EP0777728A1 - Polyzystisches nierenkrankheits-1-gen und verwendung davon - Google Patents

Polyzystisches nierenkrankheits-1-gen und verwendung davon

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Publication number
EP0777728A1
EP0777728A1 EP95924411A EP95924411A EP0777728A1 EP 0777728 A1 EP0777728 A1 EP 0777728A1 EP 95924411 A EP95924411 A EP 95924411A EP 95924411 A EP95924411 A EP 95924411A EP 0777728 A1 EP0777728 A1 EP 0777728A1
Authority
EP
European Patent Office
Prior art keywords
pkdl
nucleic acid
gene
fragment
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95924411A
Other languages
English (en)
French (fr)
Inventor
Peter Charles Harris
Belen Peral
Christopher James Ward
James Hughes
Martin Hendrik Breuning
Dorothea Johanna Maria Peters
Jeroen Hendrik Roelfsema
Julian Sampson
Dirkje Jorijntje Johanna Halley
Mark David Nellist
Lambertus Antonius Jacobus Janssen
Arjenne Ligue Wilhelma Hesseling
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University College Cardiff Consultants Ltd
Medical Research Council
Erasmus Universiteit Rotterdam
Universiteit Leiden
Original Assignee
University of Wales College of Medicine
Medical Research Council
Erasmus Universiteit Rotterdam
Universiteit Leiden
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB9411900A external-priority patent/GB9411900D0/en
Priority claimed from PCT/GB1994/002822 external-priority patent/WO1995018225A1/en
Priority claimed from GBGB9507766.5A external-priority patent/GB9507766D0/en
Application filed by University of Wales College of Medicine, Medical Research Council, Erasmus Universiteit Rotterdam, Universiteit Leiden filed Critical University of Wales College of Medicine
Publication of EP0777728A1 publication Critical patent/EP0777728A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ADPKD autosomal dominant polycystic kidney disease
  • APKD adult polycystic kidney disease
  • ESRD end stage renal disease
  • ADPKD accounts for 8-10% of all renal transplantation and dialysis patients in Europe and the USA (Gabow, 1993).
  • ADPKD also causes cystic growth in other organs (reviewed in Gabow, 1990) and occasionally presents in
  • Extrarenal manifestations include liver cysts ( ilutinovic, et al., 1980), and more rarely cysts of the pancreas (Gabow, 1993) and other organs. Intracranial aneurysms occur in approximately 5% of patients and are a significant cause of
  • ADPKD Alzheimer's disease
  • ADPKD and other animal models of cystic disease
  • Cysts in ADPKD are known to develop from outpouchings of descending or ascending kidney tubules and the early stages are characterized by a thickening and disorganization of the basement membrane, accompanied by a de-differentiation of tubular epithelial cells.
  • ADPKD epithelia altered growth responses, abnormal expression of various proteins and reversal of polarity, may be a sign of this de-differentiation and important in cyst expansion.
  • the nature of the primary defect which triggers these changes is, however, unknown and consequently much effort has been devoted to identifying the causative agent by genetic means.
  • the first step towards positional cloning of an ADPKD gene was the demonstration of linkage of one locus now designated the polycystic kidney disease 1 (PKDl) locus to the ⁇ globin cluster on the short arm of chromosome 16 (Reeders, et al., 1985). Subsequently, families with ADPKD unlinked to markers of 16p were described (Kimberling, et al., 1988; Romeo, et al., 1988) and a second ADPKD locus (PKD2 ) has recently been assigned to chromosome region 4ql3- q23 (Kimberling, et al., 1993; Peter, et al., 1993).
  • PPDl polycystic kidney disease 1 locus
  • ADPKD ADPKD
  • PKDl Peters and Sankuijl, 1992
  • PKD2 accounting for most of the remainder.
  • PKD2 appears to be milder condition with a later age of onset and ESRD (Parfrey. et al., 1990; Gabow, et al., 1992; Ravine, et al., 1992).
  • the position of the PKDl locus was refined to chromosome band 16pl3.3 and many markers were isolated from that region (Breuning, et al., 1987; Reeders, et al., 1988; Breuning, et al., 1990; Germino, et al., 1990; Hyland, et al., 1990; Himmelbauer, et al., 1991).
  • Their order, and the position of the PKDl locus has been determined by extensive linkage analysis in normal and PKDl families and by the use of a panel of somatic cell hybrids (Reeders et al., 1988; Breuning, et al., 1990; Germino, et al., 1990).
  • ADPKD is genetically heterogenous with loci mapped not only to 16pl3.3 (PKDl), but also to chromosome 4 (PKD2) .
  • PPDl 16pl3.3
  • PPD2 chromosome 4
  • TSC2 16pl3.3
  • the TSC2 gene therefore maps within the candidate region for the hitherto unidentified PKDl gene; as polycystic kidneys are a feature common to TSC and ADPKD1 (Bernstein and Robbins, 1991) the possibility of an etiological link, as proposed by Kandt et al. (1992), was considered.
  • HG area All but 3.5 kb at the 3' end of the transcript (which is about 14 kb in total) is encoded by a region which is reiterated several times elsewhere on the same chromosome (in 16pl3.1 and termed the HG area).
  • the structure of the duplication is complex, with some regions copied more times than others, and the HG region encoding three large transcripts.
  • the transcripts from the HG area are: HG-A (21 kb), HG-B (17 kb) and HG-C (8.5 kb) and although these have 3' ends which differ from PKDl, over most of their length they share substantial homology to the PKDl transcript. Consequently, cloning and characterizing a bona fide PKDl cDNA has proven difficult.
  • this invention provides an isolated, purified or recombinant nucleic acid sequence comprising: (a) a PKDl-encoding nucleic acid or its complementary strand,
  • a sequence wherein the PKDl gene has the nucleic acid sequence according to Fig. 15, or the partial sequence of Figs. 7 or 10.
  • the invention therefore includes a DNA molecule coding for a polypeptide having the amino acid sequence of Figure 15, or a polypeptide fragment thereof; and genomic DNA corresponding to a molecule as in (a) - (c) above.
  • substantially homologous refers to a nucleic acid strand that is sufficiently duplicative of the PKDl sequence presented in Fig. 15 such that it is capable of hybridizing to that sequence under moderately stringent, and preferably stringent conditions, as defined herein below.
  • substantially homologous refers to a homology of between 97 and 100%. Further, such a strand will encode or be complementary to a strand that encodes PKDl protein having the biological activity described below.
  • a "substantial portion of a molecule” refers to at least 60%, preferably 80% and most preferably 90% of the molecule in terms of its linear residue length or its molecular weight.
  • Nucleic acid refers to both DNA and RNA.
  • the PKDl gene described herein is a gene found on human chromosome 16, and the results of studies described herein form the basis for concluding that this PKDl gene encodes a protein called PKDl protein which has a role in the prevention or suppression of ADPKD.
  • the PKDl gene therefore includes the DNA sequences shown in Figure 15, and all functional equivalents.
  • functional equivalents we mean nucleic acid sequences that are substantially homologous to the PKDl nucleic acid sequence, as presented in Fig.
  • PKDl a protein that possesses one or more of the biological functions or activities of PKDl; i.e., that is involved in cell/cell adhesion, cell/cell recognition or cell/cell communication, for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane ( hether in kidneys or otherwise ) ; assist in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells.
  • the biological function of PKDl of course includes maintaining a healthy physiological state; that is, the native protein's aberrations or absence results in ADPKD or an associated disorder.
  • the PKDl gene may furthermore include regulatory regions which control the expression of the PKDl coding sequence, including promoter, enhancer and terminator regions.
  • Other DNA sequences such as introns spliced from the end-product PKDl RNA transcript are also encompassed.
  • the present invention therefore further provides a PKDl gene or its complementary strand having the sequence according to Figure 15 which gene or strand is mutated in some ADPKD patients (more specifically, PKDl patients). Therefore, the invention further provides a nucleic acid sequence comprising a mutant PKDl gene as described herein, including wherein Intron 43 as defined hereinbelow has a deletion of 18 or 20bp resulting in an intron of 55 or 57bp.
  • PTDl mutant or “mutation” encompasses alterations of the native PKDl nucleotide or amino acid sequence, as defined by Fig. 15, i.e., substitutions, deletions or additions, and also encompasses deletion of DNA containing the entire PKDl gene.
  • the invention further provides a nucleic acid sequence comprising a mutant PKDl gene, especially one selected from a sequence comprising a partial sequence according to Figures 7 and/or 10, or the corresponding sequences disclosed in Fig. 15, when: (a) [0X114] base pairs 1746-2192 as defined in Figure 7 are deleted (446bp);
  • RNA molecules comprising an RNA sequence corresponding to any of the DNA sequences set out above.
  • Such molecule may be the transcript reference PBP and identifiable with respect to the restriction map of Figure 3a and having a length of about 14 KB.
  • the invention provides a nucleic acid probe having a sequence as set out above; in particular, this invention extends to a purified nucleic acid probe which hybridizes to at least a portion of the DNA or RNA molecule of any of the preceding sequences.
  • the probe includes a label such as a radiolable, for example, a 32 P label.
  • this invention provides a purified DNA or RNA coding for a protein comprising the amino acid sequence of Figure 15, or a protein polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein.
  • the DNA molecule defined above may be incorporated in a recombinant cloning vector for expressing a protein having the amino acid sequence of Figure 15, or a protein or a polypeptide having at least one functional domain or active site in common with said protein.
  • a vector may include any vector for expression in bacteria, e.g., E. coli; yeast, insect, or mammalian cells.
  • the invention also features a nucleic acid probe for detecting PKDl nucleic acid comprising 10 consecutive nucleotides as presented in Fig. 15.
  • the probe may comprise 15, 20, 50, 100, 200, or 300, etc., consecutive nucleotides (nt) presented in Fig. 13, and may fall within the size range 15nt-13kb, 100nt-5kb, 150nt-4kb, 300nt-2kb, and 500nt-lkb.
  • Probes are used according to the invention in hybridization reactions to identify PKDl sequences, whether they be native or mutated PKDl DNA or RNA, as disclosed herein. Such probes are useful for identifying the PKDl gene or a mutation thereof, as defined herein.
  • the invention also features a synthetic polypeptide corresponding in amino acid residue sequence to at least a portion of the sequence of naturally occurring PKDl, and having a molecular weight equal to less than that of the native protein.
  • a synthetic polypeptide of the invention is useful for inducing the production of antibodies specific for the synthetic polypeptide and that bind to naturally occurring PKDl.
  • Preferred embodiments of this aspect of the invention include a group of synthetic polypeptides whose members correspond to a fragment of the PKDl protein comprising a stretch of amino acids of at least 8, and preferably 15, 30, 50, or 100 residues in length from the sequence disclosed in Fig. 15.
  • the invention provides a polypeptide encoded by a sequence as set out above, or having the amino acid sequence according to the amino acid sequence of Figure 15, or a protein or polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein.
  • an isolated, purified or recombinant polypeptide comprising a PKDl protein or a mutant or variant thereof or encoded by a sequence set out above or a variant thereof having substantially the same activity as the PKDl protein.
  • the present invention may further comprise a polypeptide having 9 or 13 transmembrane pairs instead of 11 transmembrane domains as described hereinbelow.
  • a molecule which interacts with a polypeptide as herein described which molecule synergises, causes, enhances or is necessary for the functioning of the PKDl protein as herein described.
  • the invention also encompasses recombinant expression vectors comprising a nucleic acid or isolated DNA encoding PKDl and a process for preparing PKDl polypeptide, comprising culturing a suitable host cell comprising the vector under conditions suitable for promoting expression of PKDl, and recovering said PKDl.
  • This invention also provides an in vitro method of determining whether an individual is at risk of a PKD1- associated disorder, comprising assaying a biological sample from the individual to determine the presence and/or amount of PKDl protein or polypeptide having the amino acid sequence of Figure 15.
  • biological sample includes any fluid or tissue sample from a mammal, preferably a human, including but not limited to blood, urine, saliva, any body organ tissue, cells from any body tissue, including blood cells. Additionally or alternatively, a sample may be assayed to determine the presence and/or amount of mRNA coding for the protein or polypeptide having the amino acid sequence of Figure 15, or to determine the fragment lengths of fragments of nucleotide sequences coding for the protein or polypeptide of Figure 15, or to detect inactivating mutations in DNA coding for a protein having the amino acid sequence of Figure 15 or a protein having homologous properties.
  • the screening preferably includes applying a nucleic acid amplification process, as described herein in detail, to said sample to amplify a fragment of the DNA sequence.
  • the nucleic acid amplification process advantageously utilizes at least one of the following sets of primers as identified herein: AH3 F9 : AH3 B7; 3A3 Cl : 3A3 C2; and AH4 F2 : JH14 B3.
  • the screening method may comprise digesting the sample DNA to provide EcoRI fragments and hybridizing with a DNA probe which hybridizes to the EcoRI fragment identified (A) in Figure 3(a), and the DNA probe may comprise the DNA probe CW10 identified herein.
  • Another screening method may comprise digesting the sample to provide BamHI fragments and hybridizing with a DNA probe which hybridizes to the BamHI fragment identified (B) in Figure 3(a), and the DNA probe may comprise the DNA probe 1A1H.6 identified herein.
  • a method according to the present invention may comprise detecting a PKDl-associated disorder in a patient suspected of having or having predisposition to the disorder (i.e., a carrier), the method comprising detecting the presence of and/or evaluating the characteristics of PKDl DNA, PKDl mRNA and.or PKDl protein in a sample taken from the patient.
  • Such method may comprise detecting and/or evaluating whether the PKDl DNA is deleted, missing, mutated, aberrant or not expressing normal PKDl protein.
  • One way of carrying out such a method comprises: A. taking a biological, tissue or biopsy sample from the patient; B.
  • a "PKDl-associated disorder” refers to adult polycystic kidney disease, as described herein, and also refers to tuberous sclerosis, as well as other disorders having symptoms such as cyst formation in common with these diseases.
  • a specific method according to the invention comprises extracting from a patient a sample of PKDl DNA or DNA from the PKDl locus purporting to be PKDl DNA, cultivating the sample in vitro and analyzing the resulting protein, and comparing the resulting protein with normal PKDl protein according to the well-established Protein Truncation Test. Less sensitive tests include analysis of RNA using RT PCR (reverse transcriptase polymerase chain reaction), and examination of genomic DNA.
  • RT PCR reverse transcriptase polymerase chain reaction
  • Step C of the above method may be replaced by: comparing the first set of results with a second set of results obtained using the same or similar methodology in an individual that is known to have the or at least one of the disorder(s); and if the first and second sets of results are substantially identical, this indicates that the PKDl DNA in the patient is deleted, mutated or not expressing normal PKDl protein.
  • the invention further provides a method of characterizing a mutation in a subject suspected of having a mutation in the PKDl gene, which method comprises:A amplifying each of the exons in the PKDl gene of the subject; B. denaturing the complementary strands of the amplified exons; C.diluting the denatured separate, complementary strands to allow each single-stranded DNA molecule to assume a secondary structural confirmation; D. subjecting the DNA molecule to electrophoresis under non- denaturing conditions; E.
  • the invention also extends to a diagnostic kit for carrying out a method as set out above, comprising nucleic acid primers for amplifying a fragment of the DNA or RNA sequences defined above, and packaging means therefore.
  • the kit may optionally include written instructions stating that the primers are to be used for detection of disorders associated with the PKDl gene.
  • the nucleic acid primers may comprise at least one of the following sets: AH3 F9 : AH3 B7; 3A3 Cl : 3A3 C2; and AH4 F2 : JH14 B3.
  • Another embodiment of kit may combine one or more substances for digesting a sample to provide EcoRI fragments and a DNA probe as previously defined.
  • a further embodiment of kit may comprise one or more substances for digesting a sample to provide BamHI fragments and a DNA probe as previously defined.
  • a vector (such as Bluescript (available from Stratagene) ) comprising a nucleic acid sequence set out above; and a host cell (such as E. coli strain SL-1 Blue (available from Stratagene)) transfected or transformed with the vector are also provided, together with the use of such a vector or a nucleic acid sequence set out above in gene therapy and/or in the preparation of an agent for treating or preventing a PKDl-associated disorder.
  • a method of treating or preventing a PKDl-associated disorder comprises administering to a patient in need thereof a functional PKDl gene to affected cells in a manner that permits expression of PKDl protein therein and/or a transcript produced from a mutated chromosome (such as the deleted WS-212 chromosome) which is capable of expressing functional-PKDl protein therein.
  • a mutated chromosome such as the deleted WS-212 chromosome
  • hybridization refers to conventional DNA/DNA or DNA/RNA hybridization conditions.
  • moderately stringent hybridization conditions include 10X SSC, 5X Denhardts, 0.1% SDS, at 35 - 50 degrees for 15 hours; for a probe of about 50 - 300 nucleotides, "stringent" hybridization conditions are preferred and refer to hybridization in 6X SSC, 5X Denhardts, 0.1% SDS at 65 degrees for 15 hours.
  • the present invention further provides the use of PKDl protein or polycystin or a mutant or variant thereof having substantially the same biological activity there as in therapy.
  • recognition or communication for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane (whether in kidneys or otherwise) ; assisting in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells.
  • the invention further provides the use of a PKDl protein or polycystin in the preparation of a medicament. Therefore, there is also provided a pharmaceutical formulation comprising a PKDl protein, functional PKDl gene and/or a transcript produced from a mutated chromosome which is capable of expressing functional PKDl protein, in association with a pharmaceutically acceptable carrier therefor.
  • the invention also features an immunoglobin, i.e., a polyclonal or monoclonal antibody specific for an epitope of PKDl, which epitope is found in the amino acid sequence presented in Fig. 15.
  • an immunoglobin i.e., a polyclonal or monoclonal antibody specific for an epitope of PKDl, which epitope is found in the amino acid sequence presented in Fig. 15.
  • the invention also features a method of assaying for the presence of PKDl in a sample of mammalian, preferably human cells, comprising the steps of: (a) providing an antibody specific for said PKDl; and (b) assaying for the presence of PKDl by admixing an aliquot from a sample of mammalian cells with antibody under conditions sufficient to allow for formation and detection of an immune complex of PKDl and the antibody.
  • Such method is useful for detecting disorders involving aberrant expression of the PKDl gene or processing of the protein, as described herein.
  • this method includes providing a monoclonal antibody specific for an epitope that is antigenically the same, as determined by Western blot assay, ELISA or immunocytochemical staining, and substantially corresponds in amino acid sequence to the amino acid sequence of a portion of PKDl and having a molecular weight equal to less than that of PKDl.
  • the invention thus also features a kit for detecting PKDl, the kit including at least one package containing an antibody or idiotype-containing polyamide portion of an antibody raised to a synthetic polypeptide of this invention or to a conjugate of that polypeptide bound to a carrier.
  • An indicating group or label is utilized to indicate the formation of an immune reaction between the antibody and PKDl when the antibody is admixed with tissue or cells.
  • Figure la A long range map of the terminal region of the short arm of chromosome 16 showing the PKDl candidate region defined by genetic linkage analysis. The positions of selected DNA probes and microsatellites used for haplotype, linkage or heterozygosity analyses are indicated. Markers previously described in linkage disequilibrium studies are shown in bold (from: Harris, et al., 1990; Harris, et al., 1991; Germino, et al., 1992; Somlo, et al., 1992; Peral, et al., 1994; Snarey, et al. , 1994).
  • the contig covering the 77 breakpoint region consists of the cosmids: 1, CW9D; 2, ZDS5; 3, JH2A; 4, REP59; 5, JC10.2B; 6, CW10III; 7, SM25A; 8, SMII; 9, NM17.
  • Figure lb Pedigree of family 77 which segregates a 16;22 translocation; showing the chromosomal composition of each subject. Individuals 77-2 and 77-3 have the balanced products of the exchange - and have PKDl; 77-4 is monosomic for 16pl3.3-->l6pter and 22qll.21-->22pter - and has TSC.
  • Figure lc PFGE of DNA from members of the 77 family: 77-1 (1); 77-2 (2); 77-3 (3); 77-4 (4); digested with Cla I and hybridised with SM6.
  • a proximal breakpoint fragment of approximately 100 kb is seen in individuals, 77-2, 77-3 and 77-4; concordant with segregation of the der(16) chromosome.
  • FIG. 2 FISH of the cosmid CWIOIII (cosmid 6; Figure la) to a normal male metaphase. Duplication of this locus is illustrated with two sites of hybridisation on 16p; the distal site (the PKDl region) is arrowed. The signal from the proximal site (16pl3.1) is stronger than that from the distal, indicating that sequences homologous to CWIOIII are reiterated in 16pl3.1.
  • Figure 3a A detailed map of the 77 translocation region showing the precise localisation of the 77 breakpoint and the region that is duplicated in 16pl3.1 (hatched).
  • DNA probes open boxes
  • the transcripts, PKDl and TSC2 filled boxes; with direction of transcription indicated by an arro
  • cDNAs grey boxes
  • the known genomic extent of each gene is indicated at the bottom of the diagram and the approximate genomic locations of each cDNA is indicated under the genomic map.
  • SM3 is a 2kb BamHI fragment shown at the 5' end of the gene.
  • Figure 3b Southern blots of BamH I digested DNA from individuals: 77-1 (1); 77-2 (2); and 77-4 (4) hybridised with: left panel, 8S3 and right panel, 8S1 (see a).
  • 8S3 detects a novel fragment on the telomeric side of the breakpoint (12 kb: arrowed) associated with the der(22) chromosome in 77-2, but not 77-4;
  • 8S1 identifies a novel fragment on the centromeric side of the breakpoint (9 kb: arrowed) - associated with the der(16) chromosome - in 77-2 and 77-4.
  • the telomeric breakpoint fragment is also seen weakly with 8S1 (arrowed) indicating that the breakpoint lies in the distal part of 8S1.
  • 8S3 and 8S1 loci are both duplicated; the normal BamH I fragment detected at the 16pl3.3 site by these probes is 11 kb (see a), but a similar sized fragment is also detected at the 16pl3.1 site. Consequently, the breakpoint fragments are much fainter than the normal (16pl3.1 plus 16pl3.3) band.
  • Figure 4a PBP cDNA, 3A3, hybridised to a Northern blot containing about 1 ⁇ g polyA selected mRNA per lane of the tissue specific cell lines: lane 1, MJ, EBV-transformed lymphocytes; lane 2, K562, erythroleukemia; lane 3, FS1, normal fibroblasts; lane 4, HeLa, cervical carcinoma; lane 5, G401, renal Wilm's tumour; lane 6, Hep3B, hepatoma; lane 7, HT29, colonic adenocarcinoma; lane 8, SW13, adrenal carcinoma; lane 9, G-CCM, astrocytoma.
  • a single transcript of approximately 14 kb is seen; the highest level of expression is in fibroblasts and in the astrocytoma cell line, G-CCM. Although in this comparative experiment little expression is seen in lanes 1, 4 and 7, we have demonstrated at least a low level of expression in these cell lines on other Northern blots and by RT-PCR (see later).
  • Figure 4b A Northern blot containing about 20 ⁇ g of total RNA from the cell line G-CCM hybridised with cDNAs or a genomic probe which identify various parts of the PBP gene.
  • a single about 14 kb transcript is seen with a cDNA from the single copy area, 3A3.
  • a cDNA, 21P.9 that is homologous to parts of the region that is duplicated (JH12, JH8 and JH10; see Figure 3a) hybridises to the PBP transcript and three novel transcripts; HG-A (about 21 kb), HG-B (about 17 kb) and HG-C (8.5 kb).
  • Figure 4c A Northern blot of 20 ⁇ g total fibroblast RNA from: normal control (N); 77-2 (2); 77-4 (4) hybridised with 8S1, which contains the 16;22 translocation breakpoint (see Figure 3).
  • a transcript of about 9 kb (PBP-77) is identified in the two patients with this translocation but not in the normal control.
  • PBP-77 is a chimeric PBP transcript formed due to the translocation and is not seen in 77-2 or 77-4 RNA with probes which map distal to the breakpoint.
  • FIGE of DNA from: normal (N) and ADPKD patient 0X875 (875), digested with EcoR I and hybridised with, left panel, CW10; middle panel, JH1. Normal fragments of 41 kb (plus a 31 kb fragment from the 16pl3.1 site), CW10, and 18 kb, JHI, are identified with these probes; 0X875 has an additional 53 kb band (arrowed). The EcoR I site separating these two fragments is removed by the deletion (see Figure 3a) . The right panel shows a Southern blot of BamH I digested DNA (as above) hybridised with 1A1H.6.
  • Figure 5b Northern blot of total fibroblast RNA, as (a), hybridised with the cDNAs, AH4, 3A3 and AH3.
  • a novel transcript (PBP-875) of about 11 kb is seen with AH4 (the band is reduced in intensity because the probe is partly deleted) and AH3 (arrowed), which flank the deletion, but not 3A3 which is entirely deleted (see figure 3a) .
  • the transcripts HG-A, HG-B and HG-C, from the duplicated area, are seen with AH3 (see figure 4b).
  • FIGE Left panel; FIGE of DNA from: normal (N) and ADPKD patient 0X114 (114), digested with EcoR I and hybridised with CW10; a novel fragment of 39 kb (arrowed) is seen in 0X114.
  • Middle panel DNA, as above, plus the normal mother (M) and brother (B) of 0X114 digested with BamH I and hybridised with CW21.
  • a larger than normal fragment of 19 kb (arrowed) was detected in 0X114 but not other family members due to deletion of a BamH I site; together these results are consistent with a 2 kb deletion (see Figure 3a).
  • Figure 5d RT-PCR of RNA from: ADPKD patient 0X32 (32) plus the probands, normal mother (M) and affected father (F) and sibs ( 1 ) and (2) using the C primer pair from 3A3 (see Experimental Procedures). A novel fragment of 125 bp is detected in each of the affected individuals.
  • FIG. 6 Map of the region containing the TSC2 and PBP genes showing the area deleted in patient WS-53 and the position of the 77 translocation breakpoint. Localisation of the distal end of the WS-53 deletion was described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and we have now localised the proximal end between SM6 and JH17. The size of the aberrant Mlu I fragment in WS-53, detected by JHl and JH17, is 90kb and these probes lie on adjacent Mlu I fragments of 120kb and 70kb, respectively. Therefore the WS-53 deletion is about lOOkb.
  • Mlu I M
  • Nru I R
  • Not I N
  • S BssH II
  • DNA probes open boxes
  • TSC2 and PBP transcripts filled boxes
  • the locations of the microsatellites KG8 and SM6 are also indicated.
  • Figure 7 The partial nucleotide sequence (cDNA) of the PKDl transcript extending 5631bp to the 3' end of the gene.
  • the corresponding predicted protein (also shown in SEQ ID NO: 4: ) is shown below the sequence and extends from the start of the nucleotide sequence.
  • the GT-repeat, KG8, is in the 3' untranslated region between 5430-5448 bp. This sequence corresponds to GenBank Accession No. L33243 and is shown in SEQ ID NO: 3:.
  • Figure 8 The sequence of the probe 1A1H0.6 (also shown in SEQ ID NO: 5: ) .
  • Figure 9 The sequence (SEQ ID NO: 6:) of the probe CW10 which is about 0.5kb.
  • Figure 10 The larger partial nucleotide sequence (SEQ ID NO: 1:) of the PKDl transcript (cDNA) extending from bp 2 to 13807bp to the 3' end of the gene together with the corresponding predicted protein (also shown in SEQ ID NO: 2:).
  • This larger partial sequence encompasses the (smaller) partial sequence of Figure 7 from amino acid no. 2726 in SEQ ID NO: 3: and relates to the entire PKDl gene sequence apart from its extreme 5' end.
  • Figure 11 A map of the 75bp intron amplified by the primer set 3A3C insert at position 3696 of the 3' sequence showing the positions of genomic deletions found in PKDl patients 461 and 0X1054.
  • Figure 12 A map of the region of chromosome 16 containing the TSC2 and PKDl genes showing the areas affected in patients WS-215, WS-250, WS-212, WS-194, WS-227 and WS-219; also WS-53 (but cf. Figure 6). Genomic sites for the enzymes Mlul (M), Clal (C), Pvul (P) and Nrul (R) are shown. Positions of single copy probes and cosmids used to screen for deletions are shown below the line which represents about 400kb of genomic DNA. The genomic distribution of the approximately 45kb TSC2 gene and known extent of the PKDl gene are indicated above. The hatched area represents an about 50kb region which is duplicated more proximally on chromosome 16p.
  • Figure 13 is a genomic map of the PKDl gene.
  • (Top) A restriction map of the genomic area containing the PKDl gene showing sites for Bam H1(B), EcoRI(E) and partial maps for Xbal (X) and Hind III(H), and the duplicated area (hatched). The position of genomic clones and the cosmid JH2A are shown above the map (open boxes). The positions of the 46 exons of the PKDl gene are shown below the map (solid boxes, translated areas; open boxes, untranslated regions; UTRs) . Each 5th exon is numbered and the direction of transcription arrowed. The area sequenced in Figs.
  • the cDNAs are: 1, revl; 2, S13;3, S3/4; 4, Sl/3;5, GAP e; 6, GAP d; 7, GAP g; 8, GAP a (see table 2 for details); 9, A1C; 10, AH3; 11, 3A3; 12, AH4.
  • FIG 14 (a) (Top): Map of the genomic BamH I fragment, SM3 which contains the CpG island at the 5' end of the PKDl gene, showing the probe CW45 (open box). Genomic restriction sites for the methylation sensitive enzymes: SacII (S), Notl (N), Mlul (M) and BssHII (H) are illustrated. The approximate position of the DNasel hypersensitive site is also shown (large arrow), plus the location of the first exon including the proposed transcription start site (small arrow), the 5'UTR (open box) and the translated region (solid bar). (Bottom) The GC content across the area is plotted with a window size of 50 nt.
  • Figure 15 provides the sequence of the PKDl transcript and predicted protein.
  • the full sequence of 14,148 bp from the transcription start site to the poly A tail is shown.
  • the probable signal sequence of 23 amino acids is shown after the first methionine (underlined) plus the cleavage site (arrow) .
  • the predicted transmembrane (TM) domains double underlined and numbered) and N-linked glycosylation sites (asterisk) are indicated.
  • the position of a possible hinge sequence is underlined and tyrosine kinase and protein kinase C phosphorylation sites marked with a box and circle, respectively.
  • FIG 16(a) The leucine rich repeats (LRRs) found in the PKDl protein (72-125aa) are compared with each other and to the LRR consensus (Rothberg, 1990; Kobe, 1994); a, aliphatic. A total of just over 2 full repeats are present in PKDl but they have been arranged into 3 incomplete repeats to show their similarity to those found in slit (Rothberg, 1990). The black boxes show identity to the LRR consensus and shaded boxes other regions of similarity between the repeats which have also been noted in other LRRs (Kobe, 1994).
  • LRRs leucine rich repeats
  • FIG 16(b) The amino flanking region to the LRR in the PKDl protein (33-71aa) is compared similar regions from a variety of other proteins. Black boxes shown identity with the consensus (adapted from [Rothberg, 1990 #1126]) and shaded boxes conserved amino acids. The different types of residue indicated in the consensus are: a, as above; p, polar or turn-like; h, hydrophobic. The listed proteins, with the species and Protein Identification Resource no.
  • PIR oligodendrocyte myelin glycoprotein
  • Slit Dermata; A36665
  • Chaoptin Dermata; A29943
  • GP-IB Beta platelet glycoprotein lb ⁇ chain (Human; A31929)
  • Pgl proteoglycan-1 (mouse; 520811); Biglycan (Human; A40757)
  • Trk Human; A25184
  • LH-CF lutropinchoriogonadotrophin receptor
  • the carboxy flanking region of the LRR repeat from the PKDl protein (126-180 aa) compared to similar regions in other proteins and a consensus accepted from [Rothberg, 1990 #1126] .
  • the shading and amino acid types are as above.
  • the proteins not described above are: Toll (Drosophila; A29943) and GP IX, platelet glycoprotein IX (Human; A46606).
  • Figure 17 is a sequence comparison of the C-type lectin domain.
  • the PKDl lectin domain (403-532aa) is compared to those of: BRA3, acorn barnacle lectin (JC1503); Kupffer cell carbohydrate-binding receptor (Rat; A28166), CSP, cartilage specific protoglycan (Bovine; A27752); Agp; asialoglycoprotein receptor (Human; 55283), E-Selectin (Mouse; B42755) and glycoprotein gpl20 (Human; A46274). Black squares show identify with the consensus and shaded boxes conserved residues. Amino acid types are: Very highly conserved residues are shown in bold in the consensus which is adapted from Drickamer 1987, Drickamer 1988.
  • Figure 18 is a sequence analysis of the Ig-like repeat.
  • the 16 copies of the PKDl Ig-like repeat (PKDl 273-356 aa; PKDII-XVI, 851-2145aa) are compared to each other and to: V.a. colAi, and C.p.
  • the PKD repeat IV has an extra repetition of 20 aa in the centre of the repeat while all of the others are between 84-87 aa.
  • Figure 19 reveals type Ill-related fibronectin domains.
  • the four fibronectin-related domains from the PKDl protein (2169-2573aa) are compared to similar domains in: Neuroglian (Drosophila; A32579 ) ; Ll, neural recognition molecule Ll (X59847); Fll, neural cell recognition molecule Fll (X14877); TAG 1, transiently expressed axonal surface glycoprotein-1 (Human; S28830); F3, Neuro-1 antigen (mouse; S05944); NCAM, neural cell adhesion molecule (Rat; X06564); DCC, deleted in colorectal cancer (Human; X76132) ; LAR, Leukocyte-common antigen related molecule (Human; Y00815); HPTP, ⁇ protein tyrosine phosphate beta (Human; X54131 ) and FN, fibronectin (Human; X02761 ) .
  • the consensus sequence is compiled from Borh and Doolittle (1993), Kuma et al. (1993), Baron et al. (1992) and Borh and Doolittle (1992). Black boxes show identity to highly conserved residues and shaded boxes conserved changes or similarity in less highly conserved positions. The approximate positions of the ⁇ strands are illustrated.
  • the fibronectin repeats in the PKDl protein are linked by sequences of 27aa (A-B), 22aa (B- C) and 7aa (C-D) which are not shown.
  • Figure 20 presents a proposed model of the PKDl protein, polycystin. The predicted structure of the PKDl protein is shown.
  • the son, 77-4 has the unbalanced karyotype, 45XY-16- 22+der(16) (16qter—>16pl3.3: :22qll.21—>2qter) and consequently is monosomic for 16pl3.3-->16pter as well as for 22qll.21-->22pter.
  • This individual has the clinical phenotype of TSC ( see Experimental Procedures ) ; the most likely explanation is that the TSC2 locus located within 16pl3.3 is deleted in the unbalanced karyotype.
  • the 77 family was analyzed with polymorphic markers from 16pl3.3. Individual 77-4 was hemizygous for MS205.2 and GGG1, but heterozygous for SM6 and more proximal markers, locating the translocation breakpoint between GGG1 and SM6 (see Figure la). Fluorescence in situ hybridization (FISH) of a cosmid from the TSC2 region, CW9D (cosmid 1 in Figure la) , to metaphase spreads showed that it hybridized to the der(22) chromosome of 77-2; placing the breakpoint proximal to CW9D and indicating that 77-4 was hemizygous for this region consistent with his TSC phenotype.
  • FISH Fluorescence in situ hybridization
  • Figure 2 shows that a cosmid, CWIOIII, from the duplicated region hybridized to two points on 16p; the distal, PKDl region and a proximal site positioned in 16pl3.1.
  • the structure of the duplicated area is complex with each fragment present once in 16pl3.3 re-iterated two-four times in 16pl3.1 (see Figure 2).
  • Cosmids spanning the duplicated area in 16pl3.3 were subcloned (see Figure 3a and Experimental Procedures for details) and a restriction map was generated.
  • a genomic map of the PKDl region was constructed using a radiation hybrid, Hyl45.19 which contains the distal portion of 16p but not the duplicate site in 16pl3.1.
  • To localize the 77 translocation breakpoint subclones from the target region were hybridized to 77-2 DNA, digested with Cla I and separated by PFGE. Once probes mapping across the breakpoint were identified they were hybridized to conventional Southern blots of 77 family DNA.
  • Figure 3b shows that novel BamH I fragments were detected from the centromeric and telomeric side of the breakpoint, which was localized to the distal part of the probe 8S1 ( Figure 3a) .
  • the balanced translocation was not associated with a substantial deletion, and the breakpoint was located more than 20 kb proximal to the TSC2 locus ( Figure 3a).
  • the polycystic breakpoint (PBP) gene is disrupted by the translocation Localization of the 77 breakpoint identified a precise region in which to look for a candidate or the PKDl gene.
  • TSC2 gene we identified other transcripts not associated with TSC including a large transcript (about 14 kb) partially represented in the cDNAs 3A3 and AH4 which mapped to the genomic fragments CW23 and CW21 ( Figure 3a).
  • the orientation of the gene encoding this transcript had been determined by the identification of a polyA tract in the cDNA, AH4: the 3' end of this gene lies very close to the TSC gene, in a tail to tail orientation (European Chromosome 16 Tuberous Sclerosis Consortium, 1993).
  • FIG. 4a shows that the about 14 kb transcript was identified by 3A3 in various tissue-specific cell lines. From this and other Northern blots we concluded that the PBP gene was expressed in all of the cell lines tested, although often at a low -level. The two cell lines which showed the highest level of expression were fibroblasts and a cell line derived from an astrocytoma, G-CCM. Significant levels of expression were also obtained in cell lines derived from kidney (G401) and liver (Hep3B).
  • HG-4/1.1 hybridized to all three HG transcripts, but not to the PBP transcript and on a hybrid panel it mapped to 16pl3.1 (no ⁇ the PKDl area).
  • the first rearrangement was identified in patient 0X875 (see Experimental Procedures for clinical details) who was shown to have a 5.5 kb genomic deletion without the 3 ' end of the PBP gene, producing a smaller transcript (PBP-875) (see Figures 5a, b and 3a for details).
  • This genomic deletion results in a ⁇ 3 kb internal deletion of the transcript with the ⁇ 500 bp adjacent to the polyA tail intact.
  • this family linkage of ADPKD to chromosome 16 could not be proven because although 0X875 has a positive family history of ADPKD there were no living, affected relatives. However, paraffin-embedded tissue from her affected father (now deceased) was available.
  • the second rearrangement detected by hybridization was a 2 kb genomic deletion within the PBP gene, in ADPKD patient 0X114 ( see Experimental Procedures for clinical details and Figures 5c and 3a) .
  • No abnormal PBP transcript was identified by Northern blot analysis, but using primers flanking the deletion (see Experimental Procedures) a shortened product was detected by RT-PCR ( Figure 5c). This was cloned and sequenced and shown to have a frame-shift deletion of 446 bp (between base pair 1746 and 2192 of the sequence shown in Figure 7) .
  • 0X114 is the only member of the family with ADPKD (she has no children) and ultrasound analysis of her parents at age 78 (father) and 73 years old (mother) showed no evidence of renal cysts. Somatic cell hybrids were produced from 0X114 and the deleted chromosome was found to be of paternal origin by haplotype analysis. The father of 0X114 is now deceased but analysis of DNA from the brother of 0X114 (0X984) with seven microsatellite markers from the PKDl region (see Experimental Procedures) showed that he shares the same paternal chromosome, in the PKDl region, as 0X114. Renal ultrasound revealed no cysts in 0X984 at age 53 and no deletion was detected by DNA analysis (Figure 5c).
  • the deletion in 0X114 is a de novo event associated with the development of ADPKD.
  • ADPKD- is chromosome 16-linked
  • the location of the PBP gene indicated that this is a de novo PKDl mutation.
  • PKDl associated mutations single copy regions of the PBP gene were analyzed by RT-PCR using RNA isolated from lymphoblastoid cell lines established from ADPKD patients. cDNA from 48 unrelated patients was amplified with the primer pair 3A3 C (see Experimental Procedures) and the product of 260 bp was analyzed on an agarose gel. In one patient, 0X32, an additional smaller product (125bp) was identified, consistent with a deletion or splicing mutation. 0X32 comes from a large family in which the disease can be traced through three generations. Analysis of RNA from two affected sibs of 0X32 and his parents showed that the abnormal transcript segregates with PKDl ( Figure 5d) .
  • Amplification of normal genomic DNA with the 3A3 C primers generates a product of 418 bp; sequencing showed that this region contains two small introns (5', 75 bp and 3', 83 bp) flanking a 135 bp exon.
  • the product amplified from 0X32 genomic DNA was normal in size, excluding a genomic deletion. However, heteroduplex analysis of that DNA revealed larger heteroduplex bands, consistent with a mutation within that genomic interval.
  • the abnormal 0X32, RT-PCR product was cloned and sequenced: this demonstrated that, although present in genomic DNA, the 135 bp exon was missing from the abnormal transcript.
  • the deletion called WS-53 disrupts both the TSC2 gene and the PKDl gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), although the full proximal extent of the deletion was not determined. Further study has shown that the deletion extends "100 kb (see Figure 6 for details) and deletes most if not all of the PKDl gene. This patient has TSC but also has unusually severe polycystic disease of the kidneys. Other patients with a similar phenotype have also been under investigation. Deletions involving both TSC2 and PKDl were identified and characterized in six patients in whom TSC was associated with infantile polycystic kidney disease.
  • renal ultrasound showed no cysts at four years of age but a deletion was identified which removed the entire TSC2 gene and deleted an Xbal site which is located 42 bp 5' to the polyadenylation signal of PKDl.
  • a 587bp probe from the 3' untranslated region (3'UTR) was hybridized to Xbal digested DNA.
  • a 15kb XbaL 1 breakpoint fragment was detected with an approximately equal intensity to the normal fragment of 6kb, indicating that most of the PKDl 3'UTR was preserved on the mutant chromosome.
  • PKDl transcript is produced from the deleted chromosome in WS-212 was obtained by 3 ' rapid identification of cDNA ends (RACE) with a novel, smaller product generated from WS-212 cDNA. Characterization of this product showed that polyadenylation occurs 546bp 5' to the normal position, within the 3'UTR of PKDl (231bp 3' to the stop codon at 5073bp of the described PKDl sequence 1 ) . A transcript with an intact open reading frame is thus produced from the deleted WS-212 chromosome. It is likely that a functional PKDl protein in produced from this transcript, explaining the lack of cystic disease in this patient.
  • the sequence preceding the novel site of polyA addition is:
  • the WS-212 deletion is 75kb between SM9-CW9 distally and the PKDl 3'UTR proximally.
  • the WS-215 deletion is 160kb between CW15 and SM6-JH17.
  • WS-194 has 65kb deleted between CW20 and CW10-CW36.
  • WS-227 has a 50kb deletion between CW20 and JH11 and WS-219 has a 27kb deletion between JHl and JH6.
  • the distal end of the WS-250 deletion is in CW20 but the precise location of the proximal end is not known.
  • a novel fragment of ⁇ 100kb is seen in WS-219 with probes H2 and CW10 which flank the deletion in this patient. JHl is partially deleted but detects the novel band weakly. The aberrant fragment is not detected by CW-21, which is deleted on the mutant chromosome. BamHI digested DNA of normal control (N) and WS-219 separated by conventional gel electrophoresis and hybridized to probes JHl and JH6 which flank the deletion. The same breakpoint fragment of ⁇ 3kb is seen with both probes, consistent with a deletion of ⁇ 27kb ending within the BamHI fragments seen by these probes. Two further deletions
  • a polyadenylation signal is present at nucleotides 5598-5603 and a polyA tail was detected in two independent cDNAs (AH4 and AH6 ) at position, 5620.
  • the predicted amino acid sequence from the available open reading frame extends 1614 residues, and is shown in Figure 7.
  • This proximal site contains three gene loci (HG-A, -B and -C) that each produce polyadenylated mRNAs and share substantial homology to the PKDl gene; it is not known whether these partially homologous transcripts are translated into functional proteins.
  • RNA isolated from a radiation hybrid HY145.19, that contains just the PKDl part of chromosome 16, and not the duplicate site in 16pl3.1.
  • this hybrid produces transcripts from the PKDl gene but not from the homologous genes (HG-A, HG-B and HG-C).
  • HG-A, HG-B and HG-C homologous genes
  • the PKDl cDNA contig whose sequence is shown in Figure 10 is made up of (3' -5') the original 5.7 kb of sequence shown in Figure 7, and the cDNAs: gap 22 (890 bp), gap gamma (872 bp), a section of genomic DNA from the clone JH8 (2,724 bp) which corresponds to a large exon, S1-S3 (733 bp), S3-S4 (1,589 bp) and S4-S13 (1,372 bp). Together these make a cDNA of 13,807nt.
  • HG cDNA clones identified by the DNA probes JH8 or JH13 were sequenced. Clones identified by JH8 were chosen because this genomic area is duplicated fewer times than the surrounding DNA, with only the HG-A and HG-B transcripts (not HG-C) homologous to this region.
  • the comparison of these cDNA and genomic sequences showed a characteristic intron/exon pattern and we concluded that the exons highlighted in the genomic sequence were likely to be exons of the PKDl gene.
  • RNA from a radiation hybrid, HY145.19 that contains the PKDl but not the HG loci
  • PKDl specific cDNAs were amplified by RT-PCR and cloned (see Experimental Procedures for details) . In this way, a number of overlapping cDNAs spanning the PKDl transcript, for the cDNAs at the 3' end to those homologous to JH13 were cloned ( Figure 13) .
  • HG-6 showed that a short region (-100 bp) of HG-6 lay 5' to the sequenced genomic region and this was located by hybridisation to the genomic clone SM3 (figure 13); SM3 was subsequently sequenced. The position of the cDNA in SM3 was identified and the possible 5' extent of this exon was determined in the genomic sequence; and in-frame stop codon was identified hear the 3' end of the exon.
  • FIG. 14 shows a map of the PKDl CpG island including genomic sites for several methylation sensitive enzymes, the location of the first exon and the GC content across the island.
  • the total length of the predicted protein is 4302 aa with a calculated molecular mass after excision of the signal peptide of 460 kD and an estimated isoelectric point of 6.26. However, this may be an underestimate of the total mass of the protein as many potential sites for N-linked glycosylation are present ( Figure 15). Homologies with the PKDl protein
  • LRRs leucine rich-repeats
  • flank-LRR-flank structure Surrounding the LRRs are distinctive cysteine-rich amino and carboxy flanking regions ( Figures 16b and c). This flank-LRR-flank structure is exclusively found on proteins in extracellular locations and is thought to be involved in protein-protein interactions such as adhesion to other cells or to components of the extracellular matrix or as a receptor concerned with binding or signal transduction.
  • the structure found in the PKDl protein is similar to that found in the Drosophila protein, slit, which is important for normal central nervous system development (Rothberg, 1990).
  • slit contains far more LRRs than the PKDl protein, with four blocks each consisting of 4 or 5 repeat units, the structure of each block is similar as they finish on the amino and carboxy side with shortened LRRs which are immediately flanked by the cysteine rich regions.
  • two shortened LRRs surround one complete repeat unit and immediately abut the amino and carboxy flanking regions.
  • the amino flanking region consists of four invariant cysteines and a number of other highly conserved residues in an area of 30-40 amino acids; comparison of the PKDl region to amino flanking motifs of other proteins is shown in figure 4b.
  • the carboxy flanking region extends over an area of between 50-60 residues and consists of an invariant proline and four cysteines plus several other highly conserved amino acids.
  • the similarity of the PKDl region to carboxy flanking regions from other proteins is shown in figure 4c.
  • LRR proteins such as slit (Rothberg 1990) and small proteoglycans are wholly extracellular but others including Toll (Hashimoto et al, 1990) and trkc ( Lamballe 1991) have a single transmembrane sequence, while the LH-CRG receptor and related proteins have seven trans-membrane segments and are involved in signal transduction.
  • C type lectin domain Analysis of the sequence from exons 6 and 7 showed a high level of homology with a C type lectin domain.
  • C type lectins are found in a variety of proteins in extracellular locations where they bind specific carbohydrates in the presence of Ca 2 + ion (Drickamer 1987, 1988; Weiss 1992).
  • Figure 17 illustrates the similarity of the PKDl lectin domain to those found in a number of proteins including: proteogylcans, which interact with collagens and other components of the extracellular matrix; endocytic receptors, and selectins which are involved in cell adhesion and recognition.
  • proteogylcans which interact with collagens and other components of the extracellular matrix
  • endocytic receptors and selectins which are involved in cell adhesion and recognition.
  • Three different selectins have been identified: E-selectin (endothelium) , P-selectin (platelets) and L-selectin (lymphocytes) and these work with other cell adhesion molecules to promote binding of the cell carrying the selectin to various other target cells.
  • Figure 18 shows that a highly conserved structure is maintained between the repeats although in some cases less similarity is noted with the WDFGDGS sequence. Further analysis of the most conserved residues found in the repeat units showed similarity to various immunoglobulin (Ig) domains; two Ig repeats which show particular homology to the PKDl protein are shown ( figure 18 ) . The repeat unit is most similar to that found in a number of cell adhesion and surface receptors which have recently been defined as the I set of Ig domains (Harpaz 1994). Ig repeats consist of 7-9 ⁇ strands of 5-10 residues linked by turns which are packed into two ⁇ sheets.
  • Ig immunoglobulin
  • the B, C, F and G ⁇ -strands of the I set are particularly similar to the PKDl repeat, although the highly conserved cystine residues which stabilise the two ⁇ sheets through a disulphide bond are absent.
  • the D and E ⁇ strands seem less similar and in some cases are significantly shortened or apparently absent.
  • this PKDl repeat has an Ig-like structure is found by analysis of the secondary structure with the predominant configuration found of ⁇ strands linked by turns.
  • the WDFGDS area of the Ig molecule is one that often has a specific binding function (Jones et al. , 1995) and this sequence may have a specific binding role in polycystxn.
  • Type III fibronectin-related domains Analysis of the secondary structure of the PKDl protein beyond the carboxy end of the region of Ig-like repeats showed a continuation of the ⁇ stand and turn structure. No evidence of further Ig-like repeats could be found in this area but three pairs of evenly spaced (38-40aa) tryptophan and tyrosine residues was noted which are the most highly conserved positions of the type III fibronectin repeat which has a similar secondary structure to Ig domains.
  • Ig/FNIII containing proteins such as neuroglican and NrCAM are thought to be involved in neuron-neuron interactions and the patterning of the axonal network.
  • Many cell adhesion proteins of the Ig superfamily are also involved in communication and signal transduction mediated through their cytoplasmic tails. These cytoplasmic regions are known to bind to cytoskeletal proteins and other intracellular components, and phosphorylation of this part of the molecule is also thought to affect adhesive properties of the protein; potential phosphorylation sites are found in the cytoplasmic tail and one intracellular loop of polycystin ( Figure 20) .
  • the N-terminal end is extracellular and the (highly hydrophobic) carboxy-terminal region is anchored to the membrane by 11 membrane-spanning segments, with the highly charged carboxy end located in the cytoplasm.
  • This topology is supported by the study of N-glycosylation sites with all but one site, out of a total of 61 predicted, in an extracellular location according to the model, including 11 in the two large extracellular loops between TM regions.
  • degree of hydrophobicity required to define a certain putative transmembrane region is altered within the model, the predicted number of such domains can change to 9 (excluding the most N-terminal pair) or 13 (with two new domains defined between TM7 and TM8 ) . This can be ascertained by studies with specific antibodies.
  • transmembrane proteins containing the types of cell adhesion domain found on polycystin have a single transmembrane domain.
  • the role of the multiple membrane spanning domains found in polycystin is not yet clear. Proposed structure of the PKDl protein
  • PKDl protein sequence From the detailed analysis of the predicted PKDl protein sequence a model of the likely structure of the protein can be formulated ( Figure 20) .
  • This model predicts an extracellular N-terminal region of approximately 2550 aa containing several distinctive extracellular domains and an intracellular C-terminus of approximately 225 aa.
  • the intervening region of nearly 1500 aa is associated with the membrane with 11 transmembrane regions predicted and 10 variously sized extracellular and cytoplasmic loops (see Figure 20) .
  • a proline rich hinge is found between the flank-LRR-flank region and the first Ig-like repeat.
  • Two phosphorylation sites for tyrosine kinase and protein kinase C are found in cytoplasmic locations ( Figures 15 and 20). Therefore, the PKDl protein, named polycystin, has highlighted several clear domains, plus a reiterated motif that occupies over 30% of the protein.
  • PKDl gene Characterisation of the PKDl gene has proven to be a uniquely difficult problem because most of the gene lies in a region which is reiterated elsewhere on the chromosome.
  • the high degree of similarity between the two areas ()97%) both in exons and introns has meant that a novel approach has been required to clone the full length transcript; involving extensive genomic sequencing and generating cDNAs from a cell line with the PKDl but not the HG loci. In this way a contig containing the entire PKDl transcript has now been cloned.
  • Preliminary analysis shows that the HG genes are very similar to PKDl both in terms of genomic structure and sequence over most of their length (apart from the novel 3' regions) .
  • the 5' end of the PKDl gene is at a CpG island which lies within the duplicated area. Homologous areas to this island, in the HG region, also have cleavable sites for methylation sensitive enzymes; these duplicate islands probably lie at the 5' ends of the various HG genes. Analysis for DNAase hypersensitivity also indicates that the HG, CpG islands probably contain active promoters. These results are consistent with the observation of polyadenylated mRNA from the HG genes on Northern blots and the similarity of the expression pattern of the HG and PKDl genes in different tissue specific cell lines.
  • the HG genes may have complete open reading frames and may encode functional proteins.
  • PKDl transcript is large, the overall size of the gene, at 52 kb, is not (the Duchenne muscular dystrophy (DMD) gene which encodes a slightly smaller transcript has a genomic size of over 2Mb). Indeed, if the first intron of PKDl is excluded from the analysis, 40.3% of the remainder of the gene is found in the mature mRNA. In the compact structure of the PKDl gene, some of the introns are close to or smaller than the minimal size of 80 bp thought to be required for efficient splicing, although they are presumably excised effectively.
  • DMD Duchenne muscular dystrophy
  • polycystin has many features of a cell adhesion or recognition molecule with multiple different extracellular domains. These various binding domains are likely to have different specificities so that it can be envisaged that it will bind to a variety of different proteins (and carbohydrates) both on other cells and possibly in the extracellular matrix. Although provisional evidence indicates a wide range of expression of polycystin in tissue specific cell lines, detailed analysis by in situ of the mRNA and with antibodies to determine the cells expressing this protein both in adult tissue and during development will provide further evidence.
  • ADPKD kidneys abnormal thickening and splitting of the basement membrane (BM) and simultaneous de-differentiation of associated epithelial cells at the point of tubular dilation. Similar results have been noted in the heterozygote Han:SPRD rat (Schafer et al., 1994) which is a dominant model of PKD, although it is not known if it is a rat model of PKDl.
  • Concurrent changes in cellular characteristics and the BM suggests that a disruption or alteration of communication between the cell and the BM may be the primary change in this disease.
  • Polycystin could play an important role in interaction and communication between epithelial cells and the BM.
  • ECM extracellular matrix
  • communication from the ECM to cells is required for control of cellular differentiation.
  • Communication between the ECM and cells occurs by several different means including through integrins and so polycystin may bind to integrins, although it may interact directly with components of the ECM.
  • ADPKD is generally a disease of adulthood, there is plenty of evidence that the cystic changes in the kidney may start much earlier (Milutinovic et al., 1970), even in utero (Reeders, 1986).
  • Expression of polycystin during renal development may be when its major role occurs, perhaps in assembly of the BM and it is then that the errors, which later lead to cyst development, occur.
  • proteins may make functional protein (they all produce abnormal mRNA) and it is interesting to note that, in each case, these proteins would have an intact extracellular region with disrupted cytoplasmic and transmembrane areas. Such proteins may bind to extracellular targets but are unable to communicate in a normal way.
  • PKDl mutations of the PKDl gene give rise to the typical phenotype of ADPKD.
  • the present invention therefore includes the complete PKDl gene itself and the six PKDl - associated mutations which have been described: a de novo translocation, which was subsequently transmitted with the phenotype; two intragenic deletions (one a de novo event); two further deletions; and a splicing defect. It has been argued that PKDl could be recessive at the cellular level, with a second somatic mutation required to give rise to cystic epithelium (Reeders, 1992).
  • the location of the PKDl mutations may, however, reflect some ascertainment bias as it is this single copy area which has been screened most intensively for mutations. Nevertheless, no additional deletions were detected when a large part of the gene was screened by FIGE, and studies by PFGE showed no large deletions of this area in 75 PKDl patients. It is possible that the mutations detected so far result in the production of an abnormal protein which causes disease through a gain of function. However, it is also possible that these mutations eliminate the production of functional protein from this chromosome and result in the PKDl phenotype by haploinsufficiency, or only after loss of the second PKDl homologue by somatic mutation.
  • At least one mutation which seems to delete the entire PKDl gene has been identified (WS-53) but in this case it also disrupts the- adjacent TSC2 gene and the resulting phenotype is of TSC with severe cystic kidney disease. Renal cysts are common in TSC so that the phenotypic significance of deletion of the PKDl gene in this case is difficult to assess. It is clear that not all cases of renal cystic disease in TSC are due to disruption of the PKDl gene; chromosome 9 linked TSC (TSC1 ) families also manifest cystic kidneys and we have analysed many TSC2 patients with kidney cysts who do not have deletion of the PKDl gene.
  • TSC1 chromosome 9 linked TSC
  • LRRs leucine-rich repeats
  • amino acids 29-74 flanked by characteristic amino flanking (amino acids 6-28) and carboxy flanking sequences (amino acids 76-133) (Rothberg et al., 1990).
  • LRRs are thought to be involved in protein-protein interations (Kobe and Deisenhofer, 1994) and the flanking sequences are only found in extracellular proteins.
  • Other proteins with LRRs flanked on the amino and carboxy sides are receptors or are involved in adhesion or cellular signalling.
  • 77-2 and 77-3 are 48 and 17 years old, respectively and have typical ADPKD. Both have bilateral polycystic kidneys and 77-2 has impaired renal function. Neither patient manifests any signs of TSC (apart from cystic kidneys) on clinical and ophthalmological examination or by CT scan of the brain.
  • 77-4 is 13 years old, severely mentally retarded and has multiple signs of tSC including adenoma sebaceum, depigmented macules and periventricular calcification on CT scan. Renal ultrasound reveals a small number of bilateral renal cysts.
  • ADPKD patients 0X875 developed ESRD from ADPKD, aged 46. Progressive decline in renal function had been observed over 17 years; ultrasound examinations documented enlarging polycystic kidneys with less extensive hepatic cystic disease. Both kidneys were removed after renal transplantation and pathological examination showed typical advanced cystic disease in kidneys weighing 1920g and 340g (normal average 120g) .
  • 0X114 developed ESRD from ADPKD aged 54: diagnosis was made by radiological investigation during an episode of abdominal pain aged 25. A progressive decline in renal function and the development of hypertension was subsequently observed. Ultrasonic examination demonstrated enlarged kidneys with typical cystic disease, with less severe hepatic involvement.
  • 0X32 is a member of a large kindred affected by typical
  • ADPKD in which several members have developed ESRD.
  • the patient himself has been observed for 12 years with progressive renal failure and hypertension following ultrasonic demonstration of polycystic kidneys.
  • FIGE was performed with the Biorad FIGE Mapper using programme 5 to separate fragments from 25-50 kb. High molecular weight DNA for PFGE was isolated in agarose blocks and separated on the Biorad CHEF DRII apparatus using appropriate conditions.
  • Genomic DNA probes and somatic cell hybrids Many of the DNA probes used in this study have been described previously: MS205.2 (D16S309; Royle, et al., 1992); GGG1 (D16S259; Germino, et al. , 1990); N54 (D16S139; Himmelbauer, et al., 1991); SM6 (D16S665), CW23, CW21, and JHl (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). Microsatellite probes for haplotype analysis were
  • New probes isolated during this study were: JH4, JH5, JH6, 11 kb, 6 kb and 6 kb BamH I fragments, respectively, and JH13 and JH14, 4 kb and 2.8 kb BamH I-EcoR I fragments, respectively, all from the cosmid JH2A; JH8 and JH10 are 4.5 kb and 2 kb Sac I fragments, respectively and JH12 a 0.6 Sac I-BamH I fragment, all from JH4; 8S1 and 8S3 are 2.4 kb and 0.6 kb Sac II fragments, respectively, from JH8; CW10 is a 0.5 kb Not I-Mlu I fragment of SM25A; JH17 is a 2 kb EcoR I fragment of NM17.
  • Somatic cell hybrids N-OHl (Germino, et al., 1990), P-MWH2A (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and Hyl45.19 (Himmelbauer, et al., 1991) have previously been described.
  • Cosmids were isolated from chromosome 16 specific and total genomic libraries, and a contig was constructed using the methods and libraries previously described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). To ensure that cosmids were derived from the 16pl3.3 region (not the duplicate 16pl3.1 area) initially, probes from the single copy area were used to screen libraries (e.g. CW21 and N54 ) . Two cosmids mapped entirely within the area duplicated, CWIOIII and JC10.2B. To establish that these were from the PKDl area, they were restriction mapped and hybridised with the probe CW10. The fragment sizes detected were compared to results obtained with hybrids containing only the 16pl3.3. are (Hyl45.19) or only the 16pl3.1 region (P-MWH2A) .
  • FISH FISH
  • FISH FISH was performed essentially as previously described (Buckle and Rack, 1993).
  • the hybridisation mixture contained 100 ng of biotin-II-dUTP labelled cosmid DNA and 2.5 mg human Cot-1 DNA (BRL), which was denatured and annealled at 37°C for 15 min prior to hybridisation at 42°C overnight. After stringent washes the site of hybridisation was detected with successive layers of fluorescein- con ugated avidin (5 mg/ml ) and biotinylated ani-avidin (5 mg/ML) Vector Laboratories).
  • Foetal brain cDNAs libraries in ⁇ phage were screened by standard methods with genomic fragments in the single copy area (equivalent to CW23 and CW21 ) or with a 0.8 kb Pvu II-Eco RI single copy fragment of AH3.
  • Six PBP cDNAs were characterised; AH4 (1.7 kb ) and 3A3 (2.0 kb) are described in European Chromosome 16 Tuberous Sclerosis Consortium, 1993, and four novel cDNAs AH3 (2.2 kb), AH6 (2.0 kb), A1C (2.2 kb) and B1E (2.9 kb) .
  • a Striatum library (Stratagene) was screened with JH4 and a HG-C cDNA, 11BHS21 (3.8 KB) WAS ISOLATED, 21p.9 is a 0.9 kb Pvu II-EcoR I subclone of this cDNA.
  • HG-4/1.1 is a 1.1 kb Pvu II- EcoR I fragment from the 3' end of HG-4.
  • 1A1H.6 is a 0.6 kb Hind III-EcoR I subclone of a TSC2 cDNA, 1A-1 (1.7 kb), which was isolated from the Clonetech library.
  • Each cDNA was subcloned into Bluescript and sequenced utilising a combination of sequential truncation and liigonucleotide primers using DyeDeoxy Terminators (Applied Biosystems ) and an ABI 373A DNA Sequencer (Applied Biosystems) or by hand with 'Sequenase' T7 DNA polymerase OUSB) .
  • RNA electrophoresis 0.5% agarose denaturing formaldehyde gels were used which were Northern blotted. hybridised and washed by standard procedures.
  • the 0.24 - 9.5 kb RNA (Gibco BRL) size standard was used and hybridisation of the probe (1-9B3) to the 13 kb Utrophin transcript (Love, et al. , 1989) in total fibroblast RNA was used as a size marker for the large transcripts.
  • RT-PCR was performed with 2.5 mg of total RNA by the method of Brown et al. (1990) with random hexamer primers, except that AMV-reverse transcriptase (Life Sciences) was employed.
  • AMV-reverse transcriptase Life Sciences
  • the 3A3 C primers used to amplify the 0X32 cDNA and DNA were: 3A3 C15' CGC CGC TTC ACT AGC TTC GAC 3' 3A3 C25' ACG CTC CAG AGG GAG TCC AC 3' These were employed in a PCR buffer and cycle previously described (Harris, et al., 1991) with ImM MgCl 2 and an annealing temperature of 61°C.
  • PCR products for sequencing were amplified with Pfu-1 (Stratagene) and ligated into the Srf-1 site in PCR-Script (Stratagene) in the presence of Srf-1.
  • RNA samples from normal and end-stage polycystic kidneys were immediately homogenised in guanidinium thiocyanate.
  • RNA was purified on a cesium chloride gradient and 30 mg total RNA was assayed by RNAse protection by the method of Melton, et al. , (1984) using a genomic template generated with the 3A3, C primers.
  • Heteroduplex Analysis Heteroduplex analysis was performed essentially as described by Keen et al. (1991). Samples were amplified from genomic DNA with the 3A3, C primers, heated at 95°C for 5 minutes and incubated at room temperature for at least 30 minutes before loading on a Hydrolink gel (AT Biochem) . Hydrolink gels were run for 12-18 hours at 250V and fragments observed after staining with ethidiu bromide. Extraction and amplification of paraffin-embedded DNA
  • DNA from formalin fixed, paraffin wax embedded kidney tissue was prepared by the method of Wright and Manos (1990), except that after proteinase K digestion overnight at 55°C, the DNA was extracted with phenol plus chloroform before ethanol precipitation. Approximately 50 ng of DNA was used for PCR with 1.5 mM MgCl 2 and 40 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 40 s, plus a 10 min extension at 72°C.
  • the oligonucleotide primers designed to amplify across the genomic deletion of 0X875 were: AHF2 : 5' - GGG CAA GGG AGG ATG ACA AG - 3' JH14B3 : 5' - GGG TTT ATC AGC AGC AAG CGG - 3' which produced a product of about 220 bp in individuals with the 0X875 deletion. 3' RACE analysis of WS-212
  • 3' RACE was completed essentially as described (European Polycystic Kidney Disease Consortium (1994)). Reverse transcription was performed with 5 ⁇ g total RNA with 0.5 ⁇ g of the hybrid dT : - adapter primer using conditions previously described (Fronman et al.. (1988)). A specific 3' RACE product was amplified with the primer F5 and adapter primer in 0.5mM MgCl 2 with the program: 57°C, 60s; 72°C, 15 minutes and 30 cycles of 95°C, 40s; 57°C, 60s; 72°C, 60s plus 72°C, 10 minutes. The amplified product was cloned using the TA cloning system ( Invitrogen) and sequenced by conventional methods.
  • TA cloning system Invitrogen
  • the genomic clones CW21, JH5, JH6, JH8, JH10. JH12, JH13 and JH14 and the cDNAs A1C, AH3, 3A3 and AH4 are described herein. Newly described probes are: SM3 a 2.0 kb BamH 1 subclone of the cosmid SM11, JH9, 2.4kb Sac 1 fragment and JH11, 1.2kn Sac 1 - BamHI fragment, both from JH4. See Eur. Polycystic Kidney Disease Consortium, 1994 and Eur. Chromosome 16 Tuberous clerosis Consortium 1993 for all above clones. DFS5 is a 4.2 kb Not 1-Hind 111 fragment of CW23 (Eur.
  • the cDNAs; BPG4, BPG6, BPG7C and 13-A were isolated from a fetal brain cDNA library in ⁇ phage (Stratagene ) and are 7 kb, 2 kb, 4.5 kb and 1.2 kb respectively.
  • RNA from the radiation hybrid Hyl45.19 was reverse transcribed using random hexamers (Eur. Polycystic Kidney Disease Consortium, 1994). This material was used as a templa ⁇ e for PCR using the proof reading polymerase Pfu-1 with the primer pairs described in Table 2. The resultant products were cloned into the Srf-1 site of pPCRscript (SK+ ) plasmid. Sequencing
  • Primer extension was performed on total cellular fibroblast RNA. 25 ⁇ g of RNA was annealed at 60 e C in the presence of 400mM NaCl to O.OlpM of HPLC pure oligonucleotide which had been end labelled to a specific activity of 3 x 10 cpm/pM with ;i P. Primer extension was then performed in the presence of 50mM Tris pH8.2, lOmM DTT, 6mM MgCl 2 , 25mg/ml Actino ycin D. 0.5mM dNTPs, and 8 units of AMV reverse transcriptase. The extension reaction was continued for 60 min at 42°C.
  • the extension products were compared to a sequencing ladder generated using the same primer on the genomic clone SMS.
  • the primers used were: N2765: 5 ' -GGCGCGGCGGGCGGCATCGTTAGGGCAGCG-3 ' N5496: 5 ' -GGCGGGCGGCATCGTTAGGGCAGCGCGC-3' N5495:5 '-ACCTGCTGCTGAGCGACGCCCGCTCGGGGC-3' .
  • the predicted PKDl protein was analyzed for homologies with known proteins in the SwissProt and NBRF database using the BLAST (Altschul et al. , 1990) and FASTA (Pearson et al., 1988) algorithms. Layouts were prepared by hand and using the programme Pileup. Transmembrane regions
  • transmembrane segments were identified by the method of Sipos and von Heljne (Sipos et al., 1993), using the GES hydrophobicity scale (Engelmen et al., 1986) and a trapezoid sliding window (a full window of 21 residues and a core window of 11 residues), as recommended.
  • Candidate transmembrane domains were selected on the basis of their average hydrophobicity ⁇ H>, and were classified as certain ( ⁇ H> 1.0) or putative (0.6 , ⁇ H> ⁇ 1).
  • the best topology for the protein was predicted on the basis of three different criteria: a) the net charge difference between the 15 N-terminal and the 15 C-terminal residues flanking the most N-terminal transmembrane segment (Hartmann et al., 1989); b) the difference in positively charged residues between the two sides of the membrane in loops smaller than 60 residues, and c) the analysis of the overall amino acid composition of loops longer than 60 residues by the compositional distance method (Nakashima et al., 1992).
  • the TopPred II program Sipos wt al., 1993
  • the PKDl protein may be purified according to conventional protein purification procedures well known in the art.
  • the protein may be purified from cells harboring a plasmid containing an expressible PKDl gene.
  • the protein may be expressed in an E.coli expression system and purified as follows. Cells are grown in a 10 liter volume in a Chemap Fermentor (Chemapec, Woodbury, NY) in 2% medium. Fermentation temperature may be 37'C, pH 6.8, and air as provided at 1 wm. Plasmid selection may be provided using ampicillin for a plasmid containing an ampicillin resistance gene. Typical yield (wet weight) is 30 g/1.
  • 50g wet cell weight of E.coli containing the recombinant PKDl plasmid may be resuspended in a final volume of 100ml in 50 mM Tris-HCl pH 8.0, 5 M EDTA, 5mM DTT, 15 mM mercaptoethanol, 0.5% triton X-100, and 5 mM PMSF.
  • 300 mg lysozyme is added to the suspension, and incubated for 30 min at room temperature.
  • the material is then lyzed using a BEAD BEATER (R) (Biospec Products, Bartlesville, OK) containing an equal volume of 0.1-0.15 urn glass beads.
  • the liquid is separated from the beads and the supernatant removed, the pellet dissolved in 20 mM Tris-Cl pH 8.0.
  • the protein may be purified from the supernatant using DEAE chromatography, as is well known in the art. Preparation of Antibodies.
  • Antibodies specific for PDK1 protein or a fragment thereof are prepared as follows. A peptide corresponding to at least 8 amino acid residues of the PKDl sequence of Fig. 15, are synthesized. Coupling of the peptide to carrier protein and immunizations is performed as described (Dymecki, S.M., J. Biol. Chem 267:4815-4823, 1992). Rabbit antibodies against this peptide are raised and sera are titered against peptide antigen by ELISA. The sera exhibiting the highest titer (1:27,000) are most useful.
  • monoclonal antibodies of this invention may be prepared by using the synthetic polypeptides of this invention, preferably bound to a carrier, as the immunogen as was done by Arnheiter et al., Nature, 294, 278-280 (1981).
  • Monoclonal antibodies are typically obtained from hvbridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being “raised to” or “induced by” the synthetic polypeptides of this invention or their conjugates with a carrier.
  • Antibodies are utilized along with an "indicating group” also sometimes referred to as a "label”.
  • the indicating group or label is utilized in conjunction with the antibody as a means for determining whether an immune reaction has taken place, and in some instances for determining the extent of such a reaction.
  • the indicating group may be a single atom as in the case of radioactive elements such as iodine 125 or 131, hydrogen 3 or sulfur 35, or NMR-active elements such as fluorine 19 or nitrogen 15.
  • the indicating group may also be a molecule such as a fluorescent dye like fluorescein, or an enzyme, such as horseradish peroxidase (HRP), or the like.
  • indicating group or “label” are used herein to include single atoms and molecules that are linked to the antibody or used separately, and whether those atoms or molecules are used alone or in conjunction with additional reagents. Such indicating groups or labels are themselves well-known in immunochemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel antibodies, methods and/or systems.
  • Another embodiment of this invention relates to an assay for the presence of PKDl protein in cells.
  • an above-described antibody is raised and harvested.
  • the antibody or idiotype-containing polyamide portion thereof is then admixed with candidate tissue and an indicating group.
  • the presence of the naturally occurring amino acid sequence is ascertained by the formation of an immune reaction as signaled by the indicating group.
  • Candidate tissues include any tissue or cell line or bodily fluid to be tested for the presence of PKDl.
  • Metabolic labeling immunoprecipitation. and immunolocalization assays are performed in cells as described previously (Furth, M.E., et al., Oncogene 1:47-58, 1987; Laemmli, U.K., Nature 227:680-685, 1970; Yarden, Y. , et al., EMBO J. 6:3341-3351, 1987; Konopka, J.B., et al., Mol. Cell. Biol. 5:3116-3123, 1985).
  • total lysates are prepared (using Fruth's lysis buffer) (Fruth, M.E., et al., Oncogene, 1:47-58, 1987).
  • Relative protein concentrations are determined with a colorimetric assay kit (Bio-Rad) with bovine serum albumin as the standard.
  • a protein of lysate containing approximately 0.05 mg of protein is mixed with an equal volume of 2 x SDS sample buffer containing 2 mercaptoethanol, boiled for 5 min., fractioned on 10% polyacrylamide-SDS gels (Konopka, J.B., et al., J.Virol., 51:223-232, 1984) and transferred to immunobilon polyvinyldine difluoride (Millipore Corp., Bedford, MA) filters. Protein blots are treated with specific antipeptide antibodies (see below). Primary binding of the PKDl-specific antibodies is detected using anti-igG second antibodies conjugated to horseradish peroxidase and subsequent chemiluminescence development ECL Western blotting system (Amersham International).
  • CMK cells are resuspended in 1 ml of sonication buffer ( 60mM Tris-HCl, pH 7.5, 6 mM EDTA, 15 mM EGTA, 0.75M sucrose, 0.03% leupeptin 12mM phenylmethylsulfonyl fluoride, 30 mM 2- mercaptoethanol) .
  • Cells are sonicated 6 times for 10 seconds each and centrifuged at 25,000 xg for 10 min at 4°C. The pellet is dissolved in 1 ml of sonication buffer and centrifuged at 25,000 x g for 10 min at 4°C.
  • the pellet (nucleus fraction) is resuspended in 1 ml of sonication buffer and added to an equal volume of 2 x SDS sample buffer.
  • the supernatant obtained above (after the first sonication) is again centrifuged at 100,000 x g for 40 min at 4°C.
  • the supernatant (cytosolic fraction) is removed and added to an equal volume of 2 x concentrated SDS sample buffer.
  • the remaining pellet (membrane fraction) is washed and dissolved in sonication buffer and SDS sample buffer as described above. Protein samples are analyzed by electrophoresis on 10% polyacrylamide gels, according to the Laemmli method (Konopka, J.B., supra).
  • the proteins are transferred from the gels on a 0.45- ⁇ m polyvinylidine difluoride membrane for subsequent immunoblot analysis.
  • Primary binding of the PKDl specific antibodies is detected using anti-igG second antibodies conjugated to horseradish peroxidase.
  • CMK cells or U3T3 are grown on cover slips to approximately 50% confluence and are washed with PBS (pH 7.4) after removing the medium.
  • the cells are prefixed for 1 min at 37°C in 1% paraformaldehyde containing 0.075% Triton X-100, rinsed with PBS and then fixed for 10 min with 4% paraformaldehyde. After the fixation step, cells are rinsed in PBS, quenched in PBS with o.l and finally rinsed again in PBS.
  • the cells are first blocked with a blocking solution (3% bovine serum albumin in PBS) and incubated for 1 h at 37°C.
  • the cells are then incubated for 1 h at 37°C with antiserum (1:100 dilution or with preimmune rabbit serum ( 1:100) . After the incubation with the primary antibody, the cells are washed in PBS containing 3% bovine and serum albumin and 0.1% Tween 20 and incubated for 1 h at 37 C in fluorescein-conjugated donkey anti-rabbit IgGs (Jackson Immunoresearch, Maine) diluted 1:100 in blocking solution.
  • the coverslips are washed in PBS (pH 8.0), and glycerol is added to each coverslip before mounting on glass slides and sealing with clear nail polish. All glass slides are examined with a Zeiss Axiophot microscope.
  • An indicating group or label is preferably supplied along with the antibody and may be packaged therewith or packaged separately. Additional reagents such as hydrogen peroxide and diaminobenzideine may also be included in the system when an indicating group such as HRP is utilized. Such materials are readily available in commerce, as are many indicating groups, and need not be supplied along with the diagnostic system. In addition, some reagents such as hydrogen peroxide decompose on standing, or are otherwise short-lived like some radioactive elements, and are better supplied by the end-user. Pharmaceutical Compositions of the Invention; Dosage and Administration
  • compositions comprising PKDl nucleic acid or protein, or mutants thereof, can be prepared by procedures well known in the art.
  • injectables e.g., liquid solutions or suspensions.
  • Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared.
  • the preparation also can be emulsified.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • water, saline, dextrose, glycerol, ethanol, etc. or combinations thereof are also useful. Also useful are wetting or emulsifying agenrs, pH buffering agents or adjuvants.
  • PKDl protein or DNA can be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • the active protein or the nucleic acid will be present in the range of about 0.05% to about 10%, preferably in ther ange of about 1-2% by weight.
  • the active protein or the nucleic acid will be administered at a dosage of about 10mg-2kg/kg body weight, preferably 50mg- 400mg/kg/body weight. Administration may be daily, weekly, or in a single dosage, as determined by the physician.
  • Reeders et al. Lancet i, 6-8, (1986). Reeders et al., Nature 317:542-544, (1985).

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GB9411900A GB9411900D0 (en) 1994-06-14 1994-06-14 Dna sequence encoding the polycystic kidney disease 1 gene and uses thereof
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JP2002503952A (ja) * 1996-05-24 2002-02-05 ジェンザイム・コーポレーション 多嚢胞性腎疾患遺伝子
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