WO2004074302A2 - Autosomal recessive polycystic kidney disease nucleic acids and peptides - Google Patents

Abstract

A novel gene involved in autosomal recessive polycystic kidney disease has been isolated. The nucleic acid sequence of the mouse gene, Cys1, and the human homologue, CYS1, are disclosed. In addition, a mutant of the Cys1 nucleic acid containing a tandem deletion is described. The Cys1 and CYS1 nucleic acids are associated with ARPKD. Southern analyses indicate that the Cys1 gene exists as a single-copy in the mouse and other mammals. The Cys1 nucleic encodes a novel, hydrophilic protein of 145 amino acids, termed cystin, that has no significant similarity to previously characterized proteins or protein domains. The polypeptide product of the Cys1 gene is detected in cilia and contains a sequence required for localization to the cilia.

Description

Autosomal Recessive Polycystic Kidney Disease Nucleic Acids and

Polyp eptides

This application claims the benefit of and priority to US Provisional application no. 60/448,168, filed February 18, 2003. This invention was made with government support under grant no. DK55534 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The instant disclosure relates generally to polycystic kidney disease. Specifically, the instant disclosure relates to the identification and characterization of nucleic acids and polypeptides associated with autosomal recessive polycystic kidney disease (ARPKD).

BACKGROUND

Polycystic kidney disease (PKD) represents a set of hereditary nephropathies characterized by progressive cyst formation, massive renal enlargement, and often, progression to end-stage renal disease. Autosomal dominant PKD (ADPKD) occurs in 1 in 1,000 individuals and in addition to renal cystic disease, is associated with cyst formation in other epithelial organs (most notably the liver) as well as connective tissue defects, such as intracranial aneurysms, aortic dissection, and cardiac valve defects (1). Mutations in either of two genes, PKD l and PKD 2, cause ADPKD phenotypes that are virtually indistinguishable (2). In comparison, autosomal recessive PKD (ARPKD) is much less frequent (1 in 20,000 live births). The clinical phenotype is dominated by renal collecting duct ectasia, biliary dysgenesis, and portal tract fϊbrosis (3). The principal disease locus, PKHD1 , has been mapped to chromosome 6p21.1-pl2 (4; 5). In the mouse, several distinct, recessively-acting mutations cause PKD phenotypes that mimic human disease (6). Among these models, the congenital polycystic kidney (cpk) mutation is the most extensively characterized. The cpk locus on chromosome (Chr) 12 is defined by a single recessive mutation that arose spontaneously in the C57BL/6J strain (7). The renal phenotype is fully expressed in homozygotes and strikingly similar to human ARPKD (8; 9), whereas genetic background modulates the penetrance of the corresponding defect in the developing biliary tree (10; 11). Multiple cellular and extracellular matrix abnormalities have been described in cpk/cpk kidneys. These changes include: i) enhanced expression of the proto-oncogenes, c-myc, c-fos, c- Ki-ras (12-14); ii) increased expression of the transcriptional repressor, Cux~l, a putative inhibitor of terminal differentiation (15); iii) enhanced growth factor expression (16); iv) apical mislocation of a functional EGF receptor (17); v) increased expression of basement membrane constituents, laminin βl and γl, αl/α2 chains of collagen IV, collagen I, and fibronectin (18; 19); vi) overexpression of the basement membrane remodeling enzymes, matrix metalloproteinases (MMPs) and their specific tissue inhibitors, TIMPs (20), vii) abnormal expression of epithelial cell adhesion molecules (21; 22); and viii) alterations in steroid metabolism and lipid composition (23-25). These numerous abnormalities involve a wide range of developmentally-regulated cellular processes and suggest that cpk/cpk mutant kidneys are unable to complete the terminal phases of tubulo-epithelial differentiation (26). ι

SUMMARY The present disclosure is directed to the identification, isolation, cloning, and characterization of a novel gene involved in ARPKD. The sequence of the mouse and human transcripts are disclosed, as well as the sequence of the polypeptide encoded thereby, termed "cystin". The mouse gene is termed Cysl (previously referred to as cpk), while the human gene is referred to as CYSl. In addition, a mutation in the mouse Cysl gene is disclosed, which leads to a premature truncation of the cystin polypeptide in mice homozygous for the mutation (referred to herein as cpk/cpk mice). The present disclosure relates to methods for the diagnosis and treatment of ARPKD.

One goal of the present disclosure is to provide therapeutic models and compositions for the amelioration of the symptoms of ARPKD. The mouse Cysl and human CYSl transcript may be useful in identifying compounds that modulate the expression of the wild-type gene products or the mutant gene product. Such compounds may be used in the treatment of ARPKD in humans and other subjects and/or serve as lead compounds for the development of additional therapeutic compositions.

A further goal of the present disclosure is to provide methods for the diagnosis and prognosis of ARPKD. In these methods the nucleic acids coding for the ARPKD nucleic acids and the polypeptides encoded thereby and/or reagents specific to the aforementioned are used to identify individuals carrying with ARPKD or who may be predisposed to developing ARPKD.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS, la-c illustrate the integrated genetic and physical map of the Cysl candidate interval. FIG. 1(a) illustrates the recombination analyses refining the Cysl locus to a ~100-kb interval, centered on D12MU12 and delimited by 221A10SP6 and Rrm2.

FIG. 1(b) illustrates the positioned Cysl on a single BAC, 221A10, within our YAC/BAC-based physical map. FIG. 1(c) shows a computational analysis identifying six putative transcriptional units within the

Cysl interval with each predicted gene corresponded, at least in part, to a mouse UniGene cluster.

FIGS. 2a-c show the organization of the Cysl genomic sequence and identification of the cpk mutation in the Cysl genomic sequence.

FIG. 2(a) illustrates that the alignment of the Cysl cDNA, the UniGene consensus sequences, and the BAC genomic sequence demonstrates that the Cysl gene is encoded in five exons spanning

14.4-kb of genomic DNA. The first nucleotide of the cDNA corresponds to the first nucleotide of exon 1, which spans 1184-bp and is the largest of the five exons. This exon contains an ATG start site that lies within a Kozak consensus sequence (CGCGCCαtgG). The 435-bp ORF extends into exon 3. Exons 4 and 5 are apparently untranslated and a putative cryptic splice site (gaacagCTG) within exon 5 appears to account for the 1856-bp and 1786-bp (hatched box) splice variants. An atypical polyadenylation signal (ATTAAA) lies 22-nt upstream of the poly(A ) tail. Of note, the microsatellite marker, D12MU12, lies within intron 1 of the Cysl gene.

FIG. 2(b) illustrates PCR amplification and direct sequence analysis identifying tandem 12-bp and 19-bp deletions in exon 1 of the Cysl gene. The comparative sequence is indicated in bold text. The resulting frameshift truncates the predicted protein. The position of the PCR primers is identified by arrows. The putative Kozak sequence is underlined and italicized.

FIG. 2(c) shows primers flanking the tandem deletion in the Cysl mutant allele amplify a 351bp product from wild-type (B6 and D2) DNA, a 320-bp product from 6-cpk/cpk DNA, and both bands from B6-+/cpk and Fl +lcpk heterozygotes. In the key F2 cpk/cpk recombinants (RI -R5), only the 320-bp mutant allele was amplified.

FIGS. 3a-e illustrate the expression pattern of the Cysl transcript and characterization of the protein product

FIG. 3(a) shows the tissue distribution of the 2.2-kb Cysl transcript in adult mouse tissues

(Clontech). The size of the polyadenylated transcript is consistent with the 1856-bp full-length cDNA. The size markers are indicated on the left and expressed as kb.

FIG. 3(b) shows a northern blot analysis of mouse fetal poly(A⁺) RNA revealing a 2.2-kb transcript in fetal kidney.

FIG. 3(c) shows a northern blot analysis of human fetal poly(A⁺) RNA (Clontech) revealing a

2.4-kb transcript in fetal kidney. FIG. 3(d) shows the relative expression of the 2.2-kb Cysl transcript as seen in the kidney and liver poly(A⁺) RNA from two-week old B6-+/+ (n=5 mice) and B6-cpk/cpk (n=5 mice) (top panel). Re-hybridization with Gapdh cDNA served as a loading control (middle panel). RT-PCR was performed using the deletion-flanking primer pairs on the same pofy^A^"1") RNAs (bottom panel).

FIG. 3(e) shows the amino-acid sequence of the predicted 145-AA protein product. Two potential myristoylation sites (residues 2-7; 43-48; indicated by asterisks) are predicted, the first of which is coupled to a polybasic domain (underlined). FIGS. 4a-g illustrate the localization of exogenously expressed cystin in stably transfected mCCD cells. To analyze cystin localization, wild type mouse cortical collecting duct cells (mCCD) cells were transfected with a myc and his epitope-tagged cystin construct. Stable cell cultures were established by selection in Blasticidin. Immunofluorescence analysis was conducted in cells grown on cell culture inserts for a minimum of three days post-confluence to allow cilial development. FIG. 4(a) is a schematic diagram of a principal cell with its primary apical cilium. The two focal planes used in immunofluorescence imaging are indicated. The apical focal plane was used to capture the cilium and the position of the tight junction, indicated by α-ZO-1 staining, and the nuclear focal plane identified the Hoechst-stained nuclei (blue). FIG. 4(b) shows immunofluorescence localization of cystin (green) as determined using the anti- his rabbit polyclonal antibody.

FIG. 4(c) shows localization of cystin (red) in the same mCCD cells as shown in FIG. 4(b) when probed with the anti-myc monoclonal antibody.

FIG. 4(d) shows the merged image of FIG. 4(b) and (c) demonstrating co-localization (yellow) of the myc and his epitope-tagged cystin. FIG. 4(e) shows a broad field view, stammg with the anti-his polyclonal antibody (green) indicating that cystin localized to the center of mCCD cells relative to the tight junctions stained for ZO-1 (red).

FIG. 4(f) and (g) show broad field and representative high magnification views demonstrating the co-localization of the exogenously expressed cystin (red, anti-myc monoclonal antibody) and endogenous polaris (green, rabbit polyclonal antibody) in cilia of mCCD cells.

DETAILED DESCRIPTION

As disclosed and discussed herein, the mouse Cysl is associated with ARPKD in a mouse model. The human homologue CYSl is also described and is thought to be associated with ARPKD. The Cysl and CYSl transcripts are referred to as ARPKD nucleic acids. The phrase "associated with ARPKD" when used with respect to a described gene, means that the gene, when mutated in both alleles, leads to an ARPKD phenotype. Such a mutation can lead to loss of production of the functional polypeptide encoded by the gene or a decrease in production of the functional polypeptide encoded by the gene. Furthermore, the mutation may lead to the expression of a functional polypeptide with different functional and/or structural properties as compared to the non-mutated (wild-type) functional polypeptide of the gene.

The mouse Cysl transcript is disclosed. The Cysl transcript comprises 5 exons. The isolated and purified nucleic acid molecule which encodes the full length Cysl transcript (1,856- bp) is given in SEQ ID NO. 1. The isolated and purified nucleic acid molecule which encodes the transcript of an alternative splice variant of the Cysl gene is given in SEQ ID NO. 2 (1,786- bp). The amino acid sequence of the polypeptide encoded by SEQ ID NOS. 1 and 2 is given in SEQ ID NO. 3 (termed cystin). The polypeptide encoded by SEQ ID NOS. 1 and 2 is identical, as the alternative splicing occurs in exon 5 of the Cysl transcript, which is not translated. A mutant of the Cysl gene, Cysl^c^ (also referred to herein as cpk), was isolated from cpk/cpk mice and was determined to have a tandem deletion of 12 and 19-bp in exon 1. The sequences of the 12 and 19-bp deletions are given in SEQ ID NOS. 4 and 5, respectively. The tandem deletion leads to a frameshift of the coding sequence and results in a prematurely truncated polypeptide. The nucleic acid sequence mutant Cysl transcript is given in SEQ ID NO. 6.

The human homologue of the Cysl gene, CYS1, is also disclosed. The CYS1 transcript contains 6 exons. Two alternatively spliced transcripts are predicted and the isolated and purified nucleic acid sequences corresponding to the predicted transcripts are given in SEQ ID NOS. 7 and 8. The transcript shown in SEQ ID NO. 7 is coded for by portions of exons 1, 2 and 6 while the transcript shown in SEQ ID NO. 8 is coded for by exons 3, 4, 5 and 6. The polypeptide encoded by SEQ ID NO. 7 is shown in SEQ ID NO. 9 and the polypeptides encoded by SEQ ID NO. 8 is shown in SEQ ID NO. 10.

The present disclosure is directed to nucleic acid molecules encoding the wild-type ARPKD nucleic acids (such as Cysl and CYS1 as shown in SEQ ID NOS. 1, 2, 7 and 8). In addition, the present disclosure is directed to the polypeptide product of these nucleic acids, collectively referred to as a cystin polypeptide (SEQ ID NOS. 3, 9 and 10). The present disclosure is also directed to a mutant Cysl gene (SEQ ID NO. 6) and the polypeptide product of the mutant gene Cysl^φA. Furthermore, the disclosure is also directed to functional derivatives of the ARPKD nucleic acids molecules described, and their corresponding polypeptide products. The term "functional derivatives" includes "fragments," "variants," "degenerate variants," "mutants," "analogs," and "chemical derivatives". The term "fragment" is meant to refer to any nucleic acid subset of an ARPKD nucleic acid. Such fragments may be of any length. In one embodiment, a fragment is at least 10 nucleotides in length (for example, 10, 20, 50, 75, 100, 150, 200, 250, 500, 1000, or more nucleotides in length). Other fragment lengths may be included as well. Isolated ARPKD nucleic acid fragments therefore are not required to contain all of the coding regions illustrated in SEQ ID NOS. 1, 2, 6, 7 and 8. An ARPKD nucleic acid may comprise one exon of the coding sequences, or a portion of one exon. Furthermore, in come embodiments, the ARPKD nucleic acid may undergo alternative splicing to remove a portion of an exon or one or more complete exons. The term "variant" is meant to refer to a molecule substantially similar in structure to an

ARPKD nucleic acid described above, or a fragment thereof. A molecule is "substantially similar" to an ARPKD nucleic acid in terms of structure if both molecules share at least 50% identity (for example, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical) with a corresponding portion of the wild-type ARPKD nucleic acids shown in SEQ ID NOS. 1, 2, 7 and 8 of the mutant ARPKD nucleic acid shown in SEQ ID NO. 6. Methods for determining percent homology or percent identity are discussed herein below.

It is known that there is a substantial amount of redundancy in the codons which code for specific amino acids. Therefore, this disclosure is directed to those nucleic acid sequences which contain alternative codons which do not change the predicted polypeptide product of an ARPKD nucleic acid, or fragments or variants thereof. For purposes of this specification, a sequence bearing one or more alternative codons will be defined as a "degenerate variant." Also included within the scope of this disclosure are alternations either in the nucleic acid sequence and the corresponding translated polypeptide which do not substantially alter the functional properties of a polypeptide encoded by an ARPKD nucleic acid, or a fragment or variant thereof (referred to as an "analog"). Such an analog may have alternations such as, for example, nucleic acid insertions, deletions, or substitutions that do not substantially affect the property of the translated polypeptide. For example, the analog can include conservative amino acid substitutions, such as substitutions which preserve the general charge, hydrophobicity/hydrophilicity, side chain moiety, and/or stearic bulk of the amino acid substituted, for example, Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Thr/Ser, and Phe/Trp/Tyr.

The term "chemical derivative" describes a nucleic acid or polypeptide that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington, The Science and Practice of Pharmacy, 20^l edition.

It is known vthat nucleic acid sequences coding for a polypeptide may be altered so as to code for a polypeptide having properties that are different than those of the naturally-occurring polypeptide (referred to as "mutants"). Mutant nucleic acid sequences and their corresponding amino acid sequences may be isolated from nature or produced in the laboratory. Methods of producing mutant nucleic acid sequences include, but are not limited to site directed mutagenesis. Mutants may include insertions, deletions, or substitutions that lead to missense mutations which alter the sequence of the encoded polypeptide or nonsense mutations which lead to premature truncation of the encoded polypeptide. Non-limiting examples of mutations include the 12 and 19 bp tandem deletion in Cysl that leads to premature truncation of the cystin polypeptide. Examples of altered properties include, but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand. Mutant may be derived from purification from either natural or recombinant sources, standard cloning procedures, from proteolysis of cloned or native molecules, or by induction within host cells, or by delivery from cells expressing cpk nucleic acid sequences.

The disclosure also directed to cystin polypeptides encoded by the ARPKD nucleic acids disclosed above and covered herein, or functional derivatives thereof. Therefore, the disclosure covers polypeptides coded for by fragments of the ARPKD nucleic acid disclosed in SEQ ID NOS. 1, 2, 6, 7 and 8. In addition, the disclosure covers polypeptides coded for by mutants of the cpk nucleic acid disclosed in SEQ ID NO. 6. Furthermore, the disclosure covers polypeptides coded for by variants, analogs, and degenerate variants of the cpk nucleic acid disclosed in SEQ ID NOS. 1, 2, 6, 7 and 8.

The term "nucleic acid" refers to RNA and DNA, including, but not limited to, cDNA, genomic DNA and synthetic DNA. The nucleic acid can be double stranded or single stranded

(that is a sense or an antisense single strand). The term "isolated" with respect to a molecule

^• requires that the molecule be removed from its original environment (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment. The term therefore covers, by way of example only, 1) a nucleic acid which has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both of the sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; 2) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring genomic DNA; 3) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by PCR, or a restriction fragment; and 4) a recombinant nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein).

Nucleic acid sequence coding for the expression of an ARPKD nucleic acid, or functional derivatives thereof, may be used to isolate and purify homologues of the Cysl gene from other organisms. To accomplish this, an ARPKD nucleic acid, or a functional derivative thereof, may be mixed with a sample containing nucleic acids encoding homologues of Cysl under appropriate hybridization conditions. In one embodiment, appropriate hybridization conditions are stringent hybridization conditions, such as hybridizing at 68° C in 5x SSC/5x Denhardt's solution/1.0 % SDS, and washing in 0.2x SSC/0.1% SDS at room temperature. The hybridized nucleic acid complex may be isolated and the nucleic acid encoding the homologous target may be purified there from. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar oligonucleotides. Only one member of the set will be identical to the ARPKD nucleic acid, and will be capable of hybridizing to the ARPKD nucleic acid, under stringent conditions, even in the presence of oligonucleotides with mismatches. Under alternate conditions (such as reduced stringency conditions, discussed below), the mismatched oligonucleotides may hybridize to the ARPKD nucleic acid to permit identification and isolation of cpk homologues.

Nucleic acid duplex or hybrid stability is expressed as the melting temperature, Tm, which is the temperature at which the hybridizing nucleic acid disassociates with the target nucleic acid. This melting temperature is many times used to define the required stringency conditions. If sequences are to be identified that are not exact matches to an ARPKD nucleic acid, then it is useful to establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (such as SSC or SSPE). Assuming that 1% mismatch results in a 1° C decrease in Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if a sequence having a 95% identity with ARPKD nucleic acid is sought, then the final wash temperature is decreased by 5° C. The change in Tm can be between 0.5° C and 1.5° C per 1% mismatch. Stringent conditions involve hybridizing at 68° C in 5x SSC/5x Denhardt's solution/1.0 % SDS, and washing in 0.2x SSC/0.1% SDS at room temperature. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art. The disclosure is also directed to nucleic acids that hybridize under stringent conditions (as defined herein) to at least a portion an ARPKD nucleic acid, functional derivatives thereof, or their complement. The hybridizing portion of the hybridizing nucleic acid is generally 10-50 or 15-30 nucleotides in length. The hybridizing portion of the hybridizing nucleic acid may be at least 50% to 100% identical to the sequence of at least a portion of an ARPKD nucleic acid, functional derivatives thereof or their complement. Hybridizing nucleic acids as described herein can be used for many purposes, such as, but not limited to, a cloning probe, a primer for PCR and other reactions, and a diagnostic probe. Hybridization of the hybridizing nucleic acid is typically performed under stringent conditions (as defined above). The terms "percentage of sequence identity" and "percentage homology" are used interchangeably in this disclosure to refer to comparisons among oligonucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the oligonucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Identity or similarity is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. In one embodiment, protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool ("BLAST") (e.g., Karlin and Altschul, (1990), PNAS 87:2267-2268; Altschul et al., (1997), Nuc. Acids Res. 25:3389- 3402). In particular, five specific BLAST programs may be used as described herein: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) BLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) BLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al.

(Nucleic Acids Res. 25:3389-3402, 1997). The BLAST programs may be used with the default

, parameters or with modified parameters provided by the user. Another preferred method for determining the best overall match between a query nucleotide or amino acid sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. The ARPKD nucleic acid, or functional derivatives thereof, or their complements may be recornbinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements (referred to as an "expression control sequence) and transferred into prokaryotic or eukaryotic host cells to produce recombinant molecules. Techniques for such manipulations are within the ordinary skill of one in the art, and representative techniques can be found described in Sambrook, J., et al., Molecular Cloning Second Edition, 1990, Cold Spring Harbor Press. Expression vectors are defined herein as the nucleic acid sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells, fungal cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast, or bacteria-animal cells, or bacteria-fungal cells, or bacteria- invertebrate cells. An appropriately constructed expression vector should contain, at the minimum: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

A variety of expression vectors may be used, including, but not limited to, mammalian expression vectors, bacterial expression vectors and insect expression vectors. The expression vectors may be obtained from various commercial suppliers or produced according to specific needs. The choice of the appropriate expression vector is within the ordinary skill of one in the art. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including' but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including, but not limited to, Drosophila and silkworm derived cell lines. A variety of cell lines derived from mammalian species which may be suitable for use as host cells are commercially available. The choice of host cells is within the ordinary skill of one in the art.

The expression vectors may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce the compound of interest. Identification of cells expressing the polypeptides encoded by the ARPKD nucleic acid, or a functional derivative thereof, can be accomplished by a variety of means, including but not limited to, immunological reactivity, or the presence of host cell-associated ARPKD gene activity. Expression of polypeptides coded by an ARPKD nucleic acid, or functional derivatives thereof, may also be performed using in vitro produced synthetic n RNA or isolated native mRNA. Synthetic mRNA or mRNA isolated from cells expressing an ARPKD nucleic acid can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems. Following expression of the desired polypeptides in a recombinant host cell, it may be recovered to provide purified polypeptide. Purification methods for isolated expressed protein are well known in the art .and within the ordinary skill in the art. Techniques include salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, hydrophobic interaction chromatography, immunoaffinity chromatography and affinity chromatography.

Antibodies to desired polypeptides encoded by the ARPKD nucleic acids can be produced and used in the methods described herein. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of polypeptides coded for by an ARPKD nucleic acid, or a functional derivative thereof, in a biological sample, or, alternatively, as a method for the inhibition of abnormal polypeptide activity. Thus, such antibodies may be utilized as part of ARPKD treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for the presence of polypeptides encoded by an ARPKD nucleic acid, or a functional derivative thereof.

Mono-specific antibodies to desired cystin polypeptides can be purified from mammalian antisera containing antibodies reactive against said polypeptides, or are prepared as monoclonal antibodies reactive with said polypeptides. Mono-specific antibody is defined as a single antibody species, or multiple antibody species with substantially similar binding characteristics for desired cystin polypeptides, and includes polyclonal antibodies. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope. Specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of desired cystin polypeptides, either with or without an immune adjuvant. Preimmune serum is collected prior to the first immunization to establish baseline immunoreactivity. Each animal receives between about 0.1 mg and about 1000 mg of polypeptide, either with or without an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacteήum parvum and tRNA. The initial immunization consists of desired cystin polypeptides, preferably, Freund's complete adjuvant, at multiple sites either subcutaneously (SC), intraperitoneally (IP), or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the initial antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20 degree C. Monoclonal antibodies (mAb) reactive with the desired cystin polypeptides are prepared by immunizing inbred mice, preferably Balb/c, with the appropriate antigen. The mice are immunized by the IP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of antigen in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of antigen in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay using the appropriate cystin polypeptide (depending on which antigen was used for the injections), as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive cells may be cloned by techniques well know in the art.

A variety of methods may be employed for the detection, diagnosis and prognosis in a subject. The methods may utilize reagents such as, but not limited to, an ARPKD nucleic acid sequence described herein, or functional derivatives thereof, polypeptides encoded for by an ARPKD nucleic acid, or functional derivatives thereof, and antibodies directed against desired polypeptides encoded for by an ARPKD nucleic acid, or functional derivatives thereof. Specifically, such reagents may be used for the detection of the presence of mutations in an ARPKD nucleic acid, i.e., molecules present in diseased tissue but absent from, or present in greatly reduced levels relative to, the corresponding non-diseased tissue.

The method for diagnosis and/or prognosis may involve detection of an ARPKD nucleic acid in a sample. The sample is taken from an individual in need of such diagnostic or prognostic test. RNA from the tissue to be analyzed may be isolated using procedures which are well known to those in the art. Diagnostic/prognostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of tissue obtained from biopsies or resections, such that no RNA purification is necessary. The ARPKD nucleic acid sequences, or functional derivatives thereof, may be used for such procedures.

The ARPKD nucleic acid sequences, or functional derivatives thereof, may be used in hybridization or amplification assays of biological samples to detect abnormalities of ARPKD gene expression; e.g., Southern or Northern analysis, single stranded conformational polymorphism (SSCP) analysis including in situ hybridization assays, alternatively, PCR analyses. Such analyses may reveal both quantitative abnormalities in the expression pattern of ARPKD nucleic acid in the subject, or other more qualitative aspects of an ARPKD nucleic acid abnormality in the subject. In one embodiment, the nucleic acid sequences disclosed in SEQ ID NOS. 19 and 20 may be used as primers in a PCR based reaction to determine the presence of a mutant allele. In one embodiment, a diagnostic methods for the detection of ARPKD nucleic acid, or functional derivatives thereof, may involve, for example, contacting and incubating nucleic acids derived from the target tissue (target molecules) being analyzed, with one or more labeled ARPKD nucleic acids, or functional derivatives thereof, as are described herein (detecting molecules), under conditions favorable for the specific annealing of these detecting molecules to their complementary sequences within the target molecule. The lengths of detecting molecules may be at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed. The presence of target molecules which have hybridized, if any, is then detected. The target molecules may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, detecting molecules are easily removed. Detection of the remaining, annealed, labeled detecting molecules is accomplished using standard techniques well-known to those in the art.

Alternative diagnostic methods for the detection of ARPKD nucleic acids, or functional derivatives thereof, may involve their amplification, e.g., by PCR, ligase chain reaction, self sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, or any other RNA amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of RNA molecules if such molecules are present in very low numbers. In one embodiment of such an alternate detection scheme, a cDNA molecule is obtained from the target RNA molecule (e.g., by reverse transcription of the RNA molecule into cDNA). Tissues from which such RNA may be isolated include any tissue in which an ARPKD nucleic acid is known or thought to be expressed, including, but not limited, to kidney tissue, liver tissue, brain tissue and tissues of the eye. A target sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from ARPKD nucleic acid sequences, or functional derivatives thereof, as described. The lengths of such nucleic acid reagents may be 15-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing, any other suitable nucleic acid staining method.

The method for diagnosis and/or prognosis may involve detection of polypeptides encoded by an ARPKD nucleic acid, or functional derivatives thereof. Antibodies directed against such polypeptides may be used as diagnostics/prognostic reagents. Such diagnostic/prognostic methods, may be used to detect abnormalities in the level of protein expression, or abnormalities in the tissue localization and/or trafficking, or subcellular location of protein. For example, in addition, differences in the size, electronegativity, or antigenicity of the mutant polypeptides relative to the wild-type polypeptides may also be detected.

Protein from the tissue to be analyzed may easily be isolated using techniques which are well known to those of skill in the art. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. Methods for the detection of polypeptides may involve, for example, immunoassays wherein protein and/or peptides of interest are detected by their interaction with an antibody specific for the desired polypeptide.

For example, antibodies, or fragments of antibodies, such as are known in the art and described herein, useful in the present invention may be used to quantitatively or qualitatively detect the presence of polypeptides encoded by an ARPKD nucleic acid described herein. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if wild type or mutant polypeptides, or functional derivatives thereof, are expressed on the cell surface.

The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection. In situ detection may be accomplished by removing a histological specimen from a subject, and applying thereto a labeled antibody of the present invention. The histological sample may be taken from a tissue suspected of exhibiting ARPKD. The antibody (or fragment) may be applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of cystin polypeptides, but also their distribution in the examined tissue. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays for polypeptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying the polypeptide and detecting the bound antibody by any of a number of techniques well-known in the art. The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means. By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of anti-cystin polypeptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

One of the ways in which antibody specific cystin polypeptide can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Ishikawa, E. et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate de ydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments it is possible to detect cystin polypeptides through the use of a radioimmunoassay (RIA). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. Nucleotide sequences complementary to an ARPKD nucleic acid, or functional derivatives thereof, can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-0-alkylRNA, or other antisense oligonucleotide mimetics. These antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. Antisense therapy may be particularly useful for the treatment of ARPKD or diseases related thereto.

Gene therapy may be used to introduce an ARPKD nucleic acid, or a functional derivative thereof, either alone or in combination with a modulating compound, into the cells of target organs. The gene coding for the appropriate protein can be ligated into viral vectors which mediate transfer of the DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. Alternatively, DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus- ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo, as well as in vivo gene therapy. Gene therapy may be particularly useful for the treatment of ARPKD or diseases related thereto.

Pharmaceutically useful compositions comprising an ARPKD nucleic acid, or functional derivatives thereof, and their complement, or polypeptides encoded by an ARPKD nucleic acid described herein, either alone or in combination with modulating compounds, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington The Science and Practice of Pharmacy, 20^{ edition. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the nucleic acid or polypeptide. Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat and/or diagnose disorders related to the expression of an ARPKD nucleic acid, such as, but not limited to, ARPKD. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal activity, while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable. The therapeutic or diagnostic agents discussed herein may be used with or without chemical derivatives. Examples of therapeutic and diagnostic reagents and methods of their formulation and administration are described in a variety of texts, such as Remington The Science and Practice of Pharmacy, 20^th edition.

EXAMPLES Example 1- Refinement of the Cysl candidate interval and generation of a transcript map

An integrated genetic and physical map of the 650-kb region containing the Cysl locus was constructed (27). Recombinational mapping has allowed the refinement of the Cysl locus to a ~100-kb interval, centered on D12MU12 and bounded by the markers, 221A10-SP6 and Rrm2 (Fig. la). This interval is contained within a single BAC clone, 221A10 (Fig. lb). The accession number for the genomic sequence of the BAC clone 221 AlO can be found in GenBank under the accession number AF390548, the sequence of which is incorporated by reference herein.

Computational analyses of the BAC sequence, obtained using a random shotgun method, identified six putative transcriptional units, which corresponded to seven mouse UniGene clusters (Fig. lc). Mouse Rrm2 was previously excluded as a Cysl candidate gene. Comparative sequence analysis revealed no base pair variations in the mouse orthologues of LBP-32 and TIEG2 amplified from wild-type and cpk/cpk kidney cDNA. RT-PCR indicated that the mRNAs corresponding to UniGene Mm.37574 were not expressed in kidney or liver. While similar analyses with Mm.42573 demonstrated expression in kidney and liver, no transcripts were identified by northern blotting.

Example 2- Identification of the Cysl transcript and characterization of the genomic sequence

Using RT-PCR, 5' RACE-PCR of a mouse kidney adaptor-ligated cDNA library, and direct sequence analysis, it was determined that the UniGene clusters, Mm.34424 and Mm.52265, represent the 5' and 3' ends, respectively, of a single transcript (59). A 1,856-bp full length Cysl cDNA was complied from UniGene clusters, Mm.34424 and Mm.52265 (SEQ ID NO 1.). In addition, a 1,786-bp Cysl splice variant was identified in which 70 nucleotides corresponding to residues 1422-1492 of SEQ ID NO. 1 were deleted (SEQ ID NO.2). An ATG codon situated 905- nt downstream from the first nucleotide of SEQ ID NOS. 1 and 2 lies within a Kozak consensus sequence (35), followed by an predicted ORF of 435-bp and either a 513-bp (SEQ ID NO. 1) or 443-bp (SEQ ID NO. 2) 3'UTR that contains an atypical polyadenylation signal (ATTAAA) 22- nt upstream of the poly(A ) tail.

Alignment of the candidate Cysl cDNA and BAC genomic DNA sequences demonstrated that the gene is encoded in five exons spanning 14.4-kb of genomic DNA (Fig. 2a). The exon- intron boundaries for these five exons (as shown in Table 1) conform to the consensus sequences for 5' and 3' splice-sites (36) and are listed as SEQ ID NOS. 11-18. Analysis of the genomic sequence 3000-bp upstream of the ATG codon, using the TSSG and TSSW programs, did not identify a robust transcription start site. Exon 1 contains the ATG start site and has 100% homology with all 18 ESTs contained in UniGene Mm.34424 as well as 15 of the 38 ESTs that belong to Mm.52265. The ORF extends into exon 3, whereas exons 4 and 5 are apparently untranslated. A putative cryptic splice site within exon 5 accounts for the 1856-bp (SEQ ID NO. 1) and 1786-bp (SEQ ID NO. 2) splice variant.

Table 1 cpk Intron-exDπ boundaries

Exon Exon size (bp) 3' splice sice 5' spike site 1 1 1 84 C C CG CGgegagtatgg

2 53 cctgctcaagGTGTCC CTCTGAgtaagtatgg

3 1 3 tcatccacagAGCCCC TTGCAGgtctgrgaac

4 61 cat tttccagCiTCC GG ACCAAGgtatgttetc

5 415 gtcaaaacαgGTCTCG

Example 3- Mutation analysis of the Cysl gene Comparative sequence analysis of the Cysl cDNA amplified from each of eight, two- week old B6-+/+ and B6-cpk/cpk mice revealed a tandem deletion of 12-bp (992delTCGGAGGGCGGC) (SEQ ID NO. 4) and 19-bp

(1017delGCCGCGGGGCAGGAGGAGA) (SEQ ID NO. 5) in the mutant cDNA resulting in a frameshift within exon 1 that truncates the protein (Fig. 2b). The occurrence of this tandem deletion on the B6 background provides strong supportive evidence that this candidate cDNA is indeed associated with the ARPKD phenotype. The nucleic acid encoding the mutant Cysl nucleic acid is given in SEQ ID NO. 6. The amino acid sequence coded for by SEQ ID NO. 6 can be easily deduced.

The exon 1 primer set (primers C-3F and C-3R, shown in SEQ ID NOS. 23 and 24, respectively) amplified a 351-bp product from B6-+/+ genomic DNA, whereas a 320-bp product was amplified from B6-cpk/cpk genomic DNA (Fig. 2c). Both products were amplified from DNAs of B6-+/cpk and (D2 X B6-+/cplc)¥l heterozygotes, but only the mutant 320-bp allele was amplified from all five key F2 cpk/cpk recombinants. Analysis of the sequence flanking the tandem deletion failed to identify homologous sequences at the breakpoints. Therefore, the mutation event in the Cysl gene may involve a non- homologous recombination mechanism (37).

Example 4- Expression of the Cysl transcript and characterization of the protein product

The cpk phenotype primarily involves renal cysts and biliary tract dysgenesis. In adult mice, a 2.2-kb transcript was detected that was strongly expressed in kidney (Fig. 3a). A similar 2.2-kb transcript was also detected in liver, with lower expression evident in lung, brain, and heart (Fig. 3a). The expression of Cysl in mouse and human fetal tissues was also examined. Northern blot analysis revealed that the 2.2-kb transcript was strongly expressed in mouse fetal kidney, but barely perceptible in fetal brain, lung, and liver (Fig 3b). Similarly, a somewhat larger transcript of 2.4-kb was expressed predominantly in human fetal kidney (Fig 3c).

The 2.2-kb Cysl transcript was detected in kidney and liver from both two-week old B6- +/+ and B6-cpk/cpk mice, but the band intensities were relatively diminished in tissues from mutant mice (Fig 3d). These data indicate that both the wild-type and the mutant Cysl transcripts are more abundantly expressed in the kidney than in the liver and the truncating mutation reduces transcript expression in both tissues. Loading controls and RT-PCR analysis confirmed the specificity of these northern blotting results and demonstrated the presence of small amounts of Cysl transcript in mutant liver (Fig 3d).

Example 5- Characterization of the protein product of the Cysl gene

In the present disclosure, the Cysl nucleic acid which encodes for the cystin polypeptide is described (SEQ ID NOS. 1, 2 and 6). The amino acid sequence of the cystin polypeptide is shown in FIG 3(e) and given in SEQ ID NO. 3. Secondary structural predictions for the cystin polypeptide failed to identify significant alpha helical or beta sheet structures within the protein. Motif searches have identified two myristoylation sites (AA 2-7 and AA 43-48), the first of which is coupled to a polybasic domain (AA 12-16). A potential PEST (polypeptide enriched in proline (P), glutamic acid (E) serine (S) and threonine (T)) sequence is predicted at position 101-118 (PEST score = 6.98). There is no evidence for specific DNA-binding motifs, but the protein is proline-rich (10%) and is predicted to have at least two potential nuclear localization signals (PRRRRSP at AA 12-18 and PEKKVKG at AA 96-104).

Recent in vitro studies have demonstrated that N-terminal myristoylation acts as an intracellular membrane-associating signal. However, stable anchoring of N-myristoylated proteins to membranes requires, among other factors, linkage to a second signal, such as a series of positively-charged residues adjacent or distal to the protein lipidation site (40). This combined N-myristoylation site/polybasic residue motif is used by proteins such as c-Src, K-Ras, and myristolated alanine rich C-kinase substrate (MARCKS) proteins for membrane anchoring (41- 43). This suggests that cystin may be associated with the plasma membrane and/or membranes of intracellular organelles.

Southern analyses indicate that Cysl homologues exist as single-copy genes in mammals including human, monkey, rat, dog, and cow, as well as in chicken. However, no significant homology to previously characterized proteins or protein domains was revealed in searches of human, Caenorhabditis elegans, Drosophila, or yeast databases. Of note, the genomic interval flanked by TIEG2 and RRM2 that includes the human Cysl homologue, CYS1, is not contained in the draft sequence currently available from the International Human Genome Sequence Consortium (http://www.ncbi.nlm.nih.gov/genome/seq/HsBlast.htinl^').

Northern analysis in human fetal tissues identified a 2.4-kb transcript in kidney but not liver, brain, or lung (Fig. 3c). A human fetal library-derived EST, AA446394, has 85% identity with Cysl over 88 nucleotides and 71% amino acid identity with the carboxy terminus of the Cystin protein. The present disclosure also reveals the sequence of the human CYS1 transcript.

Example 6- Transfection studies and subcellular localization

Subcellular localization of the cystin polypeptide was analyzed in mouse cortical collecting duct (mCCD) cells transfected with an expression construct containing SEQ ID NO. 1 cloned in-frame with carboxyl-terminal c-myc (myc) and histidine (his) tags, under the control of the EF-lα promoter. Stable cell cultures were established by selection in Blasticidin. The cells were then grown on cell culture inserts for a minimum of three days post-confluence to allow cilial development. These polarized cells are not a clonal cell line, but rather represent a cultured cell population that have been subjected to transfection with SEQ ID NO. 1. Because the transfection efficiency in such experiments is typically < 100%, some cells in this population did not express the transgene.

Immunofluorescence analysis revealed that the epitope-tagged cystin polypeptide localized to a small domain on the apical cell surface (Fig. 4b-d) consistent with localization in the apical cilia. In a broad field view, cystin was positioned in the center of this apical domain when compared with tight junction staining by α-ZO-1 (38) (Fig. 4e). The distribution of the cystin polypeptide overlapped with β-tubulin TV, a known component of the single apical cilium (39) as well as polaris (Fig. 4f-g), a protein that is encoded by the Tg737 gene and associated with motile and immotile cilia (34). However, unlike polaris, which is expressed primarily in the ciliary basal bodies as well as in the axoneme, the cystin polypeptide appeared to be enriched in the ciliary axonemes, with minor staining in the basal bodies (Fig. 4g). The immunofluorescence images are representative of n = 5 experiments.

The mutant Cysl gene codes for a protein having an amino acid sequence of which the first 27 amino acids are identical to the wild-type cystin polypeptide. However, residues 28 through the remainder of the mutant cystin polypeptide share no homology with the wild-type cystin polypeptide due to a shift in the reading frame, which leads to premature termination of the mutant cystin polypeptide. The mutant cystin polypeptide retains one myristoylation sites (AA 2- 7) and the polybasic domain (AA 12-16). Therefore, the mutant cystin polypeptide might also be expected to be associated with the plasma membrane and/or membranes of intracellular organelles. Mouse cortical collecting duct (mCCD) cells transfected with an expression construct containing SEQ ID NO. 6 cloned in-frame with carboxyl-terminal c-myc (myc) and histidine (his) tags, under the control of the EF-lα promoter as described. Experiments have shown that the mutant cystin polypeptide is not associated with the apical cilia as is the wild-type cystin polypeptide, but is instead associated with the apical and basolateral membranes of the mCCD cells. This suggests that the myristoylation site and the polybasic domain site are sufficient for membrane association, but not sufficient for localization to the apical cilia. Therefore, the wild- type cystin polypeptide contains a cilia localization signal between amino acids 27 and 145 of SEQ ID NO. 3, which is absent in the mutated cystin polypeptide.

This subtle difference in protein distributions may indicate that the cystin polypeptide is not involved in a ciliogenic pathway like polaris, but rather plays a novel role in ciliary function. Consistent with this hypothesis, the cilia in cpk/cpk mice do not exhibit gross structural abnormalities, suggesting that the cystin polypeptide plays a role in ciliary function and not structure. While not being limited to other explanation, the cystin polypeptide may be bound to the axonemal membrane and functions as part of a molecular scaffold that stabilizes microtubule assembly within the ciliary axoneme. In support of this speculation, we note that Woo et al. have demonstrated a significant attenuation in renal cystic disease progression in cpk/cpk mice treated with paclitaxel (Taxol) and related taxanes, which promote microtubule assembly (44). In comparison, paclitaxel treatment was ineffective in Tg737°^rp homozygotes (45), consistent with the model that polaris and cystin play distinct, perhaps sequential roles in ciliary formation and function.

There is a growing body of evidence that links ciliary dysfunction, embryonic left-right (LR) patterning defects, and cystic disease of visceral organs, e.g. kidney, liver, and pancreas. Polaris is among several proteins that appear to function in ciliogenesis and LR body axis determination (46). Cilia formation is initiated in basal bodies and involves a process called mtraflagellar transport in which ciliogenic proteins assemble into complexes that migrate up the ciliary axoneme (47). Targeted deletions of these genes in mice, including Tg737, typically cause loss of embryonic nodal cilia, disruption of LR axis specification, and embryonic lethality (34; 48). In contrast, homozygosity for the Tg737°^rp hypomorphic allele causes an ARPKD-like phenotype, with renal, biliary and pancreatic cysts (49). There are no associated defects in LR specification, although Tg737°^rpk homozygotes have attenuation of the renal epithelial cilia (50) and loss of cilia in the ventricular ependymal cell layer (34). The inversion of embryo turning (inv) gene encodes a novel, ankyrin-repeat containing protein that is ubiquitously expressed in early mouse embryos (48). Homozygous mutants have both reversal of LR visceral asymmetries, as well as severely dilated renal collecting ducts, pancreatic abnormalities, including dilatation of the acinar ducts, and anomalies of the extrahepatic biliary system (48; 51). The nodal cilia in inv mutants are present and motile, but can produce only very weak leftward nodal flow (52). The monocilia in renal, biliary, and pancreatic ductal cells have not been well examined in these mutants and their functionality remains uncharacterized.

LR patterning defects have not been identified in cpk mutants and recent ultrastructural studies of renal and biliary cysts have not demonstrated defects in ciliary structure (11). The collecting duct cysts appear to be lined by a single layer of principal-like cells that have a single apical cilium and short microvilli. Similarly, the intrahepatic biliary duct cysts are lined by epithelial cells with numerous microvilli and a single apical cilium. These data are permissive for the hypothesis that cystin may be important in cilia function, but the protein is probably is not involved in ciliogenesis, a process critical in embryo patterning. Cystin homologues appear to be present only in chordates, and not in primitive ciliated eukaryotes such as C. elegans. These data suggest that cystin has a later phylogenetic origin than proteins associated with mtraflagellar transport, such as polaris, and that this novel protein may have a very specialized function in ciliated epithelia of higher vertebrates. Single non-motile cilia are expressed on the epithelia lining much of the nephron, the biliary tract, and the pancreatic ducts (53). These cilia are less well-characterized than the motile cilium present on embryonic node cells. However, recent data suggest that the apical cilium on renal epithelial cells has a mechanosensory function (54). While cilia are expressed on a broad spectrum of mammalian cell-types, in the nematode worm, Caenorhabditis elegans, cilia are present only in the specialized neurons where they function as sensory organelles. Interestingly, the nematode homologues of PKDl and PKD2 (lov-1 and pkd-2) (55), and Tg737 (osm-5) (56; 57) are all expressed in the same subset of ciliated sensory neurons, suggesting that PKD-related proteins may function in common chemosensory or mechanosensory pathways in these cilia.

Extrapolating from these data, Barr et al. (57) have proposed that cilia are required for the function of vertebrate polycystins and that any gene mutation that causes disruption of cilia assembly or function would result in PKD. Given this model, the PKD phenotype in Tg737^orpk homozygous mice may be attributed to failure to properly target polycystins due to aberrant cilia structure, whereas a similar phenotype in cpk/cpk mice may result from ciliary dysfunction.

Finally, a number of studies have strongly implicated a role for cystin in tubulo-epithelial differentiation within the embryonic kidney, biliary tract, and pancreas (10; 11; 58). In preliminary studies, applicants have observed structural changes in basement membranes surrounding early dilating renal tubules in fetal cpk/cpk mice. Therefore, further analysis of cystin should permit a more thorough investigation of the molecular interactions between cilia function, differentiation and maintenance of the polarized epithelial phenotype, and macromolecular organization of the basement membrane, all processes that are critical for normal organogenesis as well as cystogenesis.

Example 7- Identification of the CYSl transcript and characterization of the genomic sequence The human CYSl homologue was identified within the AC 104794.3 genomic sequence. The human CYSl genomic sequence extends over 23.7 kb and is organized as at least 6 different exons. The CYSl genomic sequence is shown in SEQ ID NO. 31. The CYSl gene is predicted to encode at least two alternatively spliced transcripts (shown in SEQ ID NOS. 7 and 8). Transcript 1 (SEQ ID NO. 7) is -2.8 kb in length, comprises 3,734 bp and contains exons 1, 2, and 6. SEQ ID NO. 7 shares 71.1% identity with mouse Cysl nucleic acid (SEQ ID NO. 1). The predicted polypeptide product of SEQ ID NO. 7 contains 158 amino acids and shares 57.6% sequence identity with mouse cystin (SEQ ID NO. 3). Transcript 2 is ~2.4 kb in length, comprises 3,226 bp and contains exons 3, 4, 5, and 6. SEQ ID NO. 8 share no significant sequence homology at either nucleotide or amino acid level when compared to the Cysl nucleic acid (SEQ ID NO. 1) or the cystin polypeptide (SEQ ID NO. 3, respectively. mRNA coding for transcript 1 (SEQ ID NO. 7) has been isolated from human embryonic kidney cDNA and the predicted nucleic acid sequence was confirmed. Furthermore, a ~2.8-kb band was identified in kidney, placenta, and other tissues. Probes specific for SEQ ID NO. 7 appear to recognize more than one transcript and the 2.8-kb band is rather broad, suggesting several similarly-sized transcripts or splice variants. In addition, there is a minor band and at ~6- kb. A mRNA corresponding to SEQ ID NO. 8 has not been identified to date, suggesting the transcript may be tightly regulated in a temporal, spatial or developmental manner.

The applicants maintain an extensive DNA repository for ARPKD patients/families. This repository contains a subset of families (n=5) with no evidence for PKHD1 mutations. The CYSl gene was sequenced in affected child-parent trios. No mutations were detected. Our data extend the recent observations of Omran et al. who excluded CYS 1 mutations in a set of families with Boichis disease, an early-onset form of nephronophthisis. The CYSl nucleic acids, polypeptide products and reagents developed therefrom will allow more complete analysis of CYSl as a candidate gene in subjects with renal cystic disease and congenital hepatic fibrosis not linked to known human PKD genes.

METHODS Genotyping

Genotyping of informative meioses from our previous crosses (27) and 150 test meioses generated from a [(C57BL/6J-+/cp&) X DBA/2J ]F1 intercross was performed. The BAC end- erived markers from the Cysl critical interval, 221A10-SP6, 182L8-T7, 06H17-SP6, 49G13-T7, and 319111-T7, and the Rr 2-derived polymorphic marker have been described (27). Single- strand conformation polymorphisms were amplified in the presence of [α-³²P] dCTP, electrophoresed on non-denaturing MDE gels (AT Biochem, Malvern, PA), and detected by autoradiography. One recombinant was identified between Cysl and the proximal flanking marker, 221A10SP6, and four recombinants were detected between Cysl and the distal flanking marker, Rrm2.

Large-scale sequencing of BAC 221A10

DNA from the mouse BAC clone, 221 AlO, from the RPCI-22 Mouse BAC library (Research Genetics, Huntsville, AL) was nebulized and size-fractionated on a 0.8%) agarose gel. Fragments 2-3 kb and 3-4 kb in size, were subcloned into pUC 18, were shotgun-sequenced to produce 3 -fold coverage of the BAC clone. Library DNA preparation and sequencing on an AB1377 sequencer were performed by the Australian Genome Research Facility (Brisbane, Queensland). Assembly and analysis of genomic sequence data was performed using PHRED, PHRAP and GAP4 software (28-30).

The final sequence was analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST); REPEATMASKER (http://ftp.genome.washington.edu/cgi-bin/ReueatMasker): GENSCAN (http://CCR-081.mit.edu/GENSCAN.html); and GrailPRO

(http://www.genomix.com/grailpro.htin). The complete sequence of BAC 221 AlO and the 1.86- kb cpk cDNA sequence have been submitted to GenBank (Accession numbers AF390547 for BAC 221A10 and AF390548 for the 1,856-bp mRNA).

cDNA characterization and sequence analysis

Total RNA was extracted from kidneys and livers of two-week old B6-+/+ and B6- cpk/cpk mice as well as from fetal brain, lung, liver, and kidney of B6 mice at embryonic day 15 (El 5) using the RNAgents Total RNA Isolation System (Promega). Poly(A⁺) RNA was prepared using the Oligotex mRNA Midi kit (Qiagen).

A predicted Cysl cDNA sequence was assembled using the sequences in UniGenes

Mm.34424 and Mm.52265 as a scaffold. Primary and nested PCRs were performed using the following primers (oriented 5' to 3' in the cDNA):

C-1F: 5'-CATCTCCGGCTCTCCTTTTCTGT-3' (SEQ ID NO. 19) ;

C-1R: 5'-AGAGTAAGCGGGATGAAGAGAGG-3' (SEQ ID NO. 20);

C-2F:5'-AGATGATTCTTTCGCCCTGACTTC-3' (SEQ ID NO. 21);

C-2R: 5'-AGGGGGATTCTGGAGGAGTGAG-3' (SEQ ID NO. 22); C-3F: 5'-TCCTCCCTCCCTATCTCTCCAT-3' (SEQ ID NO. 23);

C-3R: 5'-ATCCAGCAGGCGTAGGGTCTC-3' (SEQ ID NO. 24);

C-4F: 5'-AGACCCTACGCCTGCTGGATCA-3' (SEQ ID NO. 25);

C-4R: 5'-TTGTCCAGCTCAGCGGCAGTA-3' (SEQ ID NO. 26);

C-5F: 5'-AACAGCCCCAAGAGACCCGAG-3' (SEQ ID NO. 26); and C-5R: 5'-GTTGCTAGCTCTGGGAGGTTTT-3' (SEQ ID NO. 28).

To obtain the 5' end of the cDNA sequence, we amplified cDNA from mouse kidney by 5'

RACE-PCR using the Marathon cDNA Amplification kit (Clontech).

Nucleotide comparison of the Cysl cDNA sequence with known genes and ESTs listed in the non-redundant compilation of the GenBank and EMBL databases were performed using BLAST searches (31). We also performed amino-acid comparisons with the non-redundant

Swiss-Prot databases using the BLASTP program. Protein-domain homologies and motifs were analyzed with the PredictProtein package (http://cubic.bioc.columbia.edu/predictprotein .

Reverse transcription -PCR (RT-PCR) and northern blot analysis Reverse transcription was carried out in a 40 μl reaction volume with 10 μg of total kidney RNA using the BRL Superscript RNAse H-Reverse Transcriptase kit (Gibco BRL, Gaithersburg, MD). Aliquots of 100 ng of cDNA were amplified in PCR reactions using various cDNA primer combinations under standard PCR conditions (30 cycles of 96 °C for 30 s, 54-64 °C for 30 s, 72 °C for 30 s). Northern blots prepared with either 3 μg of kidney and liver poly(A⁺) RNA pooled from

B6-+/+ and B6-cpk/cpk mice (n=5) or 3 μg of brain, lung, liver, and kidney poly(A⁺) RNA pooled from El 5 mice (n=12) were probed with a 351-bp probe that encompasses the tandem deletion (primers C-3F, SEQ ID NO. 23 and C-3R, SEQ ID NO. 24). This 351-bp probe also hybridized to a Mouse Multiple Tissue Northern blot (Clontech 7762-1) as well as a Human Fetal Northern blot (Clontech 7756-1). To evaluate differential RNA loading, housekeeping gene expression was screened using glyceraldehyde 3-phosphate dehydrogenase (Gαp /z)-specifϊc primers: forward 5'TGGAGCCAAACGGGTCATCATCT-3' (SEQ ID NO. 29) and reverse 5'- GAAGAGTGGGAGTTGCTGTTGAA-3' (SEQ ID NO. 30).

Southern blot analysis

Using primers C-3F (SEQ ID NO. 23) and C-4R (SEQ ID NO. 26), we generated a 500- bp probe from neonatal kidney cDNA that contained the complete ORE. This probe was hybridized to EcoRl-digested genomic DNAs from multiple species (Zoo-blot; Clontech 7753-1) as well as to a panel of B6-+/+ and B6cpk/cpk genomic DNAs digested with EcoRl. The blots were washed serially with 2XSSC 0.1%SDS at RT; 0.5XSSC 0.1%SDS at 65 °C; and 0.2XSSC 0.1%SDS at 65 °C.

Transgene construction and cell culture transfection

A PCR fragment (BamEI-EcoΕI) containing the Kozak consensus sequence and the complete putative ORF was amplified from wild-type kidney cDNA and subcloned into the pEF6/Myc-His A vector (Invitrogen, San Diego, CA). The construct was verified by sequence analysis.

Transfection assays for expression and localization of cystin polypeptide were performed in a cortical collecting duct cell line (mCCD) isolated from the kidney of a mouse transgenic for the early region of SV40 large T antigen (32). Transfection of mCCD cells with the wild-type cystin cDNA expression construct was performed as previously described using Lipofectamine

Plus (Life Technologies, Gaithersberg, MD) (33). Stable cell lines were established by drug selection with 15 μg ml^"1 Blasticidin.

Immunolocalization studies

MCCD cells transfected with the wild-type Cysl construct (SEQ ID NO. 1) were grown in DMEM-F12 containing Earle's balanced salt solution (Cellgro, Mediatech, Inc., Washington, DC) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 mg/ml streptomycin, in 5% C0₂/95% air at 37 °C. For expression studies, cells were plated at near confluence on 6-mm Falcon tissue culture inserts (#3104, Becton Dickinson, Franklin Lakes NJ).

Immunolocalization studies were performed on confluent cultures grown for a minimum of three days to establish full epithelial polarization. Cells were fixed in 4% paraformaldehyde, 0.2% Triton in PBS for 10 minutes, incubated in blocking buffer (1% bovine serum albumin in PBS), followed by 60-min incubation with the primary antibodies diluted in blocking buffer. The primary antibodies used in this study were the α-myc mouse monoclonal (Invitrogen, San Diego, CA, R950-25; diluted 1:250), α-his-15 rabbit polyclonal antibody (Santa Cruz, Santa Cruz, CA, SC-803; diluted 1 :250), α-beta-tubulin mouse monoclonal (Biogenex, Mul78-UC; diluted 1:200), α-ZO-1 rat monoclonal (gift of DF Balkovetz; diluted 1 :2), and α-polaris rabbit polyclonal antibody (34) (GN593; diluted 1 :200). After washing with PBS, cells were incubated for one hour with fluorochrome-conjugated secondary antibodies diluted 1:200 in blocking buffer. The secondary antibodies were goat anti-mouse IgG (Oregon Green) (Molecular Probes, Eugene, OR, 0-6380), donkey anti-rabbit IgG (TRITC) (Jackson ImmunoResearch Laboratories, West Grove, PA; 711-025-152) and donkey anti-rat IgG (TRITC) (Jackson ImmunoResearch Laboratories, West Grove, PA., 712-295-153). Nuclei were stained for five minutes using Hoechst No. 33528 (Sigma, St. Louis, MO) diluted in 1 : 1,000 in PBS.

All references to articles, books, patents, websites and other publications and in this disclosure are considered incorporated by reference.

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Claims

CLAIMS What is claimed:

I . An isolated and purified nucleic acid molecule comprising a sequence which codes for a wild-type ARPKD nucleic acid.

2. The nucleic acid molecule of claim 1 where the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 1, or the complement thereof.

3. The nucleic acid molecule of claim 2 comprising at least 10 contiguous nucleotides of SEQ ID NO. 1.

4. The nucleic acid molecule of claim 1 where the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 2, or the complement thereof.

5. The nucleic acid molecule of claim 4 comprising at least 10 contiguous nucleotides of SEQ ID NO. 1.

6. The nucleic acid molecule of claim 2 where the nucleic acid sequence comprises a functional derivative of SEQ ID NO. 1, or a complement thereof. 7. The nucleic acid molecule of claim 6 where the functional derivative is selected from the group consisting of: a variant of SEQ ID NO. 1 or a complement thereof, a fragment of SEQ ID NO. 1 or a complement thereof, a degenerate variant of SEQ ID NO. 1 or a complement thereof, an analog of SEQ ID NO. 1 or a complement thereof.

8. The nucleic acid molecule of claim 6 where the functional derivative is a fragment of SEQ ID NO. 1 or the complement thereof, said fragment having a length selected from the group consisting of: 5 to 25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-150 nucleotides, 151-200 nucleotides and 200 to 300 nucleotides.

9. The nucleic acid molecule of claim 6 where the fragment comprises the sequence of SEQ ID NO. 19 or the complement thereof.

10. The nucleic acid molecule of claim 6 where the fragment comprises the sequence of SEQ ID NO. 20 or the complement thereof.

I I . The nucleic acid molecule of claim 6 where the fragment comprises the sequence of SEQ ID NO. 4 or the complement thereof. 12. The nucleic acid molecule of claim 6 where the fragment comprises the sequence of

SEQ ID NO. 5 or the complement thereof.

13. The nucleic acid molecule of claim 4 where the nucleic acid sequence comprises a functional derivative of SEQ ID NO. 2, or a complement thereof.

14. The nucleic acid molecule of claim 13 where the functional derivative is selected from the group consisting of: a variant of SEQ ID NO. 2 or a complement thereof, a fragment of SEQ ID NO. 2 or a complement thereof, a degenerate variant of SEQ ID NO. 2 or a complement thereof, an analog of SEQ ID NO. 2 or a complement thereof.

15. The nucleic acid molecule of claim 13 where the functional derivative is a fragment of SEQ ID NO. 2 or the complement thereof, said fragment having a length selected from the group consisting of: 5 to 25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-150 nucleotides, 151-200 nucleotides and 200 to 300 nucleotides.

16. An isolated and purified nucleic acid molecule the nucleic acid sequence of which codes for a mutant ARPKD nucleic acid. 17. The nucleic acid molecule of claim 16 where the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 6, or the complement thereof.

18. The nucleic acid molecule of claim 17 where the functional derivative is selected from the group consisting of: a variant of SEQ ID NO. 6 or a complement thereof, a fragment of SEQ ID NO. 6 or a complement thereof, a degenerate variant of SEQ ID NO. 6 or a complement thereof, an analog of SEQ ID NO. 6 or a complement thereof.

19. The nucleic acid molecule of claim 1 where the nucleic acid sequence codes for a wild-type CYSl gene.

20. The nucleic acid molecule of claim 1 where the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 7, or the complement thereof. 21. The nucleic acid molecule of claim 20 comprising at least 10 contiguous nucleotides of SEQ ID NO. 7.

22. The nucleic acid molecule of claim 1 where the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 8, or the complement thereof.

23. The nucleic acid molecule of claim 22 comprising at least 10 contiguous nucleotides of SEQ ID NO. 8.

24. The nucleic acid molecule of claim 20 where the nucleic acid sequence comprises a functional derivative of SEQ ID NO. 7, or a complement thereof.

25. The nucleic acid molecule of claim 24 where the functional derivative is selected from the group consisting of: a variant of SEQ ID NO. 7 or a complement thereof, a fragment of SEQ ID NO. 7 or a complement thereof, a degenerate variant of SEQ ID

NO. 7 or a complement thereof, an analog of SEQ ID NO. 7 or a complement thereof.

26. The nucleic acid molecule of claim 24 where the functional derivative is a fragment of SEQ ID NO. 7 or the complement thereof, said fragment having a length selected from the group consisting of: 5 to 25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-150 nucleotides, 151-200 nucleotides and 200 to 300 nucleotides. 27. The nucleic acid molecule of claim 22 where the nucleic acid sequence comprises a functional derivative of SEQ ID NO. 8, or a complement thereof. 28. The nucleic acid molecule of claim 27 where the functional derivative is selected from the group consisting of: a variant of SEQ ID NO. 8 or a complement thereof, a fragment of SEQ ID NO. 8 or a complement thereof, a degenerate variant of SEQ ID NO. 8 or a complement thereof, an analog of SEQ ID NO. 8 or a complement thereof.

29. The nucleic acid molecule of claim 27 where the functional derivative is a fragment of SEQ ID NO. 8 or the complement thereof, said fragment having a length selected from the group consisting of: 5 to 25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-150 nucleotides, 151-200 nucleotides and 200 to 300 nucleotides.

30. An isolated and purified nucleic acid molecule comprising a sequence which encodes a cystin polypeptide.

31. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises an amino acid sequence of SEQ ID NO. 3.

32. The nucleic acid molecule of claim 31 where said cystin polypeptide comprises at least 5 consecutive amino acids of SEQ ID NO. 3. 33. The nucleic acid molecule of claim 31 where said cystin polypeptide comprises at least 10 consecutive amino acids of SEQ ID NO. 3.

34. The nucleic acid molecule of claim 31 where said cystin polypeptide comprises at least 20 consecutive amino acids of SEQ ID NO. 3.

35. The nucleic acid molecule of claim 31 where said cystin polypeptide comprises at least 50 consecutive amino acids of SEQ ID NO. 3.

36. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises a degenerate variant of SEQ ID NO. 3.

37. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises a variant of SEQ ID NO. 3 having at least 50% identity to SEQ ID NO. 3. 38. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises an analog of SEQ ID NO. 3.

39. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises the amino acid sequence of SEQ ID NO. 9.

40. The nucleic acid molecule of claim 40 where said cystin polypeptide comprises at least 10 consecutive amino acids of SEQ ID NO. 9.

41. The nucleic acid molecule of claim 30 where said cystin polypeptide comprises the amino acid sequence of SEQ ID NO. 10.

42. The nucleic acid molecule of claim 39 where said cystin polypeptide comprises at least 10 consecutive amino acids of SEQ ID NO. 10. 43. An isolated nucleic acid comprising a sequence that hybridizes under highly stringent conditions to a hybridization probe the nucleotide sequence of which consists of the sequences selected from the group consisting of: SEQ ID NO: 1, a complement of SEQ ID NO: 1, a fragment of SEQ ID NO: 1 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 1 at least 10 nucleotides in length, SEQ ID NO: 2, a complement of SEQ ID NO: 2, a fragment of SEQ ID NO: 2 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 2 at least 10 nucleotides in length, SEQ ID NO: 6, a complement of SEQ ID NO: 6, a fragment of SEQ ID NO: 6 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 6 at least 10 nucleotides in length, SEQ ID NO: 7, a complement of SEQ ID NO: 7, a fragment of SEQ ID NO: 7 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 7 at least 10 nucleotides in length, SEQ ID NO: 8, a complement of SEQ ID NO: 8, a fragment of SEQ ID NO: 8 at least 10 nucleotides in length, and a complement of a fragment of SEQ ID NO: 8 at least 10 nucleotides in length 44. The nucleic acid molecule of claim 2 comprising a sequence of which is at least 50% identical to SEQ ID NO: 1, or a complement thereof.

45. The nucleic acid molecule of claim 4 comprising a sequence of which is at least 50% identical to SEQ ID NO: 2, or a complement thereof.

46. The nucleic acid molecule of claim 16 comprising a sequence of which is at least 50% identical to SEQ ID NO: 6, or a complement thereof.

47. The nucleic acid molecule of claim 20 comprising a sequence of which is at least 50% identical to SEQ ID NO: 7, or a complement thereof.

48. The nucleic acid molecule of claim 22 comprising a sequence of which is at least 50% identical to SEQ ID NO: 8, or a complement thereof. 49. A.purified polypeptide comprising an amino acid sequence of SEQ ID NO: 3.

50. The purified polypeptide of claim 49 where said amino acid sequence comprises at least 5 consecutive amino acids of SEQ ID NO: 3.

51. The purified polypeptide of claim 49 where said amino acid sequence is at least 50%> identical to SEQ ID NO: 3. 52. A purified polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

53. The purified polypeptide of claim 52 where said amino acid sequence comprises at least 5 consecutive amino acids of SEQ ID NO: 9.

54. The purified polypeptide of claim 52 where said amino acid sequence is at least 50% identical to SEQ ID NO: 9. 55. A purified polypeptide comprising an amino acid sequence of SEQ ID NO: 10.

56. The purified polypeptide of claim 56 where said amino acid sequence comprises at least 5 consecutive amino acids of SEQ ID NO: 10.

57. The purified polypeptide of claim 56 where said amino acid sequence is at least 50% identical to SEQ ID NO: 10. 58. An expression vector comprising a nucleic acid coding for a wild-type ARPKD nucleic acid operably linked to an expression control sequence.

59. The expression vector of claim 58 where the wild-type ARPKD nucleic acid has a nucleic acid sequence selected from the group consisting of: SEQ ID NO. 1, a complement of SEQ ID NO. 1, a fragment of SEQ ID NO: 1 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 1 at least 10 nucleotides in length,

SEQ ID NO. 2, a complement of SEQ ID NO. 2, a fragment of SEQ ID NO: 2 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 2 at least 10 nucleotides in length, SEQ ID NO. 7, a complement of SEQ ID NO. 7, a fragment of SEQ ID NO: 7 at least 10 nucleotides in length, a complement of a fragment of SEQ ID NO: 7 at least 10 nucleotides in length, SEQ ID NO. 8, a complement of SEQ ID

NO. 8, a fragment of SEQ ID NO: 8 at least 10 nucleotides in length, and a complement of a fragment of SEQ ID NO: 8 at least 10 nucleotides in length.

60. An expression vector comprising a nucleic acid coding for a mutant ARPKD nucleic acid operably linked to an expression control sequence. 61. The expression vector of claim 60 where the mutant ARPKD nucleic acid comprises the sequence of SEQ ID NO. 6.

62. A non-human cell comprising the expression vector of claim 58.

63. A non-human cell comprising the expression vector of claim 60.

64. A method of producing a polypeptide, the method comprising culturing the cells of claim 62 under conditions permitting expression of the polypeptide.

65. The method of claim 64 further comprising purifying the polypeptide from the cell of the medium of the cell.

66. A method of producing a polypeptide, the method comprising culturing the cells of claim 63 under conditions permitting expression of the polypeptide.

7. The method of claim 66 further comprising purifying the polypeptide from the cell of the medium of the cell.