WO2004074302A2 - Acides nucleiques et polypeptides associes a une polykystose renale autosomique recessive - Google Patents

Acides nucleiques et polypeptides associes a une polykystose renale autosomique recessive Download PDF

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Publication number
WO2004074302A2
WO2004074302A2 PCT/US2004/004778 US2004004778W WO2004074302A2 WO 2004074302 A2 WO2004074302 A2 WO 2004074302A2 US 2004004778 W US2004004778 W US 2004004778W WO 2004074302 A2 WO2004074302 A2 WO 2004074302A2
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seq
nucleic acid
complement
nucleotides
acid molecule
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PCT/US2004/004778
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English (en)
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WO2004074302A3 (fr
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Lisa Guay-Woodford
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The Uab Research Foundation
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Publication of WO2004074302A2 publication Critical patent/WO2004074302A2/fr
Publication of WO2004074302A3 publication Critical patent/WO2004074302A3/fr

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    • 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

Definitions

  • 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.
  • 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
  • FIG. 3(a) shows the tissue distribution of the 2.2-kb Cysl transcript in adult mouse tissues
  • 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 ⁇ -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.
  • 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.
  • 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.
  • mutant 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • BLAST Basic Local Alignment Search Tool
  • 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.
  • Gapped BLAST is utilized as described in Altschul et al.
  • 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.
  • 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.
  • 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.
  • 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.
  • ATTAAA atypical polyadenylation signal
  • 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.
  • 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).
  • N-terminal myristoylation acts as an intracellular membrane-associating signal.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Reverse transcription -PCR and northern blot analysis Reverse transcription was carried out in a 40 ⁇ l reaction volume with 10 ⁇ g 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 ⁇ g of kidney and liver poly(A + ) RNA pooled from
  • 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.
  • Beta IV is the major beta- tubulin isotype in bovine cilia.
  • a polybasic domain or palmitoylation is required in addition to thye CAAX motif to localize p21ras to the plasma membrane.

Abstract

Selon cette invention, un nouveau gène intervenant dans une polykystose rénale autosomique récessive a été isolé. Cette invention concerne la séquence d'acide nucléique du gène de souris, Cys1, et son homologue humain, CYS1. Cette invention concerne en outre un mutant de l'acide nucléique Cys1 contenant une délétion tandem. Les acides nucléiques Cys1 et CYS1 sont associés à la polykystose rénale autosomique récessive. Des analyses de Southern indiquent que le gène Cys1 est présent comme copie unique chez la souris et chez d'autres mammifères. L'acide nucléique Cys1 code une nouvelle protéine hydrophile composée de 145 acides aminés, appelée cystine, qui ne présente aucune similitude significative avec les protéines ou les domaines de protéines préalablement caractérisés. Le produit polypeptidique du gène Cys1 est détecté dans un cil et contient une séquence nécessaire à la localisation par rapport au cil.
PCT/US2004/004778 2003-02-18 2004-02-18 Acides nucleiques et polypeptides associes a une polykystose renale autosomique recessive WO2004074302A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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KR20210117523A (ko) * 2020-03-19 2021-09-29 지니너스 주식회사 섬모 손상 질환의 진단을 위한 ick 및 klc3의 용도

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5643758A (en) * 1987-03-10 1997-07-01 New England Biolabs, Inc. Production and purification of a protein fused to a binding protein

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Publication number Priority date Publication date Assignee Title
US5643758A (en) * 1987-03-10 1997-07-01 New England Biolabs, Inc. Production and purification of a protein fused to a binding protein

Non-Patent Citations (3)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210117523A (ko) * 2020-03-19 2021-09-29 지니너스 주식회사 섬모 손상 질환의 진단을 위한 ick 및 klc3의 용도
KR102345006B1 (ko) 2020-03-19 2021-12-29 지니너스 주식회사 섬모 손상 질환의 진단을 위한 ick 및 klc3의 용도

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