AU2004212565A1 - Polycystic kidney disease gene - Google Patents

Description

Wpike I Regulation 3.2

AUSTRALIA

Patents Act 1990 DIVISIONAL APPLICATION Name of Applicant: Genzyme Corporation AND Johns Hopkins University Actual Inventor(s): KLINGER, Katherine; BURN, Timothy; CONNORS, Timothy; DACKOWSKI, William; GERMINO, Gregory and QIAN, Feng Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Invention Title: "Polycystic kidney disease gene" Details of Parent Application No: 54172/01 The following statement is a full description of this invention, including the best method of performing it known to us: Q:opcAVpa\%Septcr/Sep 2004U432061 genzyir corp divbiiol appi 261.doc -1719/04 POLYCYSTIC KIDNEY_ DISEASE

GENE

This application is a continuation-in-part of U.S.

patent application Serial No. 08/381,520, filed January 31, 1995.

FIELD OF THE INVENTION The present invention pertains to the diagnosis and treatment of polycystic kidney disease in humans, using DNA sequences derived from the human PKDI gene and the protein or proteins encoded by that gene.

BACKGROUND OF THE INVENTION Autosomal dominant polycystic kidney disease (APKD), also called adult-onset polycystic kidney disease, is one of the most common hereditary disorders in humans, affecting approximately one individual in a thousand. The prevalence in the United States is greater than 500,000, with 6,000 to 7,000. new cases detected yearly (Striker et al., Am.

J. Nephrol., 6:161-164, 1986; Iglesias et al., Am. J. Kid.

Dis., 2:630-639, 1983). The disease is considered to be a systemic disorder, characterized by cyst formation in the ductal organs such as kidney, liver, and pancreas, as well as by gastrointestinal, cardiovascular, and musculoskeletal abnormalities, including colonic diverticulitis, berry aneurysms, hernias, and mitral valve prolapse (Gabow et al., Adv. Nephrol., 18:19-32, 1989; Gabow, New Eng. J. Med., 329:332-342, 1993).

The most prevalent and obvious symptom of APKD, however, is the formation of kidney cysts, which-result in grossly enlarged kidneys and a decrease in renalconcentrating ability. Hypertension and endocrine abnormalities are also common in APKD patients, appearing even before symptoms of renal insufficiency. In approximately half of APKD patients, the disease progresses to end-stage renal disease; accordingly, APKD is responsible for 4-8% of the renal dialysis and transplantation cases in the United States and Europe (Proc. European Dialysis and Transplant Assn., Robinson and Hawkins, eds., 17:20, 1981).

Thus, there is a need in the art for diagnostic and therapeutic tools to reduce the incidence and severity of this disease.

APKD exhibits a transmission pattern typical of autosomal dominant inheritance, each offspring of an affected individual has a 50% chance of inheriting the causative gene. Linkage studies indicated that a causative gene is present on the short arm of chromosome 15, near the a-globin cluster; this locus was designated PKDI (Reeders et al., Nature, 317:542, 1985). Though other PKD-associated genes exist, such as, for example, PKD2, PKD1 defects appear to cause APKD in about 85-90% of affected families (Parfrey et al., New ng. J. Med., 323:1085-1090, 1990; Peters et al., .Contrib. Nephrol., 97:128-139, 1992).

The PKD1 gene has been localized to chromosomal position 16p13.3. Using extensive linkage analysis, in conjunction with the identification of new markers and restriction enzyme analysis, the gene has been further localized to an interval of approximately 700 kb between the markers ATPL (ATP6C) and CMM65 (D16S84). The region is rich in CpG islands that are thought to flank transcribed sequences, and it has been estimated that this interval contains at least 20 genes. The precise location of the PKDI gene was pinpointed by the finding of a PKD family whose affected members carry a translocation that disrupts a 14 kb .RNA transcript associated with this region, as reported in the European PKD Consortium (EPKDC), Cell, 77:881, 1994, describing approximately 5631 bp of DNA sequence corresponding to the 3' end of the putative PKDI cDNA sequence.

Notwithstanding knowledge of the partial PKD1 3' cDNA sequenCe, several significant impediments stand in the way of determining, the complete sequence of the PKDI gene.

For the most part, these impediments arise from the complex organization of the PK11 locus- One serious obstacle is that sequences related to the PKD1 transcript are duplicated at least three times on chromosome 16 proximal to the PKD1 locus, forming PRDlhomologues. Another obstacle is that the PKD1 genomic interval also contains repeat elements that are present in other genomic regions. Both of these types of sequence duplications interfere with "chromosome walking" techniques that are widely used for identification of genomic DNA. This is because these techniques rely on hybridization to identify clones containing overlapping fragments of genomic DNA; thus, there is a high likelihood of "walking" into clones derived from PKDI homologues instead of clones derived from the authentic PKD1 gene. In a similar manner, the PKD1 duplications and chromosome 16-specific repeats also interfere with the unambiguous determination of a complete cDNA sequence that encodes the PKD1 protein. Thus, there is a need in the art for genomic and cDNA sequences corresponding to the authentic PKDl gene. This includes identification of segments of these sequences that are unique to the expressed PKD1 and not are present in the duplicated homologous sequences also present on chromosome 16.

SUNMARY OF THE INVENTION The present invention involves an isolated normal human PKDI gene having the sequence set forth in Figure i, sequences derived therefrom such as the sequence set forth in Figure 2, an isolated nucleic acid having the PKDl cDNA sequence set forth in Figure 3, and sequences derived therefrom. The PKDl gene is a genomic DNA sequence whose altered, defective, or non-functional expression' leads to adult-onset polycystic kidney disease. The invention also encompasses DNA vectors comprising these nucleic acids, Cells transformed with the vectors, and methods for producing PKDI protein or fragments thereof.

In another aspect, the invention involves isolated oligonucleotides that hybridize only to the authentic expressed PKD1 gene, and not to PKDI homologues.

In yet another aspect, the invention involves isolated mutant PKD1 genes, and their cDNA cognates, which contain alterations in nucleotide sequence relative to the normal PKD1 gene, and whose presence in one or more copies in the genome of a human individual is associated with adultonset polycystic kidney disease.

In still another aspect, the invention involves isolated oligonucleotides that discriminate between normal and mutant versions of the PKD1 gene.

In still another aspect, the invention involves methods for identifying a human subject carrying a mutant PKDi gene in a human subject, comprising: a) obtaining a sample of biological material from the subject, and b) detecting the presence of the mutant gene or its protein product.

In still another aspect, the invention involves methods and compositions for treating APKD or disease conditions having the characteristics of APKD. Such methods encompass administering an isolated human PKDl gene, or fragments of the gene, under conditions that result in expression of therapeutically effective amounts of all, or part of, the PKD1 protein. The invention also encompasses compositions for treating APKO that comprise all or part of the PKDI DNA of Figures 1, 2 and 3, or the PKD1 protein encoded by the DNA of Figures 1, 2 or 3.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the DNA sequence of the human PKDI locus between chromosomal markers ATPL (ATP6C) and D16S84.

(SEQ ID NO:l).

Figure lB shows the DNA sequence of 53,526 bases comprising the normal human PKD1 gene. (SEQ ID NO:2).

Figure 2 shows a partial DNA sequence of 894 bases within the 5' region of normal human PKDI DNA. (SEQ ID NO:3) Figure 3 shows the full-length sequence of normal human PKDl cDNA and corresponding amino acid sequence.

(SEQ

ID Figure 4A shows a comparison of the DNA sequence of the 5' region of DNAs derived from the authentic PKDI gene (SEQ ID N0:19) and PKDI homologues (SEQ ID N0:18). -A 2 9 -base pair gap must be introduced into the sequence of the authentic gene to align the two sequences. In addition, the authentic PKDI and the PKDl homologue differ at position 418 of this figure.

Figure 4B shows the DNA sequence of an oligonucleotide that can be used to discriminate between the authentic PKDI sequence and PKDI homologues. The star denotes a polymerization-blocking modification. (SEQ ID Figure 5 shows the region of chromosome 16 containing the PKDI locus. The upper panel shows NotI restriction sites, as well as previously identified genetic markers in this region. The bottom panel shows P1 clones covering this region.

Figure 6 shows the restriction map of the 91.8B P1 clone containing the PKDI gene and flanking regions with only the relevant sites indicated (B=BamHI, C=SacI, E=EcoRI, N=NotI, S=SalI, X=XhoI and V=EcoRV). The NotI site in parenthesis is methylated in genomic DNA. The position of T the 1.9 kb BamHI-BamHI fragment is shown by the shaded box, the striped box denotes the location of the 2.5 kb polypurine/polypyrimnidine tract. The arrows indicate the position and orientation of the next most centromeric transcript (NCT), TSC-2 and PKDI genes. The location of relevant cosmid clones is shown by open boxes. Restriction fragments used to generate sequencing templates are shown at the bottom with quotation marks denoting that the site is vector derived. Pools used in fluorescence in situ hybridization (FISH) are indicated by brackets at the bottom.' Figure 7 shows a comparison between the previously reported (EPKDC) partial PKDI cDNA (SEQ ID NOS:20, 21, 24, 25,28 and 29) sequence and the sequence reported herein (SEQ ID NOS: 22,23,26, 27, 30 and 31). The upper sequence is that reported for the cDNA (EPKDC), while the lower sequence is the genomic sequence of the present invention. Discrepancies are highlighted by lower case in the cDNA (EPKDC) sequence and by boxes in the genomic sequence with the corresponding changes in amino acids denoted with X's. The altered carboxy-terminal residues resulting from the frame shift are shown above the genomic sequence and the previously predicted residues are shown in lowercase. An in-frame termination codon is indicated by an underline in the genomic sequence Figure 8 shows an illustration of the PKD1 genomic structure as predicted by GRAIL2. The predicted exons are represented as boxes along the genomic sequence. The reported cDNA is at the top right. The position of the kb GC-rich region is indicated by the striped box at the bottom. The stippled box above exons 3 and 4 in the gene model indicate the position of the predicted LRR and carboxy-flanking region. The extent of the published cDNA is shown by the open (coding region) and cross hatched boxes (3' untranslated region). The filled black box indicates the relative position of the exon which was absent in the predicted gene model, while the asterisk designates the exon which contains an unspliced intron. The position of the kb GC-rich region is marked by the striped box below the GC-content bar- Figure 9 shows a schematic structure of the predicted PKDI protein. Multiple domains are depicted based on sequence homology including two copies of a leucine-rich repeat (LRR) near the N-terminal which is flanked by a cysteine-rich cluster Three perfect copies and 12 related copies of a domain of unknown function (Pmel-17 or Ig-like repeat) are shown. The predicted 7 (or more) membrane-spanning domains are indicated. The exons encoding the various domains are listed.

Figure 10 shows the RT-PCR and cDNA products comprising the PKD1 cDNA. The EPKDC 3' cDNA sequence is shown by the striped box. The full-length cDNA is shown in black. Shaded boxes denote individual cDNAs and RT-PCR products. The cross hatched box denotes the RT-PCR products containing alternatively spliced exons and an unspliced exon which do not maintain the open reading frame. Alternatively spliced exons and insertions are designated by thin lines and inverted triangles, respectively. Open boxes designate the position of open reading frames. The stippled box denotes the 5' untranslated region.

Figure 11 shows a schematic structure of the full length PKDI cDNA in pCMV-SPORT vector. Thin line represents PKD1 cDNA with restriction sites used to assemble individual DNA clones. Thick line represents pCMV-SPORT vector which contains SP6 and T7 RNA polymerase promoters to generate

RNA

for in vitro translations, CMV promoter, SV40 origin of replication and polyadenylation signal for expression in mammalian cells.

Figure 12 shows a schematic of the full-length

PKDI

product and its truncated products. Black box represents signal peptide Leucine rich repeat (LRR) and Ig-like (Ig-like) domains are indicated by shaded boxes. The eleven predicted transmembrane regions are also indicated by black bars and numbered.

Figure 13 shows regions of homology in the PKDI gene between sequences encoded by GRAIL2-predicted exons -and proteins present in SwissProt and PIR databases. (SEQ ID NOS: 32-55) Positions where the PKDI sequence matches the consensus sequence are shaded.

Figure 14 shows the results of exon trapping within the PKD1 locus.

Figure 15 shows the regions of PKD1 protein used as fusion proteins for generation of domain specific polyclonal antibodies. The predicted structure of the PKD1 protein is shown above. Each fusion protein consists of the carrier glutathione-S-transferase (GST) or maltose binding protein (MBP) and the indicated region of PKDI polypeptide.

PKDI

corresponding residues of each fusion protein are shown.

Figure 16 shows the two constructs used for immunoprecipitation, SrfIA, which corresponds to the N-terminal half of the PKDI protein and BRASH 7, which corresponds to the C-terminal half of the PKD1 protein as shown. Epitopes for anti-fusion proteins FP-LRR, FP-46-1c and FP-46-2 polyclonal antibodies used for immunoprecipitations are also indicated.

DETAILED DESCRIPTION OF THE INVENTION All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of conflict or inconsistency, the present description, including definitions, will control.

Definitions: 1. "APKD" as used herein denotes adult-onset polycystic kidney disease, which is characterized by the development of renal cysts and, ultimately, renal failure, and may alternatively or in addition involve cysts in other organs including liver and spleen, as well as gastrointestinal, cardiovascular, and musculoskeletal abnormaliLties.

2. The term "PKDl gene" refers to a genomic

DNA

sequence which maps to chromosomal position 16pl3.3 and gives rise to a messenger RNA molecule encoding the PKDI protein The PKD1 gene encompasses the sequences shown in Figures 1 and 2, which includes introns and putative.regulatory sequences. The term "authentic" is used herein to denote the genomic sequence at this location, as well as sequences derived therefrom, and serves to distinguish these authentic sequences from "PKD1 homologues" (see below).

3. "PKD1 complementary DNA (cDNA) is defined .herein as a single-stranded or double-stranded intronless

DNA

molecule encompassing the sequence shown in Figure 3, that is derived from the authentic PKDI gene and whose sequence, or complement thereof, encodes the PKDI protein shown in Figure 3.

4. A "normal" PKD1 gene is defined herein as a PKDI gene whose altered, defective, or non-functional expression leads to adult-onset polycystic kidney disease.

A

normal PKDI gene is not associated with disease and thus is considered to be a wild-type version of the gene. Included in this category are allelic variants in the PKD1 gene, also denoted allelic polymorphisms, i.e. alternate versions Of the PXDl gene, not associated with disease, that may be represented at any frequency in the population. Also included are alterations in DNA sequence, whether recombinant or naturally occurring, that have no apparent effect on expression or function of the PKDl gene product.

5. A "mutant" PKDl gene is defined herein as a PKDI gene whose sequence has been modified by transitions, transversions, deletions, insertions, or other modifications relative to the normal PKD1 gene, which modifications cause detectable changes in the expression or function of the PKDI gene product, including causing disease. The modifications may involve from one to as many as several thousand nucleotides, and result in one or more of a variety of changes in PKDI gene expression, such as, for example, decreased or increased rates of expression, or expression of a defective RNA transcript or protein product. Mutant

PKDI

genes encompass those genes whose presence in one or more copies in the genome of a human individual is associated with

APKD.

6. A "P1D1 homologue- is a sequence which is closely related to PKD1, but which does not encode the authentic expressed PKDI gene product. Several examples of such homologues that map to chromosomal location 16p13.l have been identified and sequenced by the present inventors.

7. A "PKDI carrier" is defined herein as an individual who carries at least one copy of a diseaseproducing mutant PKD1 gene. Since the disease generally exhibits an autosomal dominant pattern of transmission,

PKDI

carriers have a high probability of developing some symptom.

of PKD. Thus, a PKDl carrier is likely to be a 'PKD patient." 8. As referred to herein, a "contig" is a continuous stretch of DNA or DNA sequence, which may be represented by multiple, overlapping, clones or sequences.

9. As referred to herein, a "cosmid" is a DNA plasmid that can replicate in bacterial cells and that accommodates large DNA inserts from about 30 to about 45 kb in length.

The term "PI clones" refers to genomic DNAs cloned into vectors based on the P1 phage replication mechanisms. These vectors generally accommodate inserts of about 70 to about 105 kb (Pierce et al., Proc. Natl. Acad.

Sci., USA, 89:2056-2060, 1992).

11. As used herein, the term "exon trapping" refers to a method for isolating genomic DNA sequences that are flanked by donor and acceptor splice sites for RNA _processing.

12. The term "single-strand conformational polymorphism analysis" (SSCP) refers to a method for detecting sequence differences between two DNAs, comprising hybridization of the two species with subsequent mismatch detection by gel electrophoresis. (Ravnik-Glavac et al., Hum.

M0l. Genet., 3:801, 1994).

13. "HOT cleavage" is defined herein as a method for detecting sequence differences between two DNAs, comprising hybridization of the two species with subsequent mismatch detection by chemical cleavage (Cotton, et al., Proc. NatIl. Acad. Sci., USA, 85:4397, 1988).

14. "Denaturing gradient gel electrophoresis, (DDGE) refers to a method for resolving two DNA fragments of identical length on the basis of sequence differences as small as a single base pair change, using electrophoresis through a gel containing varying concentrations of denaturant (Guldberg et al., Nuc. Acids Res., 22:880, 1994).

As used herein, "sequence-specific oligonucleotides" refers to related sets of oligonucleotides that can be used to detect allelic variations or mutations in the PKD1 gene.

16. As used herein, "PKDl-specific oligonucleotides" refers to oligonucleotides that hybridize to sequences present in the authentic expressed PKD1 gene and not to PKD1 homologues or other sequences.

17. "Amplification" of DNA as used herein denotes a reaction that serves to increase the concentration of a particular DNA sequence within a mixture of DNA sequences.

Amplification may be carried out using polymerase chain reaction (PCR) (Saiki et al., Science, 239:487, 1988), ligase chain reaction (LCR), nucleic acid-specific based amplification (NSBA), or any method known in the art.

18. "RT-PCR" as used herein refers to coupled reverse transcription and polymerase chain reaction. This method of amplification uses an initial step in which a specific oligonucleotide, oligo dT, or a mixture of random primers is used to prime reverse transcription of RNA into single-stranded cDNA; this cDNA is then amplified Using standard amplification techniques e.g. PCR.

19. A PKD1 gene or PKD1 cDNA, whether normal or mutant, corresponding to a particular sequence is understood to include alterations in the particular sequence that do not change the inherent properties of the sequence. It will be understood that additional nucleotides may be added to the and/or terminus of the PKDI gene shown in Fidure 1B, or the PKD1 cDNA shown in Figure 3, as part of routine recombinant DNA manipulations. Furthermore, conservative

DNA

substitutions, i.e. changes in the sequence of the proteincoding region that do not change the encoded amino acid sequence, may also be accommodated.

The present invention encompasses the human gene for .PKDI. Mutations in this gene are associated with the occurrence of adult-onset polycystic kidney disease.

A

"normal" version of the genomic sequence, corresponding to 53,526 bases of the PKD1 gene is shown in Fiqure lB.

The PKD1 gene sequence was determined using the strategy described in Example 1. Briefly, a series of cosmid and P1 DNA clones was assembled containing overlapping human genomic DNA sequences that collectively cover a 700 kilobase segment of chromosome 16 known to 'contain the PKD1 locus. To identify transcribed sequences within this 700 kb segment, including those sequences encoding PKDI, both exon trapping and cDNA selection techniques were employed. At the same time, direct DNA sequencing of the human DNA sequences contained in the genomic clones was performed, using techniques that are well-known in the art. These included the isolation of subclones from particular cosmid or Pl clones. Nested deletions were created from selected subclones, and the nested deletions were then subjected to direct DNA sequencing using the ALF M automated sequencer (Pharmacia, Uppsala, Sweden).

The full-length sequence of PKDI cDNA is shown in Figure 3.

The present invention encompasses isolated oligonucleotides corresponding to sequences within the PKDl gene, or within PKD1 cDNA, which, alone or together, can be used to discriminate between the authentic expressed

PKDI

gene and PKDI homologues or other repeated sequences. These oligonucleotides may be from about 12 to about 60 nucieotides in length, preferably about 18 nucleotides, may be single- or double-stranded, and may be labelled or modified as described below. An example of an oligonucleotide that can be used in this manner is shown in Figure 4B. The discrimination function of this oligonucleotide is based on a comparison of the sequence of the authentic PKD1 gene with three cDNAs derived from the PKD1 homologues, which revealed that homologue cDNAs contain a 29 bp insertion relative to the authentic PKD1 sequence (Figure 4A). The oligonucleotide shown in Figure 4B is modified at its 3' terminus so that it does not support polymerization reactions, and is designed to hybridize specifically to the homologue sequence and not to the authentic PKD1I sequence. When this oligonucleotide is included in amplification reactions, it selectively prevents the amplification of PKD1 homologue sequences. In this manner, authentic PKD1 sequences are selectively amplified and PKD1 homologues are not. These oligonucleotides or their functional equivalents thus provide a basis for testing for the presence of mutations in the authentic PKD1 gene in a human patient (see Example 5 below)- The present invention encompasses isolated DNA and RNA sequences, including sense and antisense sequences, derived from the sequences shown in Figures 1, 2. and 3. The particular sequences may represent "normal" alleles of PKD, including allelic variants, or "mutant" alleles, which are associated with disease symptoms. PKD1-derived sequences may also be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, and the like. Furthermore, the nucleic acids can be modified to alter stability, solubility, binding affinity, and specificity. For example, PKD-derived sequences can be selectively methylated.

The DNA may comprise antisense oligonucleotides, and may further include nuclease-resistant phosphorothioate phosphoroamidate, and methylphosphonate derivatives, as well as "protein nucleic acid" (PNA) formed by conjugating bases to an amino acid backbone as described in Nielsen et al, Science, 254: 1497, 1991. The DNA may be derivatized by linkage of the a-anomer nucleotide, or by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable Sof providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes,.

fluorescent molecules, biotin, and the like.

In general, nucleic acid manipulations according to the present invention use methods that are well known in the art, as disclosed in, for example, Molecular Cloning,

A

Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), or Current Protocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman, Smith and Struhl, Greene Publ. Assoc., Wiley-Interscience, NY NY 1992).

The invention also provides vectors comprising nucleic acids having PKD1 or PKDI-related sequences. A large number of vectors, including plasmid, phage, viral and fungal vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

Advantageously, vectors may also include a promoter operably linked to the PKDI- encoding portion, particularly when the PKD1-encoding portion comprises the cDNA shown in Figure 3 or derivatives or fragments thereof. The encoded PKD1 may be expressed by using any suitable vectors, such as pREP4, pREP8, or pCEP4 (InVitrogen, San Diego, CA), and any suitable host cells, using methods disclosed or cited herein.or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical to the operation of the invention.

Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. The inserted PKDI coding sequences may.be synthesized, isolated from natural sources, or prepared as hybrids, for example.

Ligation of the PKDI coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences may be achieved by known methods. Suitable host cells may be transformed/transfected/infected by any suitable method including electroporation, CaCI 2 mediated DNA uptake, fungal infection, microinjection, microprojectile, or other established methods.

Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. Subtilis, Saccharomyces cerevisiae, SFP9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, artificial chromosomes, and the like. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts.

Examples of these regions, methods of isolation, manner of manipulation, -and the like, are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced PKD1.

This invention also contemplates the use.of unicellular or multicellular organisms whose genome has been transfected or transformed by the introduction of PKDI coding sequences through any suitable method, in order to obtain recombinantly produced PKDI protein or peptides derived -therefrom.

Nucleic acids encoding PKD1 polypeptides may also be incorporated into the genome of recipient .cells by recombination events. For example, such a sequence can be microinjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene encoding PKD1, an analog or pseudogene thereof, or a sequence with substantial identity to a PKD1-encoding gene. Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous gene by homologous recombination, especially in pluripotent cells, may also be used.

The present invention also encompasses an isolated polypeptide having a sequence encoded by the authentic

PKDI

gene, as well as peptides of six or more amino acids derived therefrom. The polypeptide(s) may be isolated from human tissues obtained by biopsy or autopsy, or may be produced in a heterologous cell by recombinant DNA methods as described above. Standard protein purification methods may be used to isolate PKDl-related polypeptides, including but not limited to detergent extraction, and chromatographic methods including molecular sieve, ion-exchange, and affinity chromatography using e.g. PKDI-specific antibodies or ligands. When the PKDI polypeptide to be purified is produced in a recombinant system, the recombinant expression vector may comprise additional sequences that encode additional amino-terminal or carboxy-terminal amino acids; these extra amino acids act as "tags" for inmmunoaffinity purification using immobilized antibodies or for affinity purification using immobilized ligands.

Peptides comprising PKDl-specific sequences may be derived from isolated larger PKDI polypeptides described above, using proteolytic cleavages by e.g. proteases such as trypsin and chemical treatments such as cyanogen bromide that are well-known in the art. Alternatively, peptides up to residues in length can be routinely synthesized in milligram quantities using commercially available peptide synthesizers.

The present invention encompasses antibodies that specifically recognize the PKDI polypeptide(s) encoded by the gene shown in Figures 1 and 2 or the cDNA shown in Figure 3, and/or fragments or portions thereof. The antibodies may be polyclonal or monoclonal, may be produced in response to the native PKD1 polypeptide or to synthetic peptides as described above. Such antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, other references cited herein, as well as immunological and hybridoma technologies known to those in the art. Where natural or synthetic PKDl-derived peptides are used to induce a PKD-specific immune response, the peptides may be conveniently coupled to an suitable carrier such as KLH and administered in a suitable adjuvant such as Freund's. Preferably, selected peptides are coupled to a lysine core carrier substantially according to the methods of Tam, Proc.Natl.Acad.Sci,usA 85:5409-5413, 1988. The resulting antibodies may be modified to a monovalent form, such as, for example. Fab, Fab 2 FAB', or FV. Anti-idiotypic antibodies may also be prepared using known methods.

In one embodiment, normal or mutant PKD1 polypeptides are used to immunize mice, after which their spleens are removed, and splenocytes used to form cell hybrids with myeloma cells and obtain clones of antibodysecreted cells according to techniques that are standard in the art. The resulting monoclonal antibodies are screened -for specific binding to PKD1 proteins or PKDl-related peptides.

In another embodiment, antibodies are screened for selective binding to normal or mutant PKD1 sequences.

Antibodies that distinguish between normal and mutant forms of PKD1 may be used in diagnostic tests (see below) employing ELISA, EMIT, CEDIA, SLIFA, and the like. Anti-PKDl antibodies may also be used to perform subcellular and histochemical localization studies. Finally, antibodies may be used to block the function of the P1(D1 polypeptide, whether normal or mutant, or to perform rational drug design studies to identify and test inhibitors of the function using an anti-idiotypic antibody approach).

Identification of Disease-Causing Mutations in PKDl In one mode of practice of the present invention, the isolated and sequenced PKD1 gene is utilized to identify previously unknown or mutant versions of the PKDi gene.

First, human subjects with inherited polycystic kidney disease are identified by clinical testing, pedigree analysis, and linkage analysis, using standard diagnostic criteria and interview procedures, and DNA or RNA samples are obtained from the subjects (see below).

A variety of techniques are then employed to pinpoint new mutant sequences. First, PKD1 DNA may be subjected to direct DNA sequencing, using methods that are standard in the art. Furthermore, deletions may be detected using a PCR-based assay, in which pairs of oligonucleotides are used to prime amplification reactions and the sizes of the amplification products are compared with those of control products. Other useful techniques include Single-Strand Conformation Polymorphism analysis (SSCP), HOT cleavage, denaturing gradient gel electrophoresis, and two-dimensional gel electrophoresis.

A confounding and complicating factor in the detection of a PXDI mutation is the presence of PKDI homologues at several sites on chromosome 16 proximal to the transcribed gene. In analysis of mutations in PKD1, it is critical to distinguish between sequences derived from the authentic PKD1 gene and sequences derived from any of the homologues. Thus, an important feature of the present invention is the provision of oligonucleotide primers that discriminate between authentic PKDI and the homologues

A

detailed comparison of the sequences of the authentic

PKDI

gene and the homologues enables the design of primers that discriminate between the authentic PKDI gene or cDNA and the homologues. Primers that conform to this criterion, such as those disclosed in Figure 4B, may be used in conjunction with any of the analytical methods described below.

For SSCP, primers are designed that amplify

DNA

products of about 250-300 bp in length across non-duplicated segments of the PKD1 gene. For each amplification product, one gel system and two running conditions are used. Each amplification product is applied to a 10% polyacrylamide gel containing 10% glycerol. Separate aliquots of each amplimer are subjected to electrophoresis at 8W at room temperature for 16 hours and at 30W at 4°C for 5.4 hours. These conditions were previously shown to identify 98% of the known mutations in the CFTR gene (Ravnik-Glavac et al., Hum. Mol.

Genet., 3:801, 1994).

For "HOT" cleavage, amplification reactions are performed using radiolabelled PKD-specific primers. -Each radiolabelled amplification product is then mixed with a fold to 100-fold molar excess of unlabelled.amplification products produced using the identical primers and DNA from APKD-affected or -unaffected subjects. Heteroduplex formation, chemical cleavage, and gel analysis are then performed as described (Cotton, et al., Proc. Natl. Acad.

Sci., USA, 85:4397, 1988). Bands on the gel that are smaller than the homoduplex result from chemical cleavage of heteroduplexes at base pair mismatches involving cytidine or thymidine. Once a mutation has been identified by this procedure, the exact location of the mismatch(es) is determined by direct DNA sequencing.

Mutations are also identified by "broad range, DDGE (Guldberg et al., Nuc. Acids Res., 22:880, 1994). The use of GC-clamped PCR primers and a very broad denaturant gradient enables the efficient detection of mutant sequences. This method can also be combined with non-denaturing size fractionation in a two-dimensional system. An apparatus is used that permits automated two-dimensional electrophoresis, and the second dimension considerably increases the resolution of mutations.

After the presence of a mutation is detected by any of the above techniques, the specific nucleic acid alteration comprising the mutation is identified by direct DNA sequence analysis. In this manner, previously unidentified

PKDI

mutations may be defined.

Once a previously unidentified PKDI mutation is defined, methods for detecting the particular tutation in other affected individuals can be devised, using a variety of methods that are standard in the art. For example, oligonucleotide probes may be prepared that allow the detection and discrimination of the particular mutation. It will be understood. that such probes may comprise either the mutant sequence itself, or, alternatively, may flank the mutant sequence. Furthermore, the oligonucleotide sequence can be used to design a peptide immunogen comprising the mutant amino acid sequence. These peptides are then used to elicit antibodies that distinguish between normal and mutant PKDl polypeptides.

Diagnostic Tests for PKDl Mutations Mutant PKD1 genes, whether identified by the methods described above or by other means, find use in the design and operation of diagnostic tests. Tests that detect the presence of mutant PKD1 genes, including those described below and in Example 5, can be applied in the following ways: To determine donor suitability for kidney transplants. In general, it is desirable to use a close relative of the transplant recipient. When the recipient is a patient suffering from familial APKD, it is important to ascertain that the donor relative does not also carry the familial mutant PKD1 gene.

To screen for at-risk individuals in APKDaffected families- Presymptomatic individuals who have a high probability of developing APKD can be identified, allowing them to be monitored and to avail themselves of preventive therapies.

To target hypertensive patients for -antihypertensive treatment. Hypertension is also link]!d to APE]D. Screening of hypertensive patients for the presence of mutant PKD1 genes can be used to identify patients for preemptive regulation of blood pressure to prevent later kidney damage.

To perform prenatal screening. Most PKDilinked PKD is of the adult-onset type. In a small subset of families carrying a mutation in PKDI genes, however, juvenile onset is common and signifies a more severe form of the disease. In these families, Prenatal screening can be useful for genetic counselling purposes.

In general, the diagnostic tests according to the present invention involve obtaining a biological sample from a subject, and screening the sample, using all or part of the PKDl gene of this invention, for the presence of one or more mutant versions of the P101 gene or its protein product. The subject may be a fetus in utero, or a human patient of any age.

In one embodiment, a sample of genomic DNA is obtained from a human subject and assayed for the presence of one or more disease-associated P10D1 mutations. This DNA may be obtained from any cell source or body fluid. Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cer-vicovagina. cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Body fluids include blood, urine, cerebrospinall fluid, amniotic fluid, and tissue exudates at the site of infection or inflammation. DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA wi-il depend on the nature -of the source. The minimum amount .of: .DNA-to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a gen ome size of 3 x 109 base pairs).

In this embodiment, the assay used to detect the presence of mutations may comprise restriction enzyme digestion, direct DNA sequencing,'hybridization with sequence-specific oligonucleotjdes, amplification by PCR, single-stranded conformational. polymorphism analysis, denaturating gradient gel electrophoresis (DDGE), twodimensional gel electrophoresis, in situ hybridization, and combinations thereof.

In a preferred embodiment; RNA is isolated from a PKDl-expressing cell or tissue, preferably lymphocytes, using standard techniques including automated systems such as that marketed by Applied Biosystems, Inc. (Foster City, CA). The RNA is then subjected to coupled reverse-transcription and PCR amplification (RT-PCR). The resulting DNA may then be screened for the presence of mutant sequences by any of the methods outlined above (see Example 5 below).

As discussed above, any nucleic-acid-based screening method for PKDl mutations must be able to discriminate between the authentic PKD1 gene present at chromosome location 16p13.3 and PKD1 homologues present at 16pl3.1 and other locations. The oligonucleotides

SEQ

ID Nos:10 and 13-15) are examples of primers that discriminate between the authentic and homologue sequences, and these oligonucleotides or their equivalents form an important part of any such diagnostic test. Furthermore, nucleotides 43,823 through 52,887 of the PKD1 sequence of Figure IB represent a sequence that is unique to the authentic PKD1 gene and is not present in the homologues.

Thus, oligonucleotides derived from this region can be used .in a screening method to insure that the authentic PKD1 gene, and not the homologues, are detected.

In another embodiment, the assay used to detect the presence of a mutant PKDl gene involves testing for mutant gene products by an immunological assay, using one of many methods known in the art, such as, for example, radioinimmunoassay, -ELISA, immunofluorescence, and the like. In this embodiment, the biological sample is preferably derived from a PKDl-expressing tissue such as kidney. The PKDl polypeptide may be extracted from the sample. Alternatively, the sample may be treated to allow detection or visualization of specifically bound antibodies in situ as occurs in, for example, cryosectioning followed by immunofluorescent staining.

The antibodies may be monoclonal or polyclonal,-may be raised against intact PKDl protein, or natural or synthetic peptides derived from PKDI. In a preferred embodiment, the antibodies discriminate between "normal" and "mutant" PKD1 sequences, and possess a sufficiently high affinity for PKDI polypeptides so that they can be used in routine assays.

it will be understood that the particular method or combination of methods used will depend on the particular application. For example, high-throughput screening methods preferably involve extraction of DNA or RNA from an easily available tissue, followed by amplification of particular PKD1 sequences and hybridization of the amplification products with a panel of specific oligonucleotides.

Therapeutic ADplications The present invention encompasses the treatment of PKD using the methods and compositions disclosed herein. All or part of the normal PKD1 gene disclosed above can be delivered to kidney cells or other affected cells using a variety of known methods, including e.g. liposomes, viral vectors, recombinant viruses, and the like. The gene can be incorporated into DNA vectors that additionally comprise tissue-specific regulatory elements, allowing PKDI expression in a tissue-specific manner. This approach is feasible if a particular mutant PK10 allele, when present in a single COpy, merely causes the level of the PKDI protein to diminish below a threshold level necessary for normal function; in this case, increasing the gene dosage by supplementing with additional normal copies of the PKDl gene should correct the functional defect. In another embodiment, a mixture of isolated nucleic acids, such as that set forth in Figure 2 and at least a portion of the normal PKD gene, may be delivered to kidney or other affected cells in order to treat APKD. Alternatively, it may be desired to limit the expression of a mutant PKJ. gene, using, for example, antisense sequences In this embodiment, antisense oligonucleotides may be delivered to kidney or other cells-, For therapeutic uses, PKD1-related DNA may be administered in any convenient way, for example, parenterally in a physiologically acceptable carrier such as phosphate buffered saline, saline, deionized water, or the like.

Typically, the compositions are added to a retained physiological fluid such as blood or synovial fluid. The amount administered will be empirically determined using routine experimentation. Other additives, such as stabilizers, bactericides, and the like, may be included in conventional amounts.

This invention also encompasses the treatment of APKD by protein replacement. In one embodiment, protein produced by host cells transformed or transfected with DNA encoding the PKD1 polypeptide of the present invention is introduced into the cells of an individual suffering from altered, defective, or non-functional expression of the PKDI gene. This approach augments the absence of PKD1 protein, or the presence of a defective PKDI protein, by adding functional PKD1 protein. The PKD1 protein used in augmentation may comprise a subcellular fragment or fraction, or may be partially or substantially purified. In any case, the PKD1 protein is formulated in an appropriate vehicle, such as, for example, liposomes, that may additionally include conventional carriers, excipients, stabilizers, and the like- It will be understood that the therapeutic compositions of the present invention need not in themselves constitute an effective amount, since such effective amounts can be reached by administering a plurality of such therapeutic compositions The following examples are intended to illustrate the invention without limiting its scope thereof.

Example 1: Cloning and Secuencing of the Human PKD qene A. Methods: Employing an ordered sequencing approach, restriction fragments from cDEBIl and cGGG0lO.2 cosmids were subcloned into either pBLUESCRIPT (Stratagene, La Jolla, CA) or pGEM (Promega, Madison, WI). Plasmids were purified by CsCI density centrifugation in the presence of ethidium bromide. Nested deletions were generated from each plasmid using ExoIII (Henikoff, Methods Enzymol. 155: 156-165, 1987) and additional enzymatic reagents provided by the Erase-A-Base kit (Promega, Madison, WI). The resulting nested clones were analyzed electrophoretically after appropriate restriction enzyme digestion and were ordered into a nested set of templates for sequencing. A minimum tiling series of plasmids, each differing by approximately 250 bp from flanking clones, were identified and used for sequencing.

Plasmid DNAs were prepared for sequencing in one of two ways. Initially, all clones of interest were cultured in 2 mL of Super Broth (Tartof et al., BRL Focus 9: 12, 1987) for 20 hours at 37°C. Sets of 12-24 were processed simultaneously using a modified alkaline SDS procedure followed by ion-exchange chromatography as described by the manufacturer (Easy-prep, Pharmacia, Piscataway, NJ). Plasmid DNA yields ranged from 2.5 to 25 4g. Poor growing clones, or those whose plasmids generated sequence of unacceptable quality, were recultured in 100 mL of Luria's Broth and the plasmid DNA isolated using Qiagen columns (Qiagen, San Diego,

CA).

Dideoxy sequencing reactions were performed on deletion clones using the Auto-Read Sequencing Kit (Pha-macia, Piscataway, NJ) and fluorescein-labeled vector primers (M13 universal, M13 reverse, T3, T7 and SP6).

Reaction products were separated on 6% denaturing acrylamide gels using the ALF Tm DNA Sequencer (Pharmacia, Piscataway

NJ).

Second strand sequencing was performed using either an opposing set of nested deletions or primer walking. For primer walking, custom 17-mers, staggered every 250 bp, were purchased from a commercial supplier (Protogene, Palo Alto, CA). Template DNAs prepared by Qiagen or CsCI density gradients were sequenced using the unlabeled 17-mers by inclusion of fluor-dATP labeling mix in the sequencing reactions as described by the manufacturer (Pharmacia, Piscataway, NJ). In all cases, except the 2.5kb GC-rich region, single-stranded DNA was rescued from deletion clones using helper phage VCSMI3 (Stratagene) as described by the manufacturer.

Single-stranded templates from the 2.5 kb GC-rich region were sequenced using fluorescein-labeled universal primer and the Sequitherm Long Read cycle sequencing kit (Epicentre Technologies, Madison, WI) (Zimmerman et al., Biotechniques 17: 303-307, 1994). All processed sequencing data was transferred to a Quadra 700 Macintosh computer and assembled using the SEQUENCHER (Gene Codes, Ann Arbor, MI) sequencing assembly program. For differences that would not be resolved by examining the chromatograms, templates were either resequenced or primers proximal to the ambiguity were designed and used for resolution of the sequence difference.

Cycle sequencing was.performed using the Sequitherm cycle sequencing kit as described by the manufacturer (Epicentre Technologies, Madison, WI). Reaction products were separated on denaturing acrylamide gels and subsequently detected by autoradiography.

B. Sequencing Strategy: A 700 kb region of chromosome 16 containing the PKDI locus is shown in Figure 5 (top panel). A contig .covering this region was assembled from overlapping PI clones (shown in the middle panel). The contig was assembled by unidirectional chromosomal walking from the ends of the interval (ATPL and D16S84) and bidirectional walking from several internal loci (D16S139 and KG8). One of the clones, 91.8B (ATCC Accession No. 98056), spans the entire PKDI interval and includes cosmids cDEBll (ATCC Accession No.

98057), cGGG10.2 (ATCC Accession No. 98058), and substantial portions of cosmids 2H2 and 325A11 (Stallings, R.L. et al., Genomics 13:1031, 1992). The P1 clone 91.8B (shown schematically in Figure 6) was used as a second genomic template to confirm discrepancies between the published

CDNA

sequence (EPKDC, Cell, 1994, supra) and the cosmid-derived genomic sequence.

Preliminary experiments revealed the presence of multiple repetitive elements in the cGGGO-.2 cosmid.

Therefore, an ordered approach based on nested deletions, rather than random shotgun subcloning, was used to sequence the PKD1 gene. Restriction fragments derived from the inserts of both cGGG0l.2 and cDEB11 were subcloned into highcopy number plasmids as a preliminary step to the generation of nested deletions. Unidirectional deletions were prepared and sequenced, using the ALF M automated sequencing system (Pharmacia, Uppsala, Sweden).

C. Primary Structure of the PKD1 Locus: The primary sequence of the locus encompassing the PKD1 gene is 53,577 bp in length. This locus is GC-rich with a CpG/GpC dinucleotide ratio of 0.485. The primary sequence of the PKD1 gene within this locus is 53,526 bp in length. The present sequence was analyzed for transcriptional elements and CpG islands using GRAIL2 (Uberbacher, E.C. et al., Proc. NatI. Acad. Sci., USA 88:11261, 1991) and XGrail client server (Shah et al., User's Guide to GRAIL and GENQUEST, Client-Server Systems, available by anonymous ftp to arthur.epm.omi.gov (128.219.9.76) from directory pub/xgrail or pub/xgenquest, as file manual.grailgenquest, 1994). Ten CpG islands were identified (Figure 8).

Forty-eight exons were predicted on the coding strand by the GRAIL program. The quality of 39 of the 48 exons was "excellent", six were considered "good", and three were deemed "marginal". These data were analyzed using the gene model feature of GRAIL2. The final gene model contained 46 exons.

Comparison of the present genomic sequence with the previously reported partial cDNA sequence (EPKDC, Cell, 1994, supra) revealed several differences (Figure The first and most significant difference is the presence of two additional cytosine residues at position 4566 of the reported sequence. The presence of these two cytosine residues results in a frame shift in the predicted protein coding sequence, leading to the replacement of 92 carboxy-terminal amino acids with a novel 12-amino acid carboxy terminus.

Seven of the twelve amino acids of the new carboxy terminus are charged or polar. Additional sequence differences are located at positions 3639-3640 and 3708-3709 of the published EPKDC sequence (Figure A GC dinucleotide pair is present at each of these positions in the present sequence, while a CG pair is found in the reported sequence. In each case, histidine and valine residues would replace the previously predicted glutamine and leucine residues, respectively.

D. Identification of Protein Coding Regions: Exons predicted by the GRAIL2 program with an "excellent" score were used to search the SwissProt and PIR databases (Bairoch and Boeckmann, Nuc. Acids Res. 20:2019- 2022, 1992) using the BLASTP program (Altschul et al., J.

Mol. Biol. 215:403-410, 1990). Exons 3 and 4 of the gene model were predicted to encode peptides with homology to a number of leucine-rich repeat (LRR)-containing proteins involved in protein-protein interactions (Figure 13). In addition to the LRR itself, sequences amino- and carboxyflanking to the LRR may also be conserved in proteins of the leucine-rich glycoprotein (LRG) family, either singly or together.

Exon 3 encodes residues homologous to the LRR from leucine-rich a2 glycoprotein, members of the GPlb.Ix complex which comprise the von Willebrand factor receptor, as well as to the Drosophila proteins chaoptin, toll, and slit. The latter are involved in adhesion, dorsal-ventral polarity, and morphogenesis, respectively.

Sequences predicted by GRAIL2 to be encoded by exon 4 were found to have homology to the conserved region carboxy terminal to the LRR in all of the above proteins except chaoptin, which lacks this conserved region. Homology was also observed between the exon 4-encoded sequences and-the trk proto-oncogene, which encodes a receptor for nerve growth factor. Further examination of the predicted PKDI peptide revealed additional regions of weaker homology with conserved regions of the trk tyrosine kinase domain. None of the more proximal exons in the gene model appear to encode a peptide with homology to the conserved amino-flanking region seen in a subset of the LRR-containing proteins- Exon trapping, RT-PCR, and Northern blot analysis revealed that GRAL2-predicted exons 3 and 4 are present in expressed sequences. During initial exon trapping experiments using genomic P1 and cosmid clones from the PKD1 locus, an exon trap was identified that contained both of these exons. In separate experiments, the presence of the LRR-carboxy-flanking motif in transcribed sequences was confirmed by RT-PCR using as a template RNA from fetal kidney and from adult brain. On a Northern blot, an RT-PCR fragment containing this motif detected the.14kb PKD1 transcript and several other transcripts of 21 kb, 17 kb, and 8.5 kb.

A region of homology was also observed between the GRAIL2-predicted peptide and the human gplOO/Pmell7 gene products, as well as with bovine RPEI. Three copies of a 34 amino acid segment that is also present in the Pmel-17 and gplOO gene products was deduced (Kwon et al, Proc. vNatl.

Acad. Sci., USA 88:9228-9232, 1991; Adema et al., J. Biol.

Chem. 269:20126-33, 1994) within the larger context of immunoglobulin repeat motifs. The RPEI gene product has significant homology to gplOO and may represent the bovine homolog (Kim and Wistow, Exp. Eye Res. 55:657-662, 1992).

GRAIL2-predicted exons 9, 22, and 28, upstream of the 3' cDNA, showed strong homology to EST T03080 255 bp), EST T04943 189 bp) and EST T05931 233 bp).

In addition, nucleotides 10378-10625 of GRAIL-predicted intron 1 showed strong homology to a region of the Apo CII gene 263 bp).

The identification of a number of transmembrane domains and a leucine-rich repeat motif possessing conserved carboxy-flanking regions, raises interesting speculations about potential protein function. LRR motifs have been shown to be involved in protein-protein interactions, while the conserved carboxy-flanking region is associated with proteins which interact with the extracellular matrix. These data suggest that the PKD1 gene product may be a membrane glycoprotein that functions in cell-matrix or cell-cell interactions. Less commonly, LRR motifs have been identified in receptors involved in signal transduction (McFarland et al., Science 245:494-499, 1989). Thus an alternative hypothesis is that the gene product is a receptor for a soluble factor(s). In either case, PKD1 would function to mediate interactions with the extracellular environment. If so, ligands for the gene product as well as downstream intracellular effectors are obvious candidates for the non-chromosome 16-linked forms of the disease. A model of the predicted PKD1 protein structure is shown in Figure 9.

E. Repeated Sequences: The PKD1 locus was searched for known classes of repetitive DNA by FASTA comparison against the repeat database of Jurka et al., J.Mol.Evol. 35:286-291, 1992. This search identified 23 Alu repeats but no other repetitive elements. The Alu repeats are organized into three clusters of four or more Alu repeats, three clusters of two Alu repeats, and two singlet Alu repeats (Figure 8).

The PKD1 sequence interval contained two dinucleotide repeats and a single tetranucleotide repeat ((TTTA)6). The TG dinucleotide repeats are present at positions 209-224 and 52,698-52,715. The tetranucleotide repeat is located at position 7796-7819. No trinucleotide repeats >5 were identified. Only the most 3' TG8 repeat is known to be polymorphic.

In addition to the more usual repetitive elements, the PKD1 gene contains several types of repeated sequences that either do not appear in existing data bases, or do not appear in the extreme form seen at this locus. The most striking repeat is a 2.5 kb segment within the 4 kb BamHI- SacI fragment. A significantly shorter C-T rich region is also found in the adjoining 1.8 kb SaCI-BamHI fragment.

These regions proved very difficult to sequence unambiguously due to the high GC content to the purine asymmetry with respect to each strand and to the length of the repeat.

The coding strand in this region has an extreme pyrimidine bias, being 96% C-T, and could-not be sequenced using T7 DNA polymerase or Sequenase. This was true regardless of the template type (plasmid, single-stranded phage, or strandseparated single-stranded DNA) In both cases, the noncoding strand, which is G-A rich, was successfully sequenced with both T7 DNA polymerase and Sequenase, although run lengths were noticeably abbreviated compared to all other regions sequenced. Compressions on the non-coding strand were resolved by conventional and cycle sequencing using single-stranded template. The extreme purine asymmetry of strands in this segment may promote localized triple strand conformation under the appropriate conditions (pH, divalent cations, supercoiling), and may be a major cause of the difficulty in sequencing this segment.

The other unusual repeat was located in the 7.6 kb Xhol fragment. This repeat is 459 bp in length and consists of 17 tandem copies of a perfect 27 bp repeat.

Example 2: PKD1 cDNA Secuences Obtained Through Exon Trapping and cDNA Selection Technirues The 700 kb interval of chromosome 16 that includes the PKD1 gene appears to be particularly rich in CpG islands and, by association, is most likely rich in expressed sequences as well. To purify and sequence expressed PKD1 sequences, an exon-rescue vector, pSPL3, was used to recover sequences from cosmids that contain both a splice acceptor and splice donor element; this method is designated "exon trapping.-" Exon trapping is a highly efficient method for isolating expressed sequences from genomic DNA. The procedure utilizes the pSPL3 plasmid, which contains rabbit i-globin coding sequences separated by a portion of the HIvtat gene, or improved derivatives of SPL3 lacking cryptic (interfering) splice sites. Fragments of cloned PKD1 genomic DNA were cloned into the intron of the tat gene, and the resulting subclones were transfected into COS-7 cells. sequences in the vector allow for both relaxed episomal replication of the transfected vectors, as well as transcription of the cloned genomic DNAS. Exons within the subcloned genomic DNAs spliced into the globin/tat transcript were recovered using RT-PCR, using primers containing tat splice donor and acceptor sequences. A major advantage of exon trapping is that expression of the cloned DNA is directed by a viral promoter; thus, developmental or tissuespecific expression of gene products is not a concern.

PKDl-containing genomic clones, in the form of either cosmid or P1 DNA, were either double digested with BamHI and BglII or partially digested with Sau3A and shotgun cloned into BamHI-digested and dephosphorylated pSPL3 (GIBCO BRL, Bethesda, MD) or its derivatives. Plasmid minipreps were electroporated into COS-7 cells, and trapped exons were recovered by RT-PCR, followed by subcloning, using standard procedures.

Trapped exons from the PKD1 locus are shown in Figure 14 (bottom). The trapped exons were subjected to automated DNA sequencing as above, allowing their alignment with the genomic PKD1 DNA.

Example 3: Construction of Full-lenqth PKD cDNA In the case of PKD1, the identification of cDNAs which are specific for the 5' end of the PKD1 locus is particularly difficult since multiple transcribed copies of homologous sequences are also present at 16p13.1 (EPKDC, Cell, 1994 supra). Regions of both genomic DNA and cDNA derived from the homologues were sequenced and compared with the present PKD1 sequence. In this data set, the PKD1 and homologous sequences were greater than 97% identical at the nucleotide level. Therefore, direct comparisons of potential PKD1 cDNAs and genomic sequence are required to definitively map a cDNA to the PKDI locus, and to verify that the correct sequence is encoded by.'the cDNA.

Multiple approaches were required to assemble the full-length PKD1 cDNA. Seven cDNAs were used to construct the full-length cDNA. Five of these cDNAs were recovered from screening cDNA libraries: the BRL Gene-Trapper brain library, and cDNA libraries constructed from fetal brain, and constructed from the somatic cell hybrid 145.19. The 145.19 cell line contains the PKD1 locus, but does not include the PKD1 homologs in its human component.

A. cDNA Library Construction and Screening The somatic cell hybrid library was constructed using both oligo(dT) and random hexamer priming and poly (A)-containing RNA from the 145.19 cell line. The duplex cDNA was linked and then ligated into lambda ZAP EXPRESS (Stratagene, La Jolla, CA) to yield a library consisting of several million independent plaques. Fourteen clones were positive by colony hybridization using a PKDI specific probe, with inserts ranging in size from 2.6 to 9 kb. Consistent with the RT-PCR products derived from the 145.19 cell line, substantial alternative splicing or incomplete splicing was evident. Interestingly, the missing exons appeared to comprise one or more distinct protein domains.

Two additional libraries were constructed using fetal brain cDNA cloned into lambda ZAP EXPRESS and the replacement vector, lambda DASH (Stratagene, La Jolla, CA).

Additionally, a variation of.the cDNA selection methodology was used to screen oligo(dT)-primed, unidirectional cDNA libraries (in phagemids). Briefly, single-stranded library DNA was prepared from cultures of the adult brain cDNA library. A single biotinylated 1 7 -mer derived from the sense-strand from the gene-specific portion of the predicted PKDI cDNA was used for hybrid selection.

Hybrid-bound cDNAs released by denaturation were made double-stranded using the same oligonucleotide as a gene-specific primer and Klenow and then introduced into E.

coli by electroporation. Colony hybridization was used to identify the PKDI clones from the enriched brain cDNA'' population. The cloned brain inserts ranged in size from 0.7 to 2.5 kb. The sequence of the two largest cDNAs was virtually identical to each other as well as to the genomic sequence.

Example 4: Expression of Full-Length PKDI cDNA Full-length PKDI cDNA was cloned into three expression vectors, pCMV-SPORT, pcDNA3, and pCEP4 (total construct sizes ranging from 18-24.2 kb) The schematic structure of full-length PKD1 cDNA in pCMV-SPORT is shown in Figure 11.

pCMV-SPORT and pcDNA3 have small differences in cloning sites and some other small features, but share the basic features of flanking T7 and SP6 promoters, CMV enhancer-promoter sequences for high level transcription, and eukaryotic polyadenylation and transcription sequences which enhance RNA stability. The SV40 origin of replication allows growth in eukaryotic cells, while the ColEl origin allows growth in E. coli. The vector pcDNA3 confers neomycin resistance in eukaryotes, while ampicillin resistance is used for selection in E. coli.

pCEP4 is an EBV-based vector which is maintained extrachromosomally in primate cells. Like pCMV-SPORT and pcDNA3, pCEP4 contains the CMV enhancer and promoter, and the ColEl origin of replication and ampicillin resistance are used for maintenance. However, hygromycin resistance is used for selection in eukaryotic cells. The use of the EBV origin of replication and hygromycin resistance are important features for studies of PKD1 transformed cell lines, since as a function of the transformation procedure they already contain SV40 large T antigen, and are G418 resistant.

A. In vitro Expression The T7 promoter feature of pcDNA3 was used to analyze the protein product encoded by the PKD1 cDNA employing the TNT Coupled Reticulocyte Lysate System, (Promega, Madison, WI). This system enables large amounts of RNA to be synthesized from the T7 promoter, and the RNA to be translated into protein in the rabbit reticulocyte lysate.

Since conventional molecular weight standards only extend up to -216 kD, the size estimates of in vitro synthesized polycystin, -462 kD (non-glycosylated), would be speculative at best. For this reason, a series of 3' deleted PKDI cDNA plasmid templates encoding truncated proteins of predicted size were constructed (Figure 10). The protein products of these deletion clones as well as the full-length PKD1 cDNA were analyzed using the TNT system.

Newly synthesized protein was labeled by inclusion of radioactive amino acids, initially 35 S-methionine. The synthesized proteins were then resolved by electrophoresis on a 3-12% gradient SDS-PAGE gel. The mobility of the protein product produced from each of the truncated clones was consistent with its predicted molecular size. These results are consistent with assembled PKDI cDNA expression vectors directing in vitro synthesis of polycystin.

B. In vivo Expression: PKDI cDNA Transfection in Human Embryonic Kidney (HEK) 293 cells cDNA constructs containing full-length PKD1 cDNA or portions thereof were transfected into HEK 293 cells andassayed for PKD1 expression using Northern analysis, 48'hours post-transfection. An insertless vector,;pcDNA3, was used in parallel as a control for transfection. -A Northern blot was probed with a PKD1-specific probe and then subsequently re-probed with a S-actin cDNA to normalize the respective lanes. The results showed that the PKDl mRNA is increased at least two-fold in HEK 293 which received the PKD1 cDNA construct.

Example 5: Diagnostic Tests for PKD1 Mutations Whole blood samples collected in high glucose

ACD

Vacutainers T M (yellow top) were centrifuged and the buffy coat collected. The white cells were lysed with two washed of a 10:1 mixture of 14mM NH 4 C1 and ImM NaHC03, their nuclei were resuspended in nuclei-lysis buffer (10mM Tris, pH 0.4M NaCl, 2mM EDTA, 0.5% SDS, 500 gg/ml proteinase K) and incubated overnight at 37 0 C. Samples were then extracted with a one-fourth volume of saturated NaCl and the DNA was precipitated in ethanol. The DNA was then washed with ethanol, dried, and dissolved in TE buffer (10mM Tris-HCl, pH ImM EDTA).

A. Test I Long PCR conditions were used with a 4-part reaction mixture. Part 1 containing the following components: 3.3X XL Buffer 12 pa dNTPs (2mM each) 8 1I Forward primer (20pM) 1-5 gil Reverse primer (20uM) 1-5 n1 Blocking oligo (2mM) 1.5 pl Mg(OAc)2, (25mM) 4.4 al water to 40 p1 Part 1 can be assembled as a single reaction component or in batch (10, 50, 100 reaction equivalents) and then dispensed as 40gi aliquots into individual reaction tubes.

Part 2 comprises carefully adding 1 AmpliWaxPCR Gem 100 (or comparable product to each Part I reaction tube). The tubes were incubated at 75-80°C for 5 min.to melt the wax bead.

The reactions were cooled allowing the wax to solidify.

In Part 3, the following components were added to the cooled reaction mixture of Part 2: 3.3X XL Buffer 18i rTth DNA Polymerase, XL 241 In Part 4, the following components are added to the reaction mixture of Part 3: human DNA 0.2-14g water to The forward primer used in the reaction described above comprises an oligonucleotide that hybridizes to both authentic PKD1 and PKD1 homologue sequences. An example of such a primer is: -CACGACCTGTCCCAGGCAT-3' (SEQ ID NO:6) (corresponding to nucleotides 4702-4720 of SEQ ID NO:1).

The reverse primer comprises a sequence derived from a 3' region of the authentic PKD1 gene, which may or may not be present in the PKD1 homologues. Examples of such 3' regions and corresponding reverse primers are: 3' sequence: reverse primer: 5'-CTGGCGGGCGAGGAGAT-3' 5'-ATCTCCTCGCCCGCCAG-3, (SEQ ID NO:7) (SEQ ID NO:56) 5'-CTTTGACAAGCACATCT-3' 5'-AGATGTGCTTGTCAAAG-3' (SEQ ID NO:8) (SEQ ID NO:57) 5'-CAACTGGCTGGACAACA-3' 5'-TGTTGTCCAGCCAGTTG-3' (SEQ ID NO:9) (SEQ ID NO:58) The blocking oligonucleotide comprises: 5'-AGGACCTGTCCAGGCATC-3' (SEQ ID Importantly, this oligonucleotide must be incapable of supporting polymerization. One example is an oligonucleotide in which the 3' terminal nucleotide comprises a dideoxynucleotide. It will be understood that any modification that achieves this effect may be used in practicing the invention. Under appropriate conditions, the blocking oligonucleotide hybridizes efficiently to PKD1 homologues but inefficiently to the authentic PKD1 sequence.

Thus, the amplification products in this diagnostic test are derived only from the authentic PKD1 gene.

Twenty-five to thirty-eight cycles of amplification were performed, using a standard DNA thermal cycler the following primer-dependent conditions for each cycle: SEQ ID NO:56: 94 0 C, 30 seconds; 62°C, 30 seconds; and 72 0

C,

34 minutes.

SEQ ID NO:57: 94°C, 30 seconds; 56 0 C, 30 seconds; and 72 0

C,

37 minutes.

SEQ ID NO:58: 94°C, 30 seconds; 58°C, 30 seconds; and 72 0

C,

minutes.

-The 72°C extension cycle was lengthened 5 seconds each subsequent cycle. The primary PCR product can be analyzed immediately for mutations or alternatively, can be used as a template for secondary PCR using a collection of paired amplimers to generate an overlapping set of smaller amplicons. The smaller amplicons can then be analyzed for mutations.

B. Test II Long PCR conditions were used with a 4 -part reaction mixture. Part 1 containing the following components: 3.3X XL Buffer 12 pl dNTPs (2mM each) 8 Al Forward primer (20pM) 1-5 Jil Reverse primer (20gM) 1-5 Ai Mg(OAc)2, (25mM) 4.4 i water to 40 gl Part 1 can be assembled as a single reaction component or in batch (10, 50. 100 reaction equivalents) and then dispensed as 40A1 aliquots into individual reaction tubes Part. 2 comprises carefully adding 1 AmpliWaXPCRGem 100 (or comparable product to each Part 1 reaction tube. The tubes were incubated at 75-80°C for 5 min. To melt the wax bead.

The reactions were cooled allowing the wax to solidify.

In Part 3, the following components were added to the cooled reaction mixture of Part 2: 3.3x XL Buffer 18g1 rTth DNA Polymerase, XL 2il In Part 4, the following components are added to the reaction mixture of Part 3: human DNA 0.2-1ig water to Twenty-five to thirty-eight cycles of amplification were performed, using a standard DNA thermal cycler the following protocol for each cycle: 94 0 C, 30 seconds; 610°C, 30 seconds; and 72°C, 11 minutes. The 72°C extension cycle was lengthened 5 seconds each subsequent cycle. The primary

PCR

product can be analyzed immediately for mutations or alternatively, can be used as a template for secondary

PCR

using a collection of paired amplimers to generate an overlapping set of smaller amplicons. The smaller amplicons can then be analyzed for mutations.

The forward primer used in the reaction described above comprises an oligonucleotide that hybridizes to both authentic PKDI and PKD1 homologue sequences. An Example of such a primer is: CTGCACTGACCTCACGCATGT (SEQ ID NO:11) The reverse primer comprises a sequence derived from the authentic PKD1 gene and is not present in the PKD1 homologues. Thus, the amplification product in this diagnostic test is derived only from the authentic PKD1 gene.

An example of a suitable reverse primer is: (SEQ ID NO:14) C. Test III Long PCR conditions were used with a 4 -part reaction mixture. Part 1 containing the following components: 3.3X XL Buffer 12 gi dNTPs (2mM each) 8 gil Forward primer (20pM) 1-5 gl Reverse primer (2011M) 1-5 gil Mg (OAc)2, (25mM) 4.4 gl water to 40 gil Part 1 can be assembled as a single reaction component or in batch (10, 50, 100 reaction equivalents) and then dispensed as 401il aliquots into individual reaction tubes.

Part 2 comprises carefully adding 1 AmpliWaxpCR Gem 100 (or comparable product to each Part 1 reaction tube. The tubes were incubated at 75-80C for 5 min. To melt the wax bead.

The reactions were cooled allowing the wax to solidify.

In Part 3, the following components were added to the cooled reaction mixture of Part 2: 3.3X XL Buffer 1841 rTth DNA Polymerase, XL 21 In Part 4, the following components are added to the reaction mixture of Part 3: human DNA 0.

2 -14g water to 40 gil Twenty-five to thirty-eight cycles of amplification were performed, using a standard DNA thermal cycler the following protocol for each cycle: 94°C, 30 seconds; 65°C, 30 seconds; and 72°C, 11 minutes. The 72°C extension cycle was lengthened 5 seconds each subsequent cycle. The primary

PCR

product can be analyzed immediately for mutations or alternatively, can be used as a template for secondary

PCR

using a collection of paired amplimers to generate an overlapping set of smaller amplicons. The smaller amplicons can then be analyzed for mutations.

The forward primer used in the reaction described above comprises an o.ligonucleotide that hybridizes to both authentic PKD1 and PKD1 homologue sequences. An Example of such a primer is: ACGTTGGGCTCCTGGGCAACC (SEQ ID NO:12) The reverse primer comprises a sequence derived from the authentic PKD1 gene and is not present in the PKDI homologues. Thus, the amplification product in this diagnostic test is derived only from the authentic PKD1 gene.

An example of a suitable reverse primer is: 5' -AGGTCAACGTGGGCCTCCAAGTAGT (SEQ ID NO: 13) For RT-PCR, first strand cDNA synthesis is performed using the reverse primer (SEQ ID NO:14) and SuperscriptII7 according to manufacturer's recommended conditions (Life Technologies, Inc., Gaithersburg, MD). PCR is then performed using 1-50% of the first strand reaction under the reaction conditions described above, with the modification that the extension cycle is conducted at 72°C for only 6 min.(due to the smaller product size).

D. Test IV To analyze PKD1 mRNA for mutations, RNA is isolated from the white blood cells as a requisite template for RT- PCR. Whole blood samples collected in high glucose

ACD

Vacutainersm (yellow top) were centrifuged and the buffy coat collected (4-20 x 106 cells/10 ml of blood). RNA can be isolated directly from white blood cells or after standard short-term culturing of white blood cells in the presence of a mitogen such as phytohemagglutinin (48-72 hours) RNA is isolated as described using standard conditions such as guanidium isothiocyanate:acid phenol extraction (Chomczynski and Sacchi, Anal. Biochem. 162:156-159, 1987).

For RT-PCR, first strand cDNA synthesis is performed using the reverse primer (below) and a commercially available reverse transcriptase, such as, for example, SuperscriptIl T m according to manufacturer's recommended conditions (Life Technologies, Inc., Gaithersburg, MD).

PCR

is then performed using 1-50% of the first strand reaction under the reaction conditions described below.

The reverse primer comprises a sequence derived from .both the authentic PKDl gene and the PKD1 homologues- In contrast, the forward primer is specific for the authentic PKDI locus and will not allow amplification of cDNAs derived from the homologous loci. Thus, the resulting

RT-PCR

amplification product in this diagnostic test is derived only from authentic PKD1 RNA.

The forward primer used in this reaction comprises an oligonucleotide that hybridizes only to authentic PKD1 and not to homologue sequences. An example of such a primer is: AGCGCAACTACTTGGAGGCCC (SEQ ID An example of a suitable reverse primer is: GCCAAAGGGAAAGGGATTGGA (SEQ ID N0:16) The amplification aspect of the RT-PCR reactions was performed using standard conditions as described below including a "hot-start" step: Taq Buffer 8 dNTPs (2mM each) 7 ji Forward Primer (lOO1pM) 0.4-1.5 ji Reverse Primer (100gM) 0.4-1.5 p.1 DNA 0.2-1.0 jg water to 80 Al Amplification was initiated using a single "hotstart" step, followed by twenty-five to thirty-eight cycles of amplification using a standard DNA thermal cycler. The single "hot-start" step consisted of 80°C for 3-5 minutes after which time lIl of Tag polymerase was added to each reaction tube. 'Hot-start- was proceeded by 25-38 cycles with each cycle consisting of the following specifications.

94°C, 20 seconds; 64°C, 30 seconds; and 72°C, 2 minutes: The primary PCR product can be analyzed immediately for mutations or alternatively, can be used as a template for secondary PCR using a collection of paired amplimers to generate an overlapping set of smaller amplicons. The smaller amplicons can then be analyzed for mutations.

The PCR and RT-PCR products obtained above were analyzed for the presence of specific PKDI mutations as follows: 8 il of the amplified products were added to 50 gl of a denaturing solution (0.5mM NaOH, 2.0M NaCi, 25mM EDTA) and spotted onto nylon membrane filters (INC Biotrans). The denatured DNA was then fixed to the nylon filters by baking the filters at 80 0 C for 15 minutes under vacuum.

Oligonucleotides that detect PKDI mutations were chemically synthesized using an automated synthesizer and radiolabeled with 3 2 p with polynucleotide kinase, using methods that are standard in the art.

Hybridizations were carried out in plastic bags containing the filters prepared above, to which one or more labeled oligonucleotides were added in a hybridization buffer Tetramethylammonium chloride (TMAC), 0.6% SDS, imM EDTA, 10mM sodium phosphate pH 6.8, 5X Denhardt's Solution, and 40 gg/ml yeast RNA). Oligonucleotide concentrations in the pools ranged from 0.03 to 0.15 pmol/ml hybridization olution.

Hybridizations were allowed to proceed overnight at 52 0 C, with agitation. The filters were then removed from the bags and washed for 20 min. at room temperature with wash buffer (3.0M TMAC, 0.6% SDS, ImM EDTA, 10mM sodium phosphate pH followed by a second wash in the same buffer for min. at 52°C. The filters were dried and exposed to Kodak X-OMAT" film.

It will be understood that the enzymes and nucleotides used in the above reactions may be obtained from any manufacturer, such as GIBCO-BRL, Promega, New England Biolabs, and the like.

Example 6: AntiDolvcvstin Antibodies A. Production and Characterization of Polyclonal Antisera Against Synthetic C-Terminal Peptide.

A peptide (C)SRTPLRAKNKVHPSST (SEQ ID NO:17) representing the last 16 carboxy-terminal amino acids of the predicted PKD1 gene product was synthesized. A cysteine residue that is not predicted from the DNA sequence was appended to the amino terminus to facilitate coupling to KLH carrier protein. Two rabbits (A and B) were immunized with the peptide as described in Cheng et al., EMBO J. 7:3845- 3855, 1988.

Polyclonal anti-peptide antisera were diluted from 1:10 up to 1:10,000, and immunoreactivity was determined by ELISA according to conventional procedures (Cheng et al., EMBO 1988 supra.). Antisera produced by both rabbits were epitope mapped by the SPOTs method (Blankenmeyer-Menge and Frank, in INNOVATION AND PERSPECTIVES IN SOLID PHASE SYNTHESIS, Epton, R. Ed., Chapman and Hall Medical, London, 1990, pp. 1-10). Briefly, overlapping 8 amino acid long peptides were synthesized simultaneously on a cellulose membrane and assayed for immunological reactivity. Positive peptides were aligned and the epitope was identified by determining sequence homologies. Interestingly, antisera

A

and B had at least 2 non-overlapping epitopes each, thus increasing the possibility that these antibodies will recognize the PKD1 gene product.

B. Domain Specific Fusion Proteins Four fusion clones were constructed to contain different domains of polycystin such that the correct open reading frame was maintained, as shown in Figure 15. Three of the expression constructs were cloned in the pGEX vectors designed for the expression of foreign sequences as glutathione S-transferase (GST) fusion proteins in E. cold.

These are FP-LRR, which contained the leucine-rich repeat (LRR); FP-46-lc, containing 83 C-terminal amino acids and FP46-2 which has 77 amino acids internal to the FP- 46 -lc.

The fourth fusion construct was cloned into a maltose binding protein (MBP) vector, and encoded 205 amino acids at the carboxy terminus, thus overlapping two of the GST fusion proteins. The overlapping carboxy-fusion products provide an additional layer of antibody reagent confirmation. They allow one to verify that positive antibody reactions are not artifactual, since similar, if not identical, patterns of antibody reactivity should be seen with antibodies raised against these overlapping proteins. Two different 'carrier' fusion proteins also allows one to purify antibody raised against a fusion product using the alternate carrier protein as the affinity ligand. This helps to eliminate antibodies raised against the carrier protein itself.

GST fusion proteins were purified from extracts of transformed bacteria using glutathione-Sepharose (Pharmacia) as described in Smith and Johnson, Gene 67:31-40, 1988. MBP fusion proteins were purified on amylose resin (NEB, Beverly,

MA).

C. Generation and characterization of polyclonal antibodies to domain specific polycystin fusion proteins.

Antibodies against the fusion proteins were raised in rabbits using published procedures (Cheng et al., EMBO J-.

1988 supra.) with 200 jg of protein. These respective antibodies specifically recognized PKD1 protein as part of the fusion protein construct used as immunogen

FP-LRR,

FP46-1c, FP46-2 and MAL-BD-3). Further, these antibodies did not bind the irrelevant antigens GST or MBP, nor cross-react -to polycystin domains not present in the immunogen included as controls after sufficient antibody purification.

In vitro synthesized polycystin protein was used to test the domain specific antibodies. In addition to the full-length PKD1 cDNA, two shorter clones which each expressed only a subset of the PKD1 domains were constructed in expression vectors as shown in Figure 16. The BRASH 7 clone contains the carboxy terminal epitopes, as well as the transmembrane domains, while SrfIA contains the amino terminus, the LRR, and the majority of the Ig-like domains.

Both are efficiently expressed in the TNT in vitro transcription/translation system.

D. Immunoprecipitation Antipolycystin antibodies were incubated with either protein A Sepharose or Protein G Sepharose to generate antibody coupled beads. These beads were then incubated with 3 5 S-labeled protein synthesized in vitro from the expression clones. The void and retained fractions were collected and analyzed by SDS gel electrophoresis. Sepharose alone was included as a control against artifactual binding, a concern due to the large size of polycystin, the presence of the large number of Ig-like repeats, and the lectin domain.

Antibodies to irrelevant antigens were also included as controls. If the antibody specifically bound the antigen, a protein species of the correct molecular mass will be detected on the gel in the bead fraction. If not, the expressed protein will appear in the void volume on the gel.

Each of the anti-fusion protein antibodies coupled to Sepharose A specifically immunoprecipitated protein expressed by clones which contained the matching antigenic domain. The antibodies did not immunoprecipitate protein expressed from irrelevant domains of polycystin domains not used as immunogen to generate that particular antibody), nor did they recognize other irrelevant antigens luciferase). These results confirm that these polyclonal antibodies specifically recognize the carboxy terminus and LRR domains of polycystin.

ExamDle 7: Identification of Proteins that Interact with PKD Further characterization of the PKD1 protein can be accomplished through identification of other proteins which' normally interact with the PKD1 protein. Those of skill in the art are familiar with a variety of approaches useful for such purposes, including, but not limited to, immunoprecipitation of protein complexes using antipolycystin antibodies, screening of expression libraries with labeled in vitro synthesized polycystin, and use of yeast systems that exploit the interaction of DNA binding and activation domains.

For example, one such approach is the two-hybrid yeast system (Fields and Song, Nature 340:245-6, 1989; Finley and Brent, Proc. Natl. Acad. Sci., USA 91:12980-84, 1994) which enables the identification of genes which encode proteins that interact with PKDI. This technique relies on the fact that eukaryotic transcriptional activators, such as GAL4, function utilizing two essential and discrete domains, an amino terminal DNA binding domain and a carboxy terminal transcriptional activation domain (Ma and Ptashne, Cell 51:113-119, 1987).. The two-hybrid system exploits the observation that a functional transcriptional activator can be generated even when the two domains are encoded by different hybrid polypeptides, so long as the spatial relationship between the two essential domains is similar to the native transcriptional activator. The yeast two hybrid system has been used successfully to screen cDNA expression libraries in search of proteins that interact with Yin-Yang-i (Shrivastava et al., Science 262:1889-92, 1993), E12 (Staudinger et al., J. Biol. Chem. 268:4608-11, 1993), H-Ras (Vojtek et al., Cell 74:205-214, 1993), Pr55gag (Luban et al., Cell 73:1067-78; 1993), pllORB (Durfee et al., Genes Dev. 7:555-69, 1993), and p53 (Iwabuchi et al., Oncogene 8:1693-96, 1993).

A. Hybrid Construction Several constructs of the PKDI regions as fusion proteins with the GAL4 DNA binding domain were prepared. The constructs were: a BD-3 fusion between the GAL4 DNA-binding domain and the cytoplasmic tail of the PKD1 protein (amino acid residues 4097-4302) using pGBT9 vector, a BD-1 clone containing a DNA-binding domain and the LRR region of polycystin (amino acid residues 27-360), and a BD-2 clone which contains DNA-binding domain and region of ig-like repeats (amino acid residues 713-2324).

B. Transformation of constructs into yeast Competent yeast cells HF7c, containing the lacz reporter gene are obtained by the LiAc method. Briefly, overnight cultures are diluted to OD600 0.2 and continue to grow for an additional 3 hr. Cells are collected, washed in H20 and resuspended in 0.1M LiAc in TE. Competent cells (0.i ml) are mixed with 0.1 mg of plasmid-construct DNA and i00 mg of carrier DNA. 50% PEG400 (0.6 ml) is added and incubated at 0 C for lh. Following this incubation, the cells are heated to 42 0 C for 10 min. and plated on minimal medium (Difco Yeast Nitrogen Base without amino acids, supplemented with auxotrophic requirements) Yeast transformants are selected after 3 days of culture.

B. Colony lift filter assay for S-galactosidase VWR grade 410 filters are layered over agar plates containing transformants on selection medium and transferred to a pool of liquid nitrogen for 10 sec. Filters, colony side up are placed on another filter that is presoaked in X-gal solution. After two hours, filters are analyzed for the presence of blue, £-galactosidase producing colonies (not shown). Alternatively, individual colonies from different transformations can be streaked onto the same plate and processed for S-galactosidase activity.

While the present invention has been described with respect to what are presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments- To the contrary, the -59invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and-functions.

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

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims (24)

1. Isolated-gene encoding human PKD1 polypeptide.

2. Isolated human PKD1 gene according to claim 1, comprising the sequence set forth in SEQ ID NO:2.

3. Isolated RNA transcript expressed by the gene of claim 1.

4. Isolated nucleic acid comprising the sequence set forth in SEQ ID NO:4. Isolated nucleic acid fragment consisting of a sequence encoding human PKD 1 that hybridizes under stringent conditions to the gene of claim 1.

6. Isolated nucleic acid fragment according to claim 5, comprising the sequence set forth in SEQ ID NO:3.

7. Isolated polypeptide encoded by the gene of claim 1.

8. Isolated polypeptide according to claim 7 comprising the amino acid sequence set forth in SEQ ID

9. A vector comprising the isolated gene of claim 1.

10. A vector according to claim 9, further comprising a transcriptional regulatory element operably linked to said gene, said element having the ability to direct the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

11. A host cell comprising the vector of claim 9.

12. A method for producing PKD1 protein, which comprises: P.pOiVWpWVPA roEui\;12493492 |Llyetdivd imL 261 doc-l794 -170- culturing the host cell of claim 11 in a medium and under conditions suitable for expression of said protein, and isolating said expressed protein.

13. Isolated mutant human PKD 1 gene, comprising the DNA sequence set forth in SEQ ID NO:2 having modifications selected from the group consisting of: transitions, transversions, deletions and insertions.

14. The gene of claim 13, comprising a DNA sequence whose presence in one or more copies in the genome of a subject is associated with adult-onset polycystic kidney disease (APKD) in said subject. A recombinant vector comprising the gene of claim 13.

16. The vector of claim 15 further comprising a transcriptional regulatory element operably linked to said gene, said element having the ability to direct the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

17. A host cell comprising the vector of claim

18. A method for producing mutant PKD1 protein, which comprises: culturing the host cell of claim 17 in a medium and under conditions suitable for expression of said protein, and isolating said expressed protein.

19. An isolated nucleic acid comprising: 5'-AGGACCTGTCCAGGCATC-3' (SEQ ID

20. An isolated nucleic acid comprising: 5'-GCGCTTTGCAGACGGTAGGCG-3' (SEQ ID NO:14). P:Oa\VpWA P -orudow2l493492 gauy, div ,lis 261..k'c-t109O4 171

21. An isolated nucleic acid comprising: 5'-AGGTCAACGTGGGCCTCCAAGTAGT-3' (SEQ ID NO:13).

22. An isolated nucleic acid comprising: 5'-AGCGCAACTACTTGGAGGCCC-3' (SEQ ID

23. A diagnostic method for screening human subjects to identify PKDI carriers, which comprises the steps of: obtaining a sample of biological material from said subject; and assaying for the presence of mutant PKD1 genes or their protein products in said biological material.

24. The method of claim 23 wherein said biological material comprises nucleic acid, and said assaying comprises: selectively amplifying the PKD1 gene, or fragments thereof, from said biological material, and detecting the presence of a normal or a mutant gene using an analytical method selected from the group consisting of: restriction enzyme digestion, direct DNA sequencing, hybridization with sequence-specific oligonucleotides, single-stranded conformational polymorphism analysis, denaturing gradient gel electrophoresis (DDGE), two-dimensional gel electrophoresis, and combinations thereof. The method of claim 24 wherein said amplifying is performed in the presence of at least one oligonucleotide selected from the group consisting of 5'-AGGACCTGTCCAGGCATC-3' (SEQ ID 5'-GCGCTTTGCAGACGGTAGGCG-3' (SEQ ID NO:14), 5'-AGGTCAACGTGGGCCTCCAAGTAGT-3' (SEQ ID NO:13), and 5'-AGCGCAACTACTTGGAGGCCC-3' (SEQ ID P.'-pOVpWtVA P r tcutoA24J9492 uymt dilains 261 da-l7 04

172- 26. The method of claim 23, wherein said assaying step comprises an immunoassay employing an antibody specific for said PKDD1 gene product. 27. An isolated antibody immunospecific for a peptide comprising the sequence (C)SRTPLRAKNKVHPSST (SEQ ID NO: 17), or antibody fragments thereof. 28. The antibody of claim 27 immunospecific for a polypeptide encoded by the gene sequence set forth in SEQ ID NO:2. 29. An isolated antibody immunospecific for a polypeplide encoded by the gene sequence set forth in SEQ ID NO:2. An isolated antibody immunospecific for a polypeptide comprising the amino acid sequence set forth in SEQ ID 31. A method for treating a disease condition having the characteristics of APKD, which comprises administering to cells having defective PKDI function a normal human PKD 1 gene or fragments thereof, wherein said administration results in expression of therapeutically effective amounts of normal PKDI protein or fragments thereof. 32. The method of claim 31, wherein said normal human PKD1 gene comprises the DNA sequence of SEQ ID NO:2. 33. The method of claim 31, wherein said normal human PKD gene comprises the cDNA sequence of SEQ ID NO:4. 34. A method for treating a disease condition having the characteristics of APKD, which comprises administering to cells having defective PKD1 function therapeutically effective amounts of a normal PKD 1 protein or fragments thereof. PFOpcaiWplA n-cadiall 249349; tmne div clims 26t doca-I1OM

173- The method of claim 34, wherein said PKDI protein is encoded by the DNA sequence of SEQ ID NO:2. 36. The method of claim 34, wherein said PKD1 protein is encoded by the cDNA sequence of SEQ ID NO:4. 37. The method of claim 34, wherein said PKD1 protein has the amino acid sequence set forth in SEQ ID 38. A composition comprising an isolated human PKD1 gene having the DNA sequence of SEQ ID NO:2, or fragments thereof, and a pharmaceutically acceptable carrier or diluent. 39. A composition comprising a vector containing a PKD1 gene having the DNA sequence of SEQ ID NO:2, or fragments thereof, and a carrier or diluent. A composition comprising the DNA sequence of SEQ ID NO:4 or fragments thereof, and a carrier or diluent. 41. A composition comprising a normal PKDI protein encoded by the DNA sequence of SEQ ID NO:2, or fragments thereof, and a pharmaceutically acceptable carrier or diluent. 42. A composition comprising a polypeptide encoded by the DNA sequence of SEQ ID NO:4, or fragments thereof, and a carrier or diluent. 43. A non-human unicellular or multicellular organism whose genome comprises a recombinant PKD1 gene or fragments thereof. 44. The non-human organism of claim 43 wherein said recombinant PKD1 gene has the DNA sequence of SEQ ID NO:2 or fragments thereof. P:%Opeipa\VPA N n'12492 mr)=c div damm 261 dw,- 7.f -174- The non-human organism of claim 43 wherein said recombinant PKD1 gene has the cDNA sequence of SEQ ID NO:4 or fragments thereof. DATED this 17th day of September, 2004 Genzyme Corporation AND Johns Hopkins University by DAVIES COLLISON CAVE Patent Attorneys for the Applicants