and RNA 15 isolated from sporadic renal cell carcinoma (RCC) cell lines (Anglard, P. et al. Cancer Res. (1992) 52:348-356) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The primers used for RT-PCR of the RCC cell lines are shown as SEQ. ID. NO. 50 thru SEQ. ID. NO. 53: SEQ. ID. NO. CATCTTCTGC AATCGCAGTC CGCGCGT SEQ. ID. NO. 51 CAAAAGCTGA GATGAAACAG TGTAAGT SEQ. ID. NO. 52 GTTTGGTTAA CCAGAAGCCC ATCGT SEQ. ID. NO. 53 GATGGGCTTC TGGTTAACCA AACT whose SEQ. ID. NO. 50 and NO. 51 are on pair of primers and SEQ. ID. NO. 52 and SEQ. ID. NO. 53 are a second pair. The results of these analyses are shown in Table 2.

43 TABLE 2 Germ-line (VHL) and somatic (sporadic RCC) mutations in the VHL candidate gene.

Patients Mutation Consequence VHL family "VA" 8 bp (TTGTCCGT) insertion after NT714* frameshift E"9 bp in-frame deletion (NT456-464) Three amino acid 15 3 -1 54 deletion (Arg Val Va) 3 bp in-frame deletion (NT434-436) One amino acid deletion (146, le) Sporadic RCC "UOK1 18" 1 bp deletion (NT737) framneshift "UMRC5" 1 bp deletion (NT737) framneshift "UMRC6" 10 bp deletion (NT71 5-724) f rameshift "A498" 5.bp deletion (NT638-642) frameshift "UOK1 51" nonsense C (NT761) transversion stop codon NT nucleotide(s).

44 0 RCC were chosen because according to Knudson's dictum (Knudson (1971)), sporadic cancers should be associated with mutations in the same loci affected in the hereditary form of the same malignancy. So far aberrant patterns have been identified in five RCC cell lines and proved four of them have been proven to be small (1 to bp) deletions creating frameshift mutations and truncated proteins (TABLE The cell lines UMRC5 and RCC "UOK118" have the same 1 bp deletion at nucleotide 737, amino acid 246, creating 28 new amino acids followed by a stop codon.

0 Incidentally, this deletion creates a new EcoRI site, leading to two aberrant bands on Southern blots (not shown).

Line UMRC6 has a 10 bp deletion (nucleotides 715 to 724) creating a frameshift such that 32 new amino acids are present followed by a new stop codon. Finally, line A498 has a 5 bp deletion (nucleotides 638 to 642) leading to a premature stop after new 62 amino acids. In the fifth RCC cell line, UOK151, the change is a nonsense (stop codon) mutation resulting from a C to A transversion at nucleotide 761 (TCG TAG), creating a truncated protein. These data 20 suggest that the VHL disease gene plays an important role in sporodic kidney cancer. As such, RT-PCR or PCR-SSCP as described in this application can be used as diagnostic methods to distinguish primary kidney tumors from tumors that spread to the kidney from other tissues or organs and to distinguish different histological types of kidney tumors.

In the DNA of the VHL lymphoblastioid cell lines derived from VHL patients, SSCP aberrant patterns segregating with the disease were also detected using primers shown in SEQ. ID. NO. 7 thru SEQ. ID. NO. 12. 'One (patient was found to be an 8 bp (TTGTCCGT) insertion after nucleotide 714. This insertion created a shift in the reading frame and a truncated protein. The second patient had an in-frame 3 bp deletions leading to the removal of amino acid 146 (isoleucine). Finally, patient had an 45 in-frame 9 bp deletion (nucleotides 456 to 464) that resulted in the removal of three amino acids (Arg Val Val) at position 153-155. These combined results strongly support the conclusion that the g7 gene represents the VHL and the sporodic RCC tumor suppressor gene.

Example 7 Conservation of the q7 cDNA Across Species In order to determine whether the g7 cDNA is highly conserved across species ranging from mammals to Drosophila and sea urchins, Zoo blotting using g7 cDNA as a probe was performed on DNA isolated from human (Homo sapiens), chimpanzee (Pan troglodytes), macaque (Macaca fascicularis), cow, (Bovis domesticus) rat (Rattus norvigicus), mouse (Mus musculus) chicken (Gallus domesticus), frog (Xenopus laevis), fly (Drosophila melanogaster), sea urchin (Strongylocentrotus purpuratus), and yeast (Saccharomyces ceriviseae) all purchased from *BIOS Laboratories (New Haven, CT, USA). (Pre)Hybridization was done in Church buffer M. Church and W. Gilbert, 20 Proc. Natl. Acad. Sci. 81, 1991 (1984)] at 65 0 C for 18 hours. Blots were washed in 0.1 x-Church buffer at 60 0

C

for 60 min. The results of the zoo blot are shown in Figure 6. The results demonstrate an extensive evolutionary conservation which is indicative of g7 serving a basic life function and also, of g7 having a tumor suppressor role.

Example 8 Identification and characterization of the promoter of the human VHL tumor supressor gene Transcription initiation sites were located near the putative SPI/AP2 binding site. In one stably transfected clone of the renal carcinoma UMRC 6 cell line, the level of transcription from VHL minigene, containing flanking genomic DNA up to residue -647, was comparable with endogenous VHL expression. Using luciferase reporter 46 0 constructs which include 5' flanking genomic sequence (residues -467/+195) the minimal promoter was delineated within 106bp (positions -83/+23) in human embryonic kidney 293 cells. The 5' flanking DNA (residues -467/+195) were also examined for putative transcription factor binding sites and for other regulatory sequences. Several putative binding sequences for tissue specific transcription factors were located near transcription initiation sites. Among them is a core sequence for the Pax family of transcription factors which, apparently, regulates organogenesis. Pax 2 0 protein, a member of this family, is required for mesenchyme-to-epithelium conversion and is temporarily expressed during kidney development (Rothenpieler and Dressier, 1993). Since clear renal carcinomas originate from proximal tubular epithelium, Pax 2 may have an effect on VHL expression. A related gene, Pax 8, is also activated in developing kidney (Plachov, et al. 1990). Another potentially important site is a 12 bp consensus sequence for the nuclear respiratory factor 1 (NRF-1), which is involved in nuclear-mitochondrial interactions, and apparently, :20 coordinates regulation of nuclear and mitochondrial genes during organelle biogensis (Evans and Scarpulla. 1990; Virbasius and Scarpulla 1994). Identical potential binding sites were also found in several other groups of genes (Virbasius, et al. 1993), including those involved in regulation of the cell cycle (cdc 2, RCC 1) cell growth (ornithine decarboxylase, DNA polymerase alpha) and apoptosis (bcl 2).

Consistently, all observed VHL point mutations were located downstream of the first methionine codon (Latif, et al., 1993b; Crossey, et al., 1994; Gnarra, et al.

1994; Richards, et al. 1994; Shuin, et al. 1994; Brauch, et al. 1995; Chen, et al. 1995) The codons upstream of this point are rarely used in human translated sequences (Wada, et al.' 1992), whereas the downstream codons are used frequently. Finally, the region of homology between the 47 0 human VHL cDNA and its recently isolated mouse counterpart does not extend upstream of the first methionine (Latif and Duh. personal communication accession No. U12570).

To position the cloned cDNA within the full length VHL mRNA, RNase H mapping was employed (Berger, 1987).

Restricted cleavage of the VHL mRNA with RNase H was directed by antisense DNA oligomers (Figures 7A-7C). The oligomers 1 and 2 were designed to anneal with the VHL mRNA at 267 to 296 nt and 572 to 596 nt downstream of the cDNA end respectively (Figures 7A-7C). As shown on Figure 7A, 0 the cleaved 5' part of the VHL mRNA is comparable by length with the known cDNA sequence. The size difference between 5' fragments obtained when RNA was digested with different oligomers agrees with the distance calculated from the cDNA sequence. Similar results were obtained using total RNA 15 from 293, UMRC6, U2020 cell lines and human prostate poly(A) RNA. Thus, the group 7 cDNA completely (or almost completely) represents the 5' end of the VHL mRNA.

In agreement with these data, extensive screening of 155 cDNA libraries (totalling 15 million clones. 100 20 positive clones were evaluated) and the rapid amplification of 5' cDNA end (5'RACE) technique did not yield any gain upstream of the known cloned cDNA sequences. No gross genomic rearrangements were found within the region covering •60 kb upstream of the VHL cDNA in more than 100 of the VHL kindred. When hybridized to Northern blots, the cloned genomic fragments from this region did not reveal any message the length of VHL.

Mapping of the Transcription Initiation Sites Attempts to use primer extension to determine the VHL transcription starts were unsuccessful apparently because of high GC content and stable secondary structures near the 5' end of the VHL mRNA.

Thus, the transcription start sites were determined by RNase protection analysis. An antisense 48 0 riboprobe no. 1 (Figure 8A) was generated from PstI-NotI (530 nt) genomic fragment, which included a part of exon 1 from the cDNA sequence (223 nt) and the immediate flanking region (308 nt) After hybridization with poly(A) RNA from 293 cells several protected fragments 225 to 240 nt were found (Figure 8B slots 1, 2 and This result roughly agrees with the RNase H mapping data but it falls far below the predicted figure (390 nt) for the "extended" exon 1 which would presumably contain the whole open reading frame, deducted from genomic sequence downstream of the 0 putative splice acceptor site (Latif, et al., 1993b). To exclude any artifacts resulting from possible internal RNase cleavage of longer protected fragments, the experiment was repeated with probes no. 2 and no. 3. Probe no. 2, which was identical to probe no. 1 except for a shorter flanking genomic region (44 nt instead of 308 nt) did not reveal any protected fragments (Figure 8B, slots 7, 8 and The same results were obtained with poly(A)- RNA from human prostate and adult kidney (data not shown). According to these data transcription start sites were placed not more 20 than 30 nt upstream of the 5' cloned cDNA 5' border.

For precise mapping of the transcription start sites, a shorter probe (no. 5; Figure 8A).was used which included 149 nt of the exon 1 sequences from the cDNA and 104 nt of the 5' flanking genomic region. Using RNA 2 markers, the size of the protected fragments was identified as 152, 153, 161, 162, 163, 171 and 176 nts, which means that the 5' ends of the VHL mRNA were located respectively 3, 4, 12, 13, 14, 22 and 27 bp upstream of the cDNA border.

The first nucleotide of the RNA specie which was initiated 22 bp upstream of the cDNA border was assigned number +1 (Figure 8C).

A Functional Promoter is Located Around Initiation Sites To test the promoter activity a fragment from the 5' flanking genomic region (bases -467 through 195) was 49 inserted into pGL-2-enhancer luciferase reporter vector, which was transfected into 293 cells. The fragment was shown to drive transcription of luciferase. The efficiency of the full length VHL promoter (bases -467-195) in 293 cells was assigned 100% SV 40 early promoter activity comprised 60% and thymidine kinase promoter about 500 of the full VHL promoter strength. The promoter activity appeared to be unidirectional, since the activity of the fragment in reverse orientation was about seven times weaker.

To localize more precisely the minimal promoter region, a set of 5' and 3' deletion constructs was prepared (Figure The results of transfection indicated that the minimal promoter can be delineated within 106 bp, between restriction sites for EagI and SacII The 15 minimal construct retained 32±9% of the full promoter activity. No separate promoter activity was found upstream of the EagI site The region downstream of the Smal site (+30/+195) enhances transcription by about two times; however it does not possess promoter activity of its 20 own.

Because the mutations in the VHL gene apparently play a critical role in the origin of clear renal carcinoma (Latif, et al., 1993b; Gnarra et al. 1994; Shuin et al.

1994), the UMRC 6 cell line derived from this malignancy was also studied. When normalized to (-galactosidase expressed under cytomegalovirus (CMV) promoter, the luciferase activity in UMRC 6 cells was about two times lower than in 293 cells. However, the relative activity of different constructs compared to the full length construct no. 1 (Figure 3) in each cell line appeared to be similar. These data indicate that the same promoter region is active in both 293 and UMRC6 cell lines.

50 0 flanking genomic fraQment, containing VHL promoter, confers apparently normal level of transcription to VHL miniqenes To estimate the level of transcription from the native VHL promoter in VHL minigenes in renal carcinoma, three minigene constructs were used, which were based on the pRc/CMV vector (Invitrogen). In these constructs

CMV

promoter/enhancer region was substituted by a VHL flanking EcoRI-NotI genomic fragment which was fused to the rest of the VHL cDNA (Figure 10A). The final expression 1 plasmids included VHL sequences from base -647 to +710 (pRcpVHL) and from -647 to +1664 (pRcpVHL3U). To eliminate any possible effects of the native VHL protein on cell growth, a frameshift was introduced into the VHL reading frame (duplication of bases -408/-412 in exon 2) of the 15 pRcpVHL by digestion with BstEII, fill-in with Klenow fragment and relegation (plasmid pRcpVHLm). A transcript from the construct containing CMV promoter and VHL reading time (pRc-HAVHL) was used as a size marker of Northern blots. For transfection, the UMRC6 cell line was used. The cells were shown to have a 10 bp microdeletion in VHL exon 3 (Latif et al., 1993b) which would allow discrimination between endogenous and exogenous VHL mRNA by reverse transcription/polymerase chain reaction (RT-PCR). After transfection 40 to 50 geneticin positive clones were pooled and expression from VHL minigenes was assayed by Northern analyses (Figure 10B) and RT-PCR. The sizes of the exogenous VHL mRNAs indicated that transcription was initiated roughly from the same region inside the NotI-EcoRI fragment as we have shown above for endogenous VHL gene using the RNase protection assay. RT-PCR analysis confirmed expression from the VHL minigenes.

The question of whether the obvious difference in the level of expression between endo- and exogenes (Figure reflected a lack of important regulatory elements within the minigenes or just frequent rearrangements of the -51 0 VHL transgene in many of the geneticin resistant clones was next investigated. Five colonies were expanded and analysed by Southern and Northern blotting analyses (three of them were transfected by pRcpVHL3U construct, another two carried pRcpVHLm). However, only one clone (pRcpVHLm, clone 4) was shown to have nonrearranged VHL transgene (1.3 kb EcoRI fragment, Figure 11A) which expressed VHL mRNA (Figure 11B).

Both the 950 nt and about 4800 nt transcripts showed a similar signal intensity on Northern blot with apparently the same gene copy number on Southern blot. This observation may indicate that the 5' VHL genomic region confers apparently normal level of transcription in the UMRC 6 renal carcinoma cell line. However, other factors may interfere, for example, the enhancing, (silencing) activity of the DNA sequences near integration site and different 15 stability of the exogenous mRNA due to absence of a fulllength 3' UTR.

Sequence analysis of the VHL promoter The VHL promoter and exon 1 comprised a CpG 20 island. The GC content within the minimal promoter region is 72.6%. The minimal promoter harbors several GC-specific restriction sites including one for EagI, three for BssHII, one for Sail and six for Hhal. The region around minimal promoter (-467/-195) does not contain TATA and CCAAT boxes. A putative binding suite for SP-1 (KRGGCGKRRY; -1-13; Briggs, et al., 1986) and AP-2 transcription factors (YCSCCMNSS: Imagawa, et al.

1987) was found near transcription initiation sites. It appears to play a major role in the VHL transcription initiation. However, the reporter deletion analysis described above indicates that the region -83-10 is also functionally essential. Another site for SP1/AP2 was found in position +74/-83. Two sites for SP1 with a more loose recognition sequence (KRGGCKRRK; Faisst and Meyer, 1992) and one site for AP2 factor were located upstream of the minimal 52 0 promoter (Figure Other putative transcription factor binding sites include Pax core sequence (GTTCC; -56/-60; Chaiepakis, et al., 1991) sites for nuclear respiratory factor 1 (YGCGCAYGCGCR: -92/-103; Evans and Scarpulla, 1990), nuclear hormone receptor for retinoic acid H-2RIIBP (GAGCTC; -21/-26; -293/-298; Marks, et al., 1992) and several other factors.

An important feature of the region further upstream to the VHL minimal promoter is a termination polyadenylation signal for RNA polymerase II 379), 0 which may prevent continuous transcription form other putative promoters upstream. Indeed, no evidence of such promoters has been found as yet.

The contents of all citations, journal articles, patents and the like, are incorporated herein by 15 reference.

It is understood that the examples and embodiments described herein are for illustrative purposes and that various modifications and changes in light thereof to persons skilled in the art are included within the spirit 20 and purview of this application and scope of the appended claims.

53 oS THE FOLLOWING PAGE(S) APPEAR AFTER THE DESCRIPTION AND BEFORE THE CLAIMS.

-53 0 SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES (ii) TITLE OF INVENTION: PARTIAL INTRON SEQUENCE OF VHL DISEASE GENE AND ITS USE IN DIAGNOSIS OF DISEASE (iii) NUMBER OF SEQUENCES: 53 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: MORGAN FINNEGAN, L.L.P.

STREET: 345 PARK AVENUE CITY: NEW YORK STATE: NEW YORK COUNTRY: USA 15 ZIP: 10154 COMPUTER READABLE FORM: MEDIUM TYPE: FLOPPY DISK COMPUTER: IBM PC COMPATIBLE OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: WORDPERFECT 5.1 20 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: TO BE ASSIGNED FILING DATE: MARCH 27, 1997

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(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/623,428 FILING DATE: 28 MARCH 1996 CLASSIFICATION: APPLICATION (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/061,889 FILING DATE: 14 MAY 1993 CLASSIFICATION: APPLICATION (viii) ATTORNEY/AGENT INFORMATION: NAME: FEILER, WILLIAM S.

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DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1816 TYPE: nucleic acid STRANDEDNESS: single 15 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

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55

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IDENTIFICATION METHOD: OTHER INFORMATION (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CCTCGCCTCC

TTCTGCGCAC

AGCGCGTTCC

GAGGTCGACT

CCGACCCGCG

GATCGCGGAG

GAGGCCGAGG

ACGGCCCTGA

GGAGTCCGGC

GCCGAGGAGG

TGCGCTCGGT

CTGCAATCGC

AACTTCGACG

CTGGCACGGG

15, TTGGCTCTTC

GTTAACCAAA

ACGGACAGCC

TACTCTGAAA

GTCAAGCCTG.

CGCTCTACGA

AGACCTGGAG

CGGATGGGAG

TTCATCTCAG

GATACAGGAC

TTCTCAGAGT

AACTGACTTC

TCACAAAATG

TTATCAGGAG

TCAGGACAGC

CAGTGGGAAT

TTGGCATCTG

ACATCCGTAG

GGAAGACTGA

CCTCTTTGAG

GTCAGGGTTT

TAGAAAGTGC

GGTGGGAGAG

GTCTTAAAGG

ATTTTCTAAG

AGTGCTGTTT

CTACAGTTGT

CACAGAAAAT

TTAATGGACA

GTTCCTTTT

GTTTTTTGT

GG GATGGAGG GTTACAACAG CCTACGGTGC GCGCACAGCC TCCGGCCGGC ATCCTCTACC GAGCGCGCGC CGGGAGCGCG CACGCAGCTC GATCCCGCGG CGTCCGGCCC GGAATGCCCC GGAGGGCGGA TAGGCGCGGA GGAGGCAGGC AGAAGACGGC GGGGAGGAGT CCGGAAGAGT CCGGCCCGGA AGATGGAGGC CGGGCGGCCG GAACTCGCGC GAGCCCTCCC AGTCCGCGCG TCGTGCTGCC GCGAGCCGCA GCCCTACCCA CCGCCGCATC CACAGCTACC AGAGATGCAG GGACACACGA CTGAATTATT TGTGCCATCT TATTTTTGCC AATATCACAC GAGCGATGCC TCCAGG'FTGT AGAATTACAG GAGACTGGAC AGATCTGGAA GACCACCCAA CGGCTGACAC AGGAGCGCAT ATTGAAGATT TCTGTTGAAA CTTTTGATGG TACTGATGAG TGGTTCCTTC CTTAGTTTCA AAAATAGGCA CCATTGCTTA ACTAGGCATT GTGATGTTTA TAATTTAATG CCTGCCCATT AAGGTGGTGG CATTTTTGCT TTGTATGTAA GGAGGTTTAT TGCAGCATAT CGTTTAATTT

TGGAGGATCC

TATTTCCGCG

GAAGACTACG

CGCCCCGCGT

GGGTGGTCTG

GAACTGGGAC

GTCGAAGAGT

CGGGCGCCGA

GGAACTGGGC

CGGCCCGTGC

AGGTCATCTT

CGTATGGCTC

ACGCTGCCGC

GAGGTCACCT

TGGGCTTCTG

CTCAATGTTG

TGCCAGTGTA

CCGGAGCCTA

ATCGTCAGGT

ATGTGCAGAA

TGC ACATCAA

CTTACACTGT

TCTTGATCTA

AAGTGTCTCA

AAAGAAAGTT

GGGGCAAACA

AGAGAAGTAT

TCCTAGTAAG

ATAAGTAATT

TAAGAAGGCA

120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 1040 1080 1120 1160 1200 1240 1280 1320 1360 1400 1440 1480 1520 1560 1600 1640 1680 1720 1760 1800 1816 CT TTT AATGG ATGTATAATA CATCCATTCT CGGTTGGTGA CTTGTC!TGCC TCCTGCTTTG GGCATCCGTG AGGCAGGGAC AAGTCTTTCT ACCCCAGTGC CTGCACATCA TGAGCCTTCA CTCAGAGGAA CAAACCAGGG GACACTTTGT TTAGAGGTTC TGCCTCTATT TTTGTITGGGG GGGACCTTAA AATGTGTACA GTGAACAAAT GAATCA'F=r TGTAGGAAGC ATTTTTTATA TCGTGCACTT TCTCGGTCCA CTCTTGTTGA TATTACTGTT TCTAAACTAG GATTGACATT GATAATAGCA TTTTTGTAAC TTGCCATCCG ACGAGAAAAT CTGCATGTTT GATTATAGTA AATAAGTTTT TGCTAAATGT GAGTATTTCT GTAAATATGT GACATTCCTG ATTGATTTGG TGTTGTTGTT TTGTTTTGTT TTGT TTT TTT

GAATTC

a 56 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 284 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: AMINO ACID SEQUENCE (ii) (iii) HYPOTHETICAL: No (iv) (v) (vi)

ANTI-SENSE:

FRAGMENT TYPE: ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE;

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) (xi)

FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: SEQUENCE DESCRIPTION: SEQ ID NO:2: Arg Tyr Asn Ser Leu Arg Cys Trp Arg Arg Thr Arg Thr Ala Ser Gly Arg Leu Ala Arg Ser Ile Leu Tyr Arg Ala Arg 30 Pro Arg Leu Ile Leu Leu Phe Pro Arg 57 Al a Gin Arg Pro Gly Glu Ser Ala Val Val 145 Pro 15 Tyr His Al a Glu 205 Pro Leu Val Arg Val 265 Ile Lys Leu Pro Arg Ala Giu Gly 110 Glu Leu Ile Val Pro 170 Se r Gly Leu Ile Lys 230 Lys Ser Gin Al a Thr Arg Ala Arg Giu Asp Pro Glu Arg 135 Phe Trp Thr Tyr Thr 195 Phe Phe Glu Pro Leu 255 Lys His Thr Pro Arg Ala Giu Gly 100 Glu Giu Ser Cys Leu 160 Leu Arg His Val Al a 220 Arg Giu Tyr Asp Gin 280 Glu Val Ala Ser Val Val Glu Asn Ala Gly 90 Gly Glu Glu Ser Met Glu 125 Val Asn Asn Arg 150 Asn Phe Pro Pro Gly His 185 Asp Gly Pro Ser 210 Asn Ile Cys Leu Asn Tyr 245 Glu Asp Leu Glu 270 Arg Met Asp Asp 55 Trp, Trp, Val Giu Gly 115 Ala Ser Ser Asp Gly 175 Leu Leu Leu Ser Pro Ilie Asp Glu Ser Pro Gly Arg 140 Pro Gly Thr Trp Leu 200 As n Gly Arg Ala Glu Glu Gly 105 Glu Arg Glu Arg Glu 165 Gly Leu Val1 Val Pro 225 Val1 Leu Asp Thr Ala Ilie Giu Ala Tyr Ala Glu Pro 130 Pro Val Pro Arg Phe 190 Asn Asp Val Arg Asp 250 His Gin Arg Pro Gly Glu Gly Giu Leu Arg Ser Val 155 Gin Arg Arg Gin Gly 215 Tyr Ser Ile Pro Giu 275 Thr Arg Met Val Pro Giu Gly 120 Pro Gin Leu Pro Il.e 180 Asp Thr Gin Thr Leu 240 Val Asn Arg Thr Leu Gin Val 235 Arg Arg Leu Glu 260 Arg Leu Gly Asp INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 169 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA -58 0 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION: S" UNITS: (ix) FEATURE: S 2 NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TACCCAACGC TGCCGCCTGG CACGGGCCGC CGCATCCACA GCTACCGAGG TACGGGCCCG GCGCTTAGGC CCGACCCAGC AGGACGATAG CACGGTCTAA GCCCCTCTAC CGCCCCGGGG 120 TCCATTCAGA CGGGGAACTA GGCCCCTTGA GGCAGGACAC 160 ATCCAGGGT 169 3 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 403 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 59 (iii) HYPOTHETICAL: No 0 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

C.

*00 0

CTCCTGACCT

GCTGGGATTA

TGTGGCTCTT

CTTTGGCTCT

TGGTTAACCA

TGACGGACAG

ACTGACGTTT

AAGTACAGGA

CAATTTTTGC

GTATCCTATT

TTT

CTATGATCCG CCTGCCTCGG CCTCCAAAGT CAGGTGTGGG CCACCGTGCC CAGCCACCGG TAACAACCTT TGCTTGTCCC GATAGGTCAC TCAGAGATGC AGGGACACAC GATGGGCTTC AACTGAATTA TTTGTGCCAT CTCTCAATGT CCTATTTTTG CCAATATCAC ACTGCCAGGT TACTTTTTAA AAAGATAAGG TTGTTGTGGT TAGACCACTT GAAAAATTAA GCCCAGTTCT CTGATGTCAG GCACGGTATC CAATCTTTTT CTCTACCATA AATAAAATGG AAGTGATGAT 120 160 200 240 280 320 360 400 403 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 60 6 LENGTH: 193 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: 15. CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

0 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

S*

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CTACAGAAGG CATGAACACC ATGAAGTGTC CATAGGGGCC ACAGCATACA CACTGCCACA TACATGCACT CACTTTTTTT CTTTAACCTA AAAGTGAAGA TCCATCAGTA GTACAGGTAG 120 TTGTTGGCAA AAGCCTCTTG TTCGTTCCTT GTACTGAGAC 160 CCTAGTCTGC CACTGAGGAT TTGGTTTTTG CCC 193 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 663 4 Cl 61 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) (v) (vi)

ANTI-SENSE:

FRAGMENT TYPE: ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

AGAGGCCAAG

AGACCAGCCT

ACAAATAAAA

'ACAGTACCAA

TTGAGCCAGG

GCCACTGCAC

CTCCAAAAAA

AAAAAAATTT

AGCCTCGCCT

CCTTCTGCGC

CGAGCGCGTT

CGGAGGTCGA

GCAGGAGGAT

AGGCAACATA

ATAATTAGTC

CTACTCGGGA

GAGGTCAAGG

TCCAGCCCGG

AAAAAAAAAC

TATAGTGGAA

CCGTTACAAC

ACGCGCACAG

CCATCCTCTA

CTCGGGAGCG

CACTTGAACC CAGGAGTTCG GCGAGACTCC GTTTCAAACA GGGCATGGTG GTGCGCGCCT GGCTGAGGCG AGACGATCGC CTGCAGTGAG CCAAGCTCGC GCGACAGAGT GAGACCCTGT ACCAAACCTT AGAGGGGTGA ATACAGTAAC GAGTTGGCCT AGCCTACGGT GCTGGAGGAT CCTCCGGCCG GCTATTTCCG CCGAGCGCGC GCGAAGACTA CGCACGCAGC TCCGCCCCGC 120 160 200 240 280 320 360 400 440 480 62 0 GTCCGACCCG CGGATCCCGC GGCGTCCGGC CCGGGTGGTC 520 TGGATCGCGG AGGGAATGCC CCGGAGGGCG GAGAACTGGG 560 ACGAGGCCGA GGTAGGCGCG GAGGAGGCAG GCGTCGAAGA 600 GTACGGCCCT GAAGAAGACG GCGGGGAGGA GTCGGGCGCC 640 GAGGAGTCCG GCCCGGAAGA GTC 663 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 37 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv)

ANTI-SENSE:

15 FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATAGTGGAAA TACAGTAACG AGTTGGCCTA GCCTCGC f 63 0 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 33 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv)

ANTI-SENSE:

FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

15 INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

20 (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CCCAGCTGGG TCGGGCCTAA GCGCCGGGCC CGT 33 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: -64 0 LENGTH: 33 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: S(G) CELL TYPE: 15 CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 20 20.

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTGGCTCTTT AACAACCTTT GCTTGTCCCG ATA 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 33 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: S(I) ORGANELLE: (vii) IMMEDIATE SOURCE: 15 LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix)

FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CAAGTGGTCT ATCCTGTACT TACCACAACA CCT 33 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 31 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii-) HYPOTHETICAL: No 66 0 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(Vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 15

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

20 IDENTIFICATION METHOD: *0 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TGTATACTCT GAAAGAGCGA TGCCTCCAGG T 31 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 33 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: (v) FRAGMENT

TYPE:

67 0 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

15 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TACCATCAAA AGCTGAGATG AAACAGTGTA AGT 33 INFORMATION FOR SEQ ID NO:13: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: 68 0 DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

0 MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: 5 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGTGGAAATA CAGTAACGAG TTGGCCT 27 S(2) INFORMATION FOR SEQ ID NO:14: 20 SEQUENCE CHARACTERISTICS: LENGTH: 26 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: 69 0 ORGANELLE (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

0 LOCATION: IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GAAATACAGT AACGAGTTGG CCTAGC 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single 20 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No S (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

70 0 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID GTCCCAGTTC TCCGCCCTCC GGGGCAT 27 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: 15 LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

i' II n

II

71 0 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGGGTCGGGC CTAAGCGCCG GGCCCGT 27 1 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 15 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: 20 (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii: POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: 72 0 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTTTAACAAC CTTTGCTTGT CCCGATA 27 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

20 DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

S• (vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix)

FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 73 0 GTGGCTCTTT AACAACCTTG C 21 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA e (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: 15 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: 20 CELL LINE: 66. ORGANELLE: (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GTCTATCCTG TACTTACCAC AACACCT 27 -74 0 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv)

ANTI-SENSE:

FRAGMENT TYPE:

SO

(vi) ORIGINAL SOURCE:

ORGANISM:

0

STRAIN:

15 INDIVIDUAL ISOLATE: DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CCTGTACTTA CCACAACACC TTAT 24 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: 75 LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE: 0

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 20 CHROMOSOME/SEGMENT: MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CTGAGACCCT AGTCTGCCAC TGAGGAT 27 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single 1q TOPOLOGY: linear

I

(I

I

I 76 0 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE: 15 LIBRARY: S*

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

20 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTCCTTGTAC TGAGACCCTA GT 22 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No i' 77 0 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: 0 CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: 20 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGAAATACAG TAACGAGTTG GCCT 24 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: (v) FRAGMENT TYPE: S I.

78 (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

15 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGAAATACAG TAACGAGTTG GCCTAGC 27 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

i Ir -79 0 DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

l0 MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: 1(D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID ACGGGCCCGG CGCTTAGGCC CGACCCA 27 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 32 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: 80 0

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ACGGGCCCGG CGCTTAGGCC CGACCCAGCA GG 32 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 33 TYPE: nucleic acid 0 STRANDEDNESS: single 0 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv)

ANTI-SENSE:

FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

81 0 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GTGGCTCTTT AACAACCTTT GCTTGTCCCG ATA 33 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: 15 LENGTH: .16 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

82 0 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CTTTAACAAC CTTTGC 16 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 15 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: 20 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

Sooo TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

83 0 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GATAAGGTTG TTGTGGTAAG TACAGGA 27 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No o (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

S(B) STRAIN: INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE: 20

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID 84 0 AGGTTGTTGT GGTAAGTACA GGATAGC 27 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT

TYPE:

15 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: 0 CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CTCCTTGTAC TGAGACCCTA GT 22 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv)

ANTI-SENSE:

FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

5 INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

20 (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GTGAGACCCT AGTCTGCCAC TGAGGAT 27 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS:

I

I'

It i' -86 LENGTH: 8 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 2 CHROMOSOME/SEGMENT: MAP POSITION: S(C) UNITS: (ix) FEATURE: S(A)

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GAGGTCAC 8 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -87 0 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE: 0

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: S* CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

20 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GATTGGTCAC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: AMINO ACID SEQUENCE (iii) HYPOTHETICAL: No -88 0 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 5

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

0 IDENTIFICATION METHOD: 2: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Glu Tyr Gly Pro Glu Glu Asp Gly Gly Glu Glu 5 S* 25 Ser Gly INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 13 0 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: AMINO ACID SEQUENCE (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: 4 I* -89 FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

1 0 (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: **2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Gly Thr Gly Arg Arg Ile His Ser Tyr Arg Gly His 5 *Leu INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 29 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: L1 90 0 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

15 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ACGACGCGCG GACTGCGATT GCAGAAGAT 29 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: 91 91 0 DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

0 MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: AGCGACCTGA CGATGTCCAG TCTC 24 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 19 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: -92 0

ORGANELLE:

(vii) IMMEDIATE

SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CCTCGCCTCC GTTACAACA 19 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 44 TYPE: nucleic acid *0 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

-93 0 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCTAAT ACGACTCACT ATAGGGAGGC GCCCGACTCC TCCC 44 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 20 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION: 94 0

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CTATCTAGAG GCCAAGGCAG GAGGATC 27 10 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: ORGANELLE (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

0 IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CATTCTAGAT TCCCTCCGCG ATCCAGA 27 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: S 20 DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: c SEQUENCE DESCRIPTION: SEQ ID NO:43: -96 0 CATTCTAGAC TCTTCCGGGC CGGACTC 27 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 10 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CACAAGTGAT GCCTTGTAGC TG 22 97 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No 10 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CAGTAGTGTC CTGTATTTAG TG 22 INFORMATION FOR SEQ ID NO:46:

CHARACTERISTICS:

SEQUENCE CHARACTERISTICS: -98 0 LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

S:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

S" (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: 20

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GTTGGCTATG GGTAGAATTG G 21 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

I

Ir -99 0 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CAGGGTAGCC TTGATCTAAG T 21 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No 100 0 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GGAGGTCCTG AGAATATGTG TCC 23 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: (v) FRAGMENT TYPE: 101 (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

15 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: TGTTCAGGCA CACAGTAGAT G 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: 102 0 DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CATCTTCTGC AATCGCAGTC CGCGCGT 27 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL

SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL

ISOLATE:

DEVELOPMENTAL

STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE: 103 0

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

0 LOCATION: IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CAAAAGCTGA GATGAAACAG TGTAAGT 27 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No Osee*: (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

104 0 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION

METHOD:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GTTTGGTTAA CCAGAAGCCC ATCGT INFORMATION FOR SEQ ID NO:53: SEQUENCE

CHARACTERISTICS:

15 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 20 (iii) HYPOTHETICAL: No 2 (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

-105- 0 (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GATGGGCTTC TGGTTAACCA AACT 24

Claims (15)

1. A purified and isolated nucleic acid, wherein said nucleic acid is able to hybridize under high stringency conditions to a nucleic acid comprising a nucleic acid sequence according to: a) SEQ ID NO:3; or b) SEQ ID NO:4; or c) SEQ ID NO:5; or .0 d) any one of SEQ ID NOS:3 to 5 in combinatiol with SEQ ID NO: 1. n

2. A purified and isolated nucleic a nucleic acid sequence according to: a) SEQ ID NO:3, or b) SEQ ID NO:4, or c) SEQ ID NO:5, or d) any one of SEQ ID NOS:3 to 5 with SEQ ID NO: 1. acid, comprising in combination

3. A method for detecting a mutant allele of a wild- type VHL gene in a subject suspected of having VHL disease or a disease related to the presence of a mutation in the wild-type VHL gene, said method comprising the step of analyzing a nucleic acid sequence from the subject for the presence of said mutant allele, in which said method utilizes a nucleic acid probe or nucleic acid primer, wherein said probe or primer has a sequence consisting of, or said probe or primer has a sequence which is fully complementary to a full length nucleic acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NOS: 23 to 28, SEQ ID NO: 52 and SEQ ID NO: 53.

4. A method for detecting a mutant allele of a wild-- type VHL gene in a subject suspected of having VHL disease or a disease related to the presence of a mutation in the 93 1- 9

7.doc 5/04/01 107 wild-type VHL gene, said method comprising the step of analyzing a nucleic acid sequence from the subject for the presence of said mutant allele, in which the method utilizes a nucleic acid probe or nucleic acid primers, wherein said probe or primers have a sequence consisting of, or said probe or primers have a sequence which is fully complementary to a full length nucleic acid sequence selected from the group consisting of: a probe or primers having SEQ ID NO: 1 or complement thereof used in combination with a probe or primer having any one of SEQ ID NOS:3 to 5 or complement thereof, and a probe or primer having any one of SEQ ID 15 NOS: 3 to 5, Or complement thereof. 5. A method according to claim 4, in which the probe or primer have a sequence consisting of, or the probe or primers has a sequence which is fully complementary to a full-length nucleic acid sequence selected from the group consisting of nucleotides 1-146, 169-391, 291-501, 585- 940, 921-1231 and 1277-1600 of SEQ ID NO: 1. 6. A method according to any one of claims 3 to 25 in which the step of analyzing comprises Southern blot analysis. 7. A method according to any one of claims 3 to in which the step of analyzing comprises PCR-SSCP.

8. A method according to any one of claims 3 to in which the step of analyzing comprises PCR.

9. A method according to any one of claims 3 to in which the step of analyzing comprises Northern blotting. H:\janel ,Keep\Speci\25931-9 7 .doc 5/04/01 108 A method according to any one of claims 3 to in which the step of analyzing comprises carrying out an RT-PCR reaction on the sample to produce RT-PCR products.

11. A method according to any one of claims 3 to in which the step of analyzing comprises RT-P.CR-SSCP.

12. A method according to any one of claims 3 to in which the mutant allele is associated with VHL disease or VHL-related diseases selected from the group consisting of: sporadic renal cancer, uterine cancer, breast cancer, testicular cancer, bladder cancer, pancreatic cancer, ovarian cancer, lung cancer, adrenal tumors, brain tumors and lung tumors.

13. A method according to any one of claims 3 to in which the nucleic acid sequence of the mutant allele has a deletion, insertion or point mutation.

14. A method according to any one of claims 3 to in which the subject is a carrier of the VHL disease gene. A primer or probe having a nucleic acid sequence consisting of, or a nucleic acid sequence fully complementary to, a full length nucleic acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ. ID NO:20, SEQ ID NOS: 23 to 34, SEQ. ID NOS: 37 to 38, and SEQ ID NOS: 52 to 53.

16. A primer or probe having a nucleic acid sequence consisting of, or fully complementary to, a full length nucleic acid sequence selected from the group consisting of: a) a primer or probe having a nucleic acid sequence according to SEQ ID NO: 1 or complement thereof in combination with a primer or probe having a nucleic acid sequence according to any one of SEQ ID NOS: 3 to H:\janel\Keep\Speci\259J1-97.doc 5/04/01 109 and b) a primer or probe having a nucleic acid sequence according to any one of SEQ ID NOS: 3 to 5, or complement thereof.

17. A primer or probe according to claim 16, in which the primer or probe has a nucleic acid sequence consisting of, or fully complementary to, a full length sequence selected from the group consisting of: nucleotides 1-146,

169-391, 291-501, 585-940, 921-1231 and 1277-1600 of SEQ ID NO: 1. 18. A diagnostic kit for use in detecting carriers or for use in detecting mutant alleles of a wild-type VHL 15 gene, the kit consisting of primers or probes, wherein said primers or probes have nucleic acid sequences consisting of, or said primers or probes have a sequence which is fully complementary to, a full length sequence S" selected from the group consisting of SEQ ID NO: 16, SEQ. ID NO: 20, SEQ ID NO: 23 through SEQ ID NO: 34, SEQ. ID NO: 37, SEQ. ID NO: 38, and SEQ ID NO: 52 through SEQ ID NO: 53. 19. A diagnostic kit for use in detecting carriers or for use in detecting mutant alleles of a wild-type VHL gene, said kit comprising primers or probes, wherein said primers or probes have a nucleic acid sequence consisting of, or fully complementary to a full length sequence selected from the group consisting of primers or. probes having SEQ ID NO: 1 or complement thereof used in combination with primers or probes having any one of SEQ ID NOS: 3 to 5, or complement thereof and primers or probes having any one of SEQ ID NOS: 3 to 5, or complement thereof. The kit of claim 19, wherein said primers or probes have a sequence consisting of, or fully H:\janel\Keep\Speci\25931-97.doc 5/04/01 110 complementary to a full length sequence selected from the group consisting of nucleotides 1-146, 169-391, 291-501,

585-940, 921-1231 and 1277-1600 of SEQ ID NO: 1. 21. A purified and isolated nucleic acid according to claim 1, substantially as herein described with reference to the examples and figures. 22. A method according to claim 3, substantially as herein described with reference to the examples and figures. 23. A method according to claim 4, substantially as herein described with reference to the examples and 15 figures. 24. A method according to claim 5, substantially as herein described with reference to the examples and o figures. 25. A primer or probe according to any one of claims 15 to 17, substantially as herein described with reference to the examples and figures. Dated this 5th day of April 2001 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\janel\Keep\Speci\2593-97.doc 5/04/01