EP1060184A1 - Gene de la dystrophie corneenne de best - Google Patents

Gene de la dystrophie corneenne de best

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Publication number
EP1060184A1
EP1060184A1 EP99908345A EP99908345A EP1060184A1 EP 1060184 A1 EP1060184 A1 EP 1060184A1 EP 99908345 A EP99908345 A EP 99908345A EP 99908345 A EP99908345 A EP 99908345A EP 1060184 A1 EP1060184 A1 EP 1060184A1
Authority
EP
European Patent Office
Prior art keywords
seq
cgice
protein
dna
amino acid
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Withdrawn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
EP99908345A
Other languages
German (de)
English (en)
Other versions
EP1060184A4 (fr
Inventor
Konstantin Petrukhin
C. Thomas Caskey
Michael Metzker
Claes Wadelius
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.)
Uppsala University
Merck and Co Inc
Original Assignee
Uppsala University
Merck and Co Inc
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Publication date
Application filed by Uppsala University, Merck and Co Inc filed Critical Uppsala University
Publication of EP1060184A1 publication Critical patent/EP1060184A1/fr
Publication of EP1060184A4 publication Critical patent/EP1060184A4/fr
Withdrawn legal-status Critical Current

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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

  • the present invention is directed to novel human and mouse DNA sequences encoding a protein which, when present in mutated form, results in the occurrence of Best's Macular Dystrophy.
  • Macular dystrophy is a term applied to a heterogeneous group of diseases that collectively are the cause of severe visual loss in a large number of people.
  • a common characteristic of macular dystrophy is a progressive loss of central vision resulting from the degeneration of the pigmented epithelium underlying the retinal macula.
  • the end stage of the disease results in legal blindness.
  • macular dystrophy More than 20 types of macular dystrophy are known: e.g., age-related macular dystrophy, Stargardt's disease, atypical vitelliform macular dystrophy (VMD1), Usher Syndrome Type IB, autosomal dominant neovascular inflammatory vitreoretinopathy, familial exudative vitreoretinopathy, and Best's macular dystrophy (also known as hereditary macular dystrophy or Best's vitelliform macular dystrophy (VMD2)).
  • VMD1 atypical vitelliform macular dystrophy
  • VMD2 Best's macular dystrophy
  • Best's macular dystrophy also known as hereditary macular dystrophy or Best's vitelliform macular dystrophy (VMD2)
  • BMD Best's Macular Dystrophy
  • Age- related macular dystrophy is an extraordinarily difficult disease to study genetically, since by the time patients are diagnosed, their parents are usually no longer living and their children are still asymptomatic. Thus, family studies which have led to the discovery of the genetic basis of many other diseases have not been practical for age-related macular dystrophy. As there are currently no widely effective treatments for age- related macular dystrophy, it is hoped that study of BMD, and in particular the discovery of the underlying genetic cause of BMD, will shed light on age-related macular dystrophy as well.
  • Recombination breakpoint mapping in a large Swedish pedigree limited the minimum genetic region containing the BMD gene to a 980 kb interval flanked by the microsatellite markers D11S4076 and UGB (Graff et al., 1997, Hum. Genet. 101: 263-279).
  • a diagnostic test that relies on a blood sample from a patient suspected of being an asymptomatic carrier of BMD would be ideal.
  • the present invention is directed to novel human and mouse DNA sequences that encode the gene CGICE, which, when mutated, is responsible for Best's macular dystrophy.
  • the present invention includes genomic CGICE DNA as well as cDNA that encodes the CGICE protein.
  • the human genomic CGICE DNA is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:l.
  • the human cDNA encoding CGICE protein is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:2 or SEQ.ID.NO.:4.
  • the mouse cDNA encoding CGICE protein is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:28. Also provided is CGICE protein encoded by the novel DNA sequences. The human CGICE protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.ID.NO.:3 or SEQ.ID.NO.:5. The mouse CGICE protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.ID.NO.:29. Methods of expressing CGICE protein in recombinant systems are provided. Also provided are diagnostic methods that detect carriers of mutant CGICE genes.
  • Figure 1A-F shows the genomic DNA sequence of human CGICE (SEQ.ID.NO.:l). Underlined nucleotides in capitals represent exons. The start ATG codon in exon 2 and the stop TAA codon in exon 11 are shown in bold italics. The consensus polyadenylation signal
  • exon 7 is shown in underlined italics. The exact lengths of two gaps between exons 1 and 2 and between exons 7 and 8 are unknown; these gaps are presented as runs of ten Ns for the sake of convenience.
  • the portion of exon 11 beginning at position 15,788 represents the 3' untranslated region; 132 base pairs downstream of the polyadenylation signal of the CGICE gene are multiple ESTs, representing the 3'- untranslated region of the ferritin heavy chain gene (FTH).
  • FTH ferritin heavy chain gene
  • the FTH gene was later shown to be a part of the smallest minimum genetic region containing the BMD gene, as determined by recombination breakpoint mapping in a 12 generation Swedish pedigree (Graff et al ., 1997, Hum. Genet. 101: 263-279).
  • Figure 2 shows the complete sequence of the short form of human CGICE cDNA (SEQ.ID.NO.:2).
  • the ATG start codon is at position 105; the TAA stop codon is at position 1,860.
  • Figure 3 shows the complete amino acid sequence of the long form of human CGICE protein (SEQ.ID.NO.:3). This long form of the human CGICE protein is produced by translation of the short form of CGICE cDNA.
  • Figure 4 shows the complete sequence of the long form of human CGICE cDNA (SEQ.ID.NO.:4). This long form of the human CGICE cDNA is produced when an alternative splice donor site is utilized in intron 7. The ATG start codon is at position 105; the TGA stop codon is at position 1410.
  • Figure 5 shows the complete amino acid sequence of the short form of the human CGICE protein (SEQ.ID.NO.:5). This short form of the human CGICE orotein is produced by translation of the long form of CGICE cDNA.
  • Figure 6 shows the results of sequencing runs of PCR fragments that represent exon 4 and adjacent intronic regions from three individuals from the Swedish pedigree SI, two of whom are affected with BMD. From top to bottom, the runs are: patient Sl-5 (homozygous affected with BMD), sense orientation; patient Sl-4 (heteroozygous affected with BMD), sense orientation; patient Sl-3 (normal control, unaffected sister of Sl-4), sense orientation; patient Sl-5 (affected with BMD), anti-sense orientation; patient Sl-4 (affected with
  • CGICE protein with partial sequences of related proteins from C. elegans.
  • Related proteins from C. elegans were identified by BLASTP analysis of non-redundant GenBank database. This figure shows that two amino acids mutated in two different Swedish families with BMD (families SI and SL76) are evolutionarily conserved. 15 of 16 related proteins from C. elegans contain a tryptophan at the position of the mutation in family SI, as does the wild-type CGICE gene. Only one C. elegans protein does not have a tryptophan at the position of the mutation.
  • tryptophan is changed for isofunctional phenylalanine (phenylalanine is highly similar to tryptophan in that it also is a hydrophobic aromatic amino acid).
  • Mutation in the BMD family SL76 changes a tyrosine to histidine.
  • all 16 related proteins from C. elegans contain tyrosine or isofunctional phenylalanine in this position (tyrosine is highly similar to phenylalanine in that it also is an aromatic amino acid).
  • FIG 8 A -C shows the complete sequence of mouse CGICE cDNA (SEQ.ID.NO.:28) and mouse CGICE protein (SEQ.ID.NO.:29).
  • Figure 9A-B shows an alignment of the amino acid sequences of the long form of human CGICE protein (SEQ.ID.NO.:3) and mouse CGICE protein (SEQ.ID.NO.:29). In this figure, CGICE is refe ⁇ ed to as "bestrophin.”
  • Figure 10A-C shows the results of in situ hybridization experiments demonstrating that mouse CGICE mRNA expression is localized to the retinal pigmented epithelium cells (RPE).
  • Figure 10A shows the results of using an antisense CGICE probe. The antisense probe hybridizes to mouse CGICE mRNA present in the various cell layers of the retina, labeling with dark bands the cells containing CGICE mRNA. The antisense probe strongly hybridized to the RPE cells and not to the cells of the other layers of the retina.
  • Figure 10B shows the results using a sense CGICE probe as a control. The sense probe does not hybridize to CGICE mRNA and does not label the RPE cells.
  • FIG. 10A is a higher magnification of the RPE cells from Figure 10A.
  • Human CGICE mRNA shows a similar distribution, being confined to the RPE cells of the human retina.
  • substantially free from other proteins means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.
  • a CGICE protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non- CGICE proteins.
  • Whether a given CGICE protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Substantially free from other nucleic acids means at least 90%, preferably 95%, more preferably 99%, and even more preferably
  • a CGICE DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non- CGICE nucleic acids. Whether a given CGICE DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g. , ethidium bromide staining, or by sequencing.
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid); substitution of one aromatic amino acid (tryptophan, tyrosine, or phenylalanine) for another.
  • the present invention relates to the identification and cloning of CGICE, a gene which, when mutated, is responsible for Best's macular dystrophy. That CGICE is the Best's macular dystrophy gene is supported by various observations:
  • CGICE maps to the genetically defined region of human chromosome Ilql2-ql3 that has been shown to contain the Best's macular dystrophy gene. CGICE is present on two PAC clones, 759J12 and 466A11, that lie precisely in the most narrowly defined region that has been shown to contain CGICE (Cooper et al., 1997, Genomics 41:185- 192; Stihr et al, 1997, Genome Res. 8:48-56; Graffs al ., 1997, Hum. Genet. 101: 263-279).
  • CGICE is expressed predominately in the retina. 3. In patients having Best's macular dystrophy, CGICE cont ⁇ rins mutations in evolutionarily conserved amino acids.
  • the CGICE genomic clones contain another gene (FTH) that has been physically associated with the Best's macular dystrophy region (Cooper et al., 1997, Genomics 41:185-192; Stihr et al., 1997, Genome Res. 8:48-56; Graff et al, 1997, Hmn. Genet. 101:263-279).
  • the FTH and CGICE genes are oriented tail-to-t ⁇ l; the distance between their polyadenylation signals is 132 bp.
  • the present invention provides DNA encoding CGICE that is substantially free from other nucleic acids.
  • the present invention also provides recombinant DNA molecules encoding CGICE.
  • the present invention provides DNA molecules substantially free from other nucleic acids comprising the nucleotide sequence shown in Figure 1 as SEQ.ID.NO.:l. Analysis of SEQ.ID.NO.:l revealed that this genomic sequence defines a gene having 11 exons. These exons collectively have an open reading frame that encodes a protein of 585 amino acids. If an alternative splice donor site is utilized in exon 7, a cDNA containing an additional 203 bases is produced.
  • this cDNA contains a shorter open reading frame of 1,305 bases (due to the presence of a change in reading frame that introduces a stop codon) that encodes a protein of 435 amino acids.
  • the present invention includes two cDNA molecules encoding two forms of CGICE protein that are
  • the present invention includes DNA molecules substantially free from other nucleic acids comprising the coding regions of SEQ. ID. NO.: 2 and SEQ.ID.NO.:4. Accordingly, the present invention includes DNA molecules substantially free from other nucleic acids having a sequence comprising positions 105-1,859 of SEQ.ID.NO.:2 and positions 105-1,409 of SEQ.ID.NO.:4. Also included are recombinant DNA molecules having a nucleotide sequence comprising positions 105- 1,859 of SEQ.ID.NO.:2 and positions 105-1,409 of SEQ.ID.NO.:4.
  • CGICE in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which CGICE is not naturally linked, to form "recombinant DNA molecules" encoding CGICE.
  • Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, promoters for RNA polymerase II, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, sequences that confer antibiotic resistance, or sequences that encode a polypeptide "tag” such as, e.g. , a polyhistidine tract or the myc epitope.
  • the novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, PI artificial chromosomes, or yeast artificial chromosomes.
  • DNA sequences that hybridize to at least one of SEQ.ID.NOs.:l, 2, or 4 under stringent conditions are included in the present invention.
  • a procedure using conditions of high stringency is as follows: Prehybridization of filters
  • - 8 - containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65 °C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 ⁇ cpm of 32p.i a eled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
  • Denhardt's solution 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.
  • the degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid. This .allows for the construction of synthetic DNA that encodes the CGICE protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequences of SEQ.ID.NOs.:2 or 4, but still encodes the same CGICE protein as SEQ.ID.NOs.:2 or 4.
  • Such synthetic DNAs are intended to be within the scope of the present invention.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 7,259 of SEQ.ID.NO.:l is T, A, or C rather than G, so that the codon at positions
  • - 9 - 7,257-7,259 encodes either cysteine or is a stop codon rather than encoding tryptophan.
  • a DNA molecule having a nucleotide sequence that is identical to SEQ. ID. NO. :1 except that at least one of the nucleotides at position 7,257 or 7,258 has been changed so that the codon at positions 7,257-7,259 does not encode tryptophan.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 383 is T, A, or C rather than G, so that the codon at positions 381-383 encodes either cysteine or is a stop codon rather than encoding tryptophan. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that at least one of the nucleotides at position 381 or 382 has been changed so that the codon at positions 381-383 does not encode tryptophan.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that the nucleotide at position 383 is T, A, or C rather than G, so that the codon at positions 381-383 encodes either cysteine or is a stop codon rather than encoding tryptophan. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that at least one of the nucleotides at position 381 or 382 has been changed so that the codon at positions 381-383 does not encode tryptophan.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 7,233 of SEQ.ID.NO.:l is C, A, or G rather than T, so that the codon at positions 7,233-7,235 does not encode tyrosine. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that at least one of the nucleotides at position 7,234 or 7,235 has been changed so that the codon at positions 7,233-7,235 does not encode tyrosine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of
  • nucleotide at position 357 is C, A, or G rather than T, so that the codon at positions 357-359 does not encode tyrosine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that the nucleotide at position 357 is C, A, or G rather than T, so that the codon at positions 357-359 does not encode tyrosine. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that at least one of the nucleotides at position 358 or 359 has been changed so that the codon at positions 357-359 does not encode tyrosine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 3,330 is C rather than A. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 3,330 of SEQ.ID.NO.:l is G, C, or T rather than A, so that the codon at positions 3,330-3,332 does not encode threonine.
  • DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that at least one of the nucleotides at position 3,330 or 3,331 has been changed so that the codon at positions 3,330-3,332 does not encode threonine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 120 is C rather than A. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 120 is G, C, or T rather than A, so that the codon at positions 120-122 does not encode threonine. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of
  • SEQ.ID.NO.:4 except that the nucleotide at position 120 is C rather than A. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that the nucleotide at position 120 is G, C, or T rather than A, so that the codon at positions 120-122 does not encode threonine. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that at least one of the nucleotides at position 120 or 121 has been changed so that the codon at positions 120-122 does not encode threonine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 8,939 is A rather than T. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 8,939 of SEQ.ID.NO.:l is A, G, or C, rather than T, so that the codon at positions 8,939-8,941 does not encode tyrosine.
  • Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that at least one of the nucleotides at position 8,939-8,941 has been changed so that the codon at positions 8,939- 8,941 does not encode tyrosine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 783 is A rather than T, Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 783 is A, G, or C rather than T so that the codon at positions 783-785 does not encode tyrosine. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that at least one of the nucleotides at position 783-
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that the nucleotide at position 783 is A rather than T. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,409 of SEQ.ID.NO.:4 except that the nucleotide at position 783 is A, G, or C rather than T, so that the codon at positions 783-785 does not encode tyrosine.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to SEQ.ID.NO.:l except that the nucleotide at position 11,241 is A rather than G.
  • the present invention includes a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 1,000 is A rather than G. Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that the nucleotide at position 1,000 is A, C, or T rather than G, so that the codon at positions 999-1,001 does not encode glycine.
  • Also included in the present invention is a DNA molecule having a nucleotide sequence that is identical to positions 105-1,859 of SEQ.ID.NO.:2 except that at least one of the nucleotides at position 999 or 1,000 has been changed so that the codon at positions 999-1,001 does not
  • Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding CGICE protein. Such recombinant host cells can be cultured under suitable conditions to produce CGICE protein. An expression vector containing DNA encoding CGICE protein can be used for expression of CGICE protein in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • mammalian expression vectors can be used to express recombinant CGICE in mammalian cells.
  • Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, ⁇ CR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146).
  • CGICE can be purified by conventional techniques to a level that is substantially free from other proteins.
  • the present invention includes CGICE protein substantially free from other proteins.
  • the amino acid sequence of the full-length CGICE protein is shown in Figure 3 as SEQ.ID.NO.:3.
  • the present invention includes CGICE protein substantially free from other proteins having the amino acid sequence SEQ.ID.NO.:3.
  • Also included in the present invention is a CGICE protein that is produced
  • Mutated forms of CGICE proteins are intended to be within the scope of the present invention.
  • mutated forms of SEQ.ID.NOs.:3 and 5 that give rise to Best's macular dystrophy are within the scope of the present invention.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 93 is cysteine rather than tryptophan.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 93 is not tryptophan.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.ID.NO.:5 except that the amino acid at position 93 is not tryptophan.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 85 is histidine rather than tyrosine.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.ID.NO.:5 except that the amino acid at position 85 is histidine rather than tyrosine.
  • the present invention includes a protein having the amino arid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 85 is not tyrosine.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.LD.NO.:5 except that the amino acid at position 85 is not tyrosine.
  • the present invention includes a protein having the amino arid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 6 is proline rather than threonine.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.ID.NO.:5 except that the amino arid at position 6 is proline rather than threonine.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 6 is not threonine.
  • the present invention also includes a protein having the amino acid sequence shown
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 227 is asparagine rather than tyrosine.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.ID.NO.:5 except that the amino acid at position 227 is asparagine rather than tyrosine.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 227 is not tyrosine.
  • the present invention also includes a protein having the amino acid sequence shown in Figure 5 as SEQ.ID.NO.:5 except that the amino acid at position 227 is not tyrosine.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 299 is glutamate rather than glycine.
  • the present invention includes a protein having the amino acid sequence shown in Figure 3 as SEQ.ID.NO.:3 except that the amino acid at position 299 is not glycine.
  • the present invention includes modified CGICE proteins which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as CGICE. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g.
  • the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NOs.:3 or 5 wherein the polypeptides still retain substantially the same biological activity as CGICE.
  • the present invention also includes polypeptides where two amino acid substitutions have been made in SEQ.ID.NOs.:3 or 5 wherein the polypeptides still retain substantially the same biological activity as CGICE.
  • the present invention includes embodiments where the above-described substitutions are conservative substitutions.
  • CGICE proteins of the present invention may contain post- translational modifications, e.g., covalently linked carbohydrate.
  • the present invention also includes chimeric CGICE proteins.
  • Chimeric CGICE proteins consist of a contiguous polypeptide sequence of at least a portion of a CGICE protein fused to a polypeptide sequence of a non- CGICE protein.
  • the present invention also includes isolated forms of CGICE proteins and CGICE DNA.
  • isolated CGICE protein or “isolated CGICE DNA” is meant CGICE protein or DNA encoding CGICE protein that has been isolated from a natural source. Use of the term “isolated” indicates that CGICE protein or CGICE DNA has been removed from its normal cellular environment. Thus, an isolated CGICE protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally.
  • isolated does not imply that an isolated CGICE protein is the only protein present, but instead means that an isolated CGICE protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the CGICE protein.
  • non-amino acid material e.g., nucleic acids, lipids, carbohydrates
  • a cDNA fragment encoding full-length CGICE can be isolated from a human retinal cell cDNA library by using the polymerase chain reaction (PCR) employing suitable primer pairs.
  • PCR polymerase chain reaction
  • primer pairs can be selected based upon the cDNA sequence for CGICE shown in Figure 2 as SEQ.ID.NO.:2 or in Figure 4 as SEQ.ID.NO.:4.
  • Suitable primer pairs would be, e.g. :
  • CAGGGAGTCCCACCAGCC (SEQ.ID.NO.:6) and TCCCCATTAGGAAGCAGG (SEQ.ID.NO.:7) for SEQ.ID.NO.:2; and CAGGGAGTCCCACCAGCC (SEQ.ID.NO.:6) and
  • thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, or Vent polymerase.
  • AmpliTaq reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM MgC , 200 ⁇ M for each dNTP, 50 mM
  • KC1 0.2 ⁇ M for each primer, 10 ng of DNA template, 0.05 units/ ⁇ l of AmpliTaq.
  • the reactions are heated at 95°C for 3 minutes and then cycled 35 times using the cycling parameters of 95 °C, 20 seconds, 62°C, 20 seconds, 72°C, 3 minutes.
  • PCR Protocols A Guide to Methods and Applications. Michael et al., eds., 1990, Academic Press .
  • a suitable cDNA library from which a clone encoding CGICE can be isolated would be Human Retina 5'-stretch cDNA library in lambda gtlO or lambda gtll vectors (catalog numbers HL1143a and HL1132b, Clontech, Palo Alto, CA).
  • the primary clones of such a library can be subdivided into pools with each pool containing approximately 20,000 clones and each pool can be amplified separately.
  • a cDNA fragment encoding an open reading frame of 585 amino acids (SEQ.ID.NO.:3) or an open reading frame of 435 amino acids (SEQ.ID.NO.:5) can be obtained.
  • This cDNA fragment can be cloned into a suitable cloning vector or expression vector.
  • the fragment can be cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, Ca).
  • CGICE protein can then be produced by transferring an expression vector encoding CGICE or portions thereof into a suitable host cell and growing the host cell under appropriate conditions. CGICE protein can then be isolated by methods well known in the art.
  • a cDNA clone encoding CGICE can be isolated from a cDNA library using as a probe oligonucleotides specific for CGICE and methods well known in the art for screening cDNA libraries with oligonucleotide probes. Such methods are described in, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A
  • Oligonucleotides that are specific for CGICE and that can be used to screen cDNA libraries can be readily designed based upon the cDNA sequence of CGICE shown in Figure 2 as SEQ.ID.NO.:2 or in Figure 4 as SEQ.ID.NO.:4 and can be synthesized by methods well-known in the art.
  • Genomic clones containing the CGICE gene can be obtained from commercially available human PAC or BAC libraries available from Research Genetics, Huntsville, AL.
  • PAC clones containing the CGICE gene are commercially available from Research Genetics, Huntsville, AL (Catalog number for individual PAC clones is RPCI.C).
  • genomic libraries especially in PI artificial chromosome vectors, from which genomic clones containing the CGICE can be isolated, using probes based upon the CGICE sequences d sclosed herein. Methods of preparing such libraries are known in the art (Ioannou et al., 1994, Nature Genet. 6:84-89).
  • the novel DNA sequences of the present invention can be used in various diagnostic methods relating to Best's macular dystrophy.
  • the present invention provides diagnostic methods for determining whether a patient carries a mutation in the CGICE gene that predisposes that patient toward the development of Best's macular dystrophy.
  • such methods comprise determining the DNA sequence of a region of the CGICE gene from the patient and comparing that sequence to the sequence from the corresponding region of the CGICE gene from a normal person, i.e., a person who does not suffer from Best's macular dystrophy.
  • one embodiment comprises:
  • the PCR primers are from the coding region of the CGICE gene, i.e., from the coding region of SEQ.ID.NOs.:l, 2, or 4.
  • the DNA sample from the patient is cDNA that has been prepared from an RNA sample from the patient.
  • the DNA sample from the patient is genomic DNA.
  • the nucleotide sequences of the PCR fragment from the patient and the control PCR fragment are determined by DNA sequencing.
  • the nucleotide sequences of the PCR fragment from the patient and the control PCR fragment are compared by direct comparison after DNA sequencing.
  • the comparison is made by a process that includes hybridizing the PCR fragment from the patient and the control PCR fragment and then using an endonuclease that cleaves at any mismatched positions in the hybrid but does not cleave the hybrid if the two fragments match perfectly.
  • an endonuclease is, e.g., SI.
  • the conversion of the PCR fragment from the patient to smaller fragments after endonuclease treatment indicates that the patient carries a mutation in the CGICE gene.
  • the present invention provides a method of diagnosing whether a patient carries a mutation in the CGICE gene that comprises:
  • RT-PCR reverse transcription-PCR
  • step (d) treating the hybrid produced in step (c) with an endonuclease that cleaves at any mismatched positions in the hybrid but does not cleave the hybrid if the two fragments match perfectly;
  • the present invention provides a method of diagnosing whether a patient carries a mutation in the CGICE gene that comprises:
  • the present invention provides a method of diagnosing whether a patient carries a mutation in the CGICE gene that comprises:
  • the primers are selected so that they amplify a portion of SEQ.ID.NOs.:2 or 4 that includes at least one position selected from the group consisting of: positions 120, 121, 122, 357, 358, 359, 381, 382, 383, 783, 784, and 785.
  • the primers are selected so that they amplify a portion of SEQ.ID.NOs.:2 or 4 that includes at least one position selected from the group consisting of: positions 384, 385, and 386.
  • the primers are selected so that they amplify a portion of SEQ.ID.NO.:2 that includes at least one position selected from the group consisting of: positions 999, 1,000, and 1,001.
  • the primers are selected so that they amplify a portion of SEQ.ID.NOs.:2 or 4 that includes at least one codon that encodes an amino acid present in CGICE that is also present in the corresponding position in at least one of the C. elegans proteins whose partial amino acid sequence is shown in Figure 7.
  • the present invention provides a diagnostic method for determining whether a person carries a mutation of the CGICE gene in which the G at position 383 of SEQ.ID.NO.:2 has been changed to a C. This change results in the creation of a Fnu4HI restriction site.
  • PCR primer pair 5'-CTCCTGCCCAGGCTTCTAC-3' SEQ.ID.NO.:30
  • 5'-CTTGCTCTGCCTTGCCTTC-3' SEQ.ID.NO.:31
  • Heterozygotes for the G383C mutation have three Fnu4HI digestion products: 125 bp, 85 bp, and 40 bp; homozygotes have two: 85 bp and 40 bp; and wild-type individuals have a single fragment of 125 bp.
  • the present invention provides a diagnostic method for determining whether a person carries a mutation of the CGICE gene in which the T at position 783 of SEQ.ID.NO.:2 has been changed to an A. This change results in the creation of a PflMI restriction site.
  • the present invention also provides oligonucleotide probes, based upon the sequences of SEQ.ID.NOs.:l, 2, or 4, that can be used in diagnostic methods related to Best's macular dystrophy.
  • the present invention includes DNA oligonucleotides comprising at least 18 contiguous nucleotides of at least one of a sequence selected from the group consisting of: SEQ.ID.NOs.:l, 2 and SEQ.ID.:N0.4.
  • corresponding RNA oligonucleotides are also provided by the present invention.
  • the DNA or RNA oligonucleotide probes can be packaged in kits.
  • the present invention makes possible the recombinant expression of the CGICE protein in various cell types. Such recombinant expression makes possible the study of this protein so that its biochemical activity and its role in Best's macular dystrophy can be elucidated.
  • the present invention also makes possible the development of assays which measure the biological activity of the CGICE protein.
  • assays using recombinantly expressed CGICE protein are especially of interest.
  • Assays for CGICE protein activity can be used to screen libraries of compounds or other sources of compounds to identify
  • Such identified compounds can serve as 'leads" for the development of pharmaceuticals that can be used to treat patients having Best's macular dystrophy.
  • mutant CGICE proteins are used and inhibitors or activators of the activity of the mutant CGICE proteins are discovered.
  • assays comprise:
  • the present invention also includes antibodies to the CGICE protein.
  • Such antibodies may be polyclonal antibodies or monoclonal antibodies.
  • the antibodies of the present invention are raised against the entire CGICE protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art. Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.
  • CGICE protein or an antigenic fragment, coupled to a suitable carrier is injected on a periodic basis into an appropriate non-human host animal such as, e.g. , rabbits, sheep, goats, rats, mice. The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen.
  • the injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
  • CGICE protein or an antigenic fragment, coupled to a suitable carrier is
  • the animal is generally a mouse.
  • the animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497.
  • For a fuller description of the production of monoclonal antibodies see Antibodies: A Laboratory Manual. Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
  • Gene therapy may be used to introduce CGICE polypeptides into the cells of target organs, e.g. , the pigmented epithelium of the retina or other parts of the retina.
  • Nucleotides encoding CGICE polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells.
  • Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors.
  • nucleotides encoding CGICE polypeptides can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with CGICE polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate CGICE activity.
  • the present invention includes DNA comprising nucleotides encoding mouse CGICE. Included within such DNA is the DNA sequence shown in Figure 8A-C (SEQ. ID. NO.:28). Also included is DNA comprising positions 11-1,663 of SEQ. ID. NO.:28. Also included are mutant versions of DNA encoding mouse CGICE. Included is DNA comprising nucleotides that are identical to positions 11-1,663 of SEQ. ID.
  • nucleotides at positions 26-28, positions 263-265, positions 287-289, positions 689-691, and/or positions 905-907 differs from the corresponding nucleotide at positions 26-28, positions 263-265, positions 287-289, positions 689-691, and/or positions 905-907 of SEQ. ID. NO.:28.
  • Particularly preferred versions of mutant DNAs are those in which the nucleotide change results in a change in the corresponding encoded amino acid.
  • - 25 - CGICE can be in isolated form, can be substantially free from other nucleic acids, and/or can be recombinant DNA.
  • the present invention includes mouse CGICE protein (SEQ. ID. NO.:29).
  • This mouse CGICE protein can be in isolated form and/or can be sustantially free from other proteins.
  • Mutant versions of mouse CGICE protein are also part of the present invention. Examples of such mutant mouse CGICE proteins are proteins that are identical to SEQ. ID. NO.:29 except that the amino acid at position 6, position 85, position 93, position 227, and/or position 299 differs from the corresponding amino acid at position 6, position 85, position 93, position 227, and/or position 299 in SEQ. ID. NO.:29.
  • cDNA encoding mouse CGICE can be amplified by PCR from cDNA libraries made from mouse eye or mouse testis.
  • Suitable primers can be readily designed based upon SEQ. ID. NO.:28.
  • cDNA encoding mouse CGICE can be isolated from cDNA libraries made from mouse eye or mouse testis by the use of oligonucleotide probes based upon SEQ. ID. NO.:28.
  • the present invention allows for the generation of an animal model of Best's macular dystrophy.
  • This animal model can be generated by making "knockout” or “knockin” mice containing altered CGICE genes.
  • Knockout mice can be generated in which portions of the mouse CGICE gene have been deleted.
  • Knockin mice can be generated in which mutations that have been shown to lead to Best's macular dystrophy when present in the human CGICE gene are introduced into the mouse gene.
  • mutations resulting in changes in amino acids 6, 85, 93, 227, or 299 of the mouse CGICE protein are contemplated.
  • knockout and knockin mice will be valuable tools in the study of the Best's macular dystrophy disease process and will provide important model systems in which to test potential pharmaceuticals or treatments for Best's macular dystrophy.
  • Methods of producing knockout and knockin mice are well known in the art. For example, the use of gene-targeted ES cells
  • a targeting vector i.e., a plasmid containing part of the genetic region it is desired to mutate.
  • a targeting vector contains a selectable marker gene as well.
  • homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10-6 and 10-3 (Lin et al., 1985, Proc. Natl. Acad. Sci. USA 82:1391-1395; Smithies et al., 1985, Nature 317: 230-234; Thomas et al., 1986, Cell 44:419-428).
  • Nonhomologous plasmid-chromosome interactions are more frequent, occurring at levels 105-fold (Lin et al., 1985, Proc. Natl. Acad. Sci. USA 82:1391-1395) to 102-fold (Thomas et al., 1986, Cell 44:419-428) greater than comparable homologous insertion.
  • PNS positive-negative selection
  • Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with herpes drugs such as gancyclovir (GANC) or FIAU (l-(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5- iodouracil).
  • HSV-TK Herpes Simplex virus thymidine kinase
  • GANC gancyclovir
  • FIAU l-(2-deoxy 2-fluoro-B-D-arabinofluranosyl
  • the minimum tiling path between markers D11S4076 and UGB that represents the minimum genetic region containing the BMD gene includes the following nine PAC clones: 363M5 (140 kb), 519013(120 kb), 527E4 (150 kb), 688P12 (140 kb), 741N15 (170 kb), 756B9 (120 kb), 759J12 (140 kb), 1079D9 (170 kb), and 363P2 (160 kb).
  • PAC DNA was purified by equilibrium centrifugation in cesium chloride-ethidium bromide gradient (Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press). Purified PAC DNA was brought to 50 mM Tris pH 8.0, 15 mM MgCl2, and 25% glycerol in a volume of 2 ml and placed in a AERO-MIST nebulizer (CIS-US, Bedford, MA). The nebulizer was attached to a nitrogen gas source and the DNA was randomly sheared at 10 psi for 30 sec.
  • the sheared DNA was ethanol precipitated and resuspended in TE (10 mM Tris, 1 mM EDTA). The ends were made blunt by treatment with Mung Bean Nuclease (Promega, Madison, WI) at 30°C for 30 min, followed by phenol/chloroform extraction, and treatment with T4 DNA polymerase (GIBCO/BRL, Gaithersburg, MD) in multicore buffer (Promega, Madison, WI) in the presence of 40 uM dNTPs at 16°C.
  • TE 10 mM Tris, 1 mM EDTA
  • BstX I adapters (Invitrogen, Carlsbad, CA) were ligated to the fragments at 14°C overnight with T4 DNA ligase (Promega, Madison, WI). Adapters and DNA fragments less than 500 bp were removed by column chromatography using a cDNA sizing column (GIBCO/BRL, Gaithersburg, MD) according to the instructions provided by the manufacturer. Fractions containing DNA greater than 1 kb were pooled and concentrated by ethanol precipitation.
  • the DNA fragments containing BstX I adapters were ligated into the BstX I sites of pSHOT II which was constructed by subcloning the BstX I sites from pcDNA II (Invitrogen, Carlsbad, CA) into the BssH II sites of pBlueScript (Stratagene, La Jolla, CA).
  • pSHOT II was prepared by digestion with BstX I restriction endonuclease and purified by agarose gel electrophoresis. The gel purified vector DNA was extracted from the agarose by following the Prep-A-Gene (BioRad, Richmond, CA) protocol.
  • the digested vector was treated with calf intestinal phosphatase (GIBCO/BRL, Gaithersburg, MD.
  • Ligation reactions of the DNA fragments with the cloning vector were transformed into ultra-competent XL-2 Blue cells (Stratagene, La Jolla, CA), and plated on LB agar plates supplemented with 100 ⁇ g/ml ampicillin. Individual colonies were picked into a 96 well plate containing 100 ⁇ l/well of LB broth supplemented with ampicillin and
  • plasmid DNA Glycerol stocks were used to inoculate 5 ml of LB broth supplemented with 100 ⁇ g/ml ampicillin either manually or by using a Tecan Genesis RSP 150 robot (Tecan AG, Hombrechtikon, Switzerland) programmed to inoculate 96 tubes containing 5 ml broth from the 96 wells. The cultures were grown overnight at 37°C with shaking to provide aeration. Bacterial cells were pelleted by centrifugation , the supernatant decanted, and the cell pellet stored at -20°C. Plasmid DNA was prepared with a QIAGEN Bio Robot 9600 (QIAGEN, Chatsworth, CA) according to the Qiawell Ultra protocol.
  • plasmid DNA was digested with the restriction endonuclease Pvu II.
  • the size of the restriction endonuclease products was examined by agarose gel electrophoresis with the average insert size being 1 to 2 kb.
  • PRISMTM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS (Perkin Elmer, Norwalk, CT). DNA sequence analysis was performed with M13 forward and reverse primers. Following amplification in a Perkin-Elmer 9600, the extension products were purified and analyzed on an ABI PRISM 377 automated sequencer (Perkin Elmer, Norwalk, CT). Approximately 4 sequencing reactions were performed per kb of DNA to be examined (384 sequencing reactions per each of nine PACs).
  • Phred/Phrap was used for DNA sequences assembly. This program was developed by Dr. Phil Green and licensed from the University of Washington (Seattle, WA). Phred Phrap consists of the following programs: Phred for base-calling, Phrap for sequence assembly, Crossmatch for sequence comparisons, Consed and
  • the sublibraries were plated on agar plates, and colonies were transfered to nylon membranes and probed with randomly primed polynucleotide, (dC-dA) n (dG-dT) n , Hybridization was performed overnight in a solution containing 6X SSC, 20 mM sodium phosphate buffer (pH 7.0), 1% bovine serum albumin, and 0,2% sodium dodecyl sulfate at 65°C. Filters were washed four times for 15 min each in 2X SSC and 0.2% SDS at 65°C. CA-positive subclones were identified for all but one PAC clone (527E4). DNA from these subclones was isolated and sequenced as descrobed above for the shotgun library clones.
  • DNA sequence at the final stage of assembly was checked for the presence of micro satellite repeats using a Consed visualization tool of the Phred/Phrap package.
  • Sequence fragments containing CA repeats were analyzed using the PRIMER program; oligonucleotide pairs flanking each of the CA repeats were synthesized.
  • the forward primer was kinase-labeled with [gamma-32p]-ATP. Amplification of the genomic DNA was
  • EM dNTPs 0.2 pmol/El of both primers; 0.025 unit/El of Taq polymerase.
  • the PCR program consisted of 94° C for 3 min followed by 30 cycles of 94°C for 1 min, 55°C for 2 min, 72°C for 2 min and a final elongation step at 72°C for 10 min.
  • samples were mixed with 2 vol of a formamide dye solution and run on a 6% polyacrylamide sequencing gel.
  • Two newly identified markers detected two recombination events in disease chromosomes of individuals from family SI. This limited the minimum genetic region to the interval covered by 6 PAC clones: 519013, 759J12, 756B9, 363M5, 363P2, and 741N15.
  • a CA-positive subclone (pCA759J12-2) was identified in the shotgun library generated from the PAC 759J12 DNA by hybridization to the (dC-dA)n (dG-dT) n probe. DNA sequence from pCA759J12-2 was queried against the EST sequences in the GenBank database using the BLAST algorithm (S.F. Altschul, et al, 1990, J. Mol. Biol. 215:403-410).
  • the BLAST analysis identified a high degree of similarity between the DNA sequence obtained from the clone pCA759J12-2 and a retina- specific human EST with GenBank accession number AA318352.
  • BLASTX analysis of EST AA318352 revealed a strong homology of the corresponding protein to a group of C. elegans proteins with unknown function (RFP family).
  • the RFP family is known only from C. elegans genome and EST sequences (e.g., C. elegans C29F4.2 and B0564.3) and is named for the amino arid sequence RFP that is invariant among 15 of the 16 family members; members share a conserved 300-400 amino acid sequence including 25 highly conserved aromatic residues.
  • a human gene partially represented in pCA759J12-2 and
  • EST AA318352 was dubbed CGICE (Candidate £ene #1 with the homology to the C_. ⁇ legans g . roup of genes) and selected for detaled analysis.
  • Genbank database entries for PACs 466A11 and 363P2 were analyzed with the use of the same AceDB package.
  • PAC clones 466A11 and 363P2 represent parts of the PAC contig across the BMD region (Cooper et al., 1997, Genomics 41:185-192); both clones map to the minimum genetic region containing the BMD gene that was determined by recombination breakpoint analysis in a 12-generation Swedish pedigree (Graff et al ., 1997, Hum. Genet. 101: 263-279).
  • Datbase entries for PACs 466A11 and 363P2 represent unordered DNA pieces genereated in Phase 1 High Throughput Genome Sequence Project (HTGS phase 1) by Genome Science and Technology Center, University of Texas Southwestern Medical Center at Dallas.
  • Genomic DNA sequences from PACs 466A11 and 759J12 were compared with the CGICE cDNA sequence from EST AA318352 using the program Crossmatch which allowed for a rapid and sensitive detection of the location of exons. The identification of intron/exon boundaries was then accomplished by manually comparing visualized genomic and cDNA sequences by using the AceDB package. This analysis allowed the identification of exons 8, 9, and 10 that are represented in EST AA318352. To increase the accuracy of the analysis, the DNA sequence of EST AA318352 was verified by comparison with genomic sequence obtained from pCA759J12-2, PAC 466A11, and shotgun PAC 759J12 subclones.
  • the verified EST AA318352 sequence was reanalyzed by BLAST; two new ESTs (accession numbers AA307119 and AA205892) were found to partially overlap with EST AA318352. They were assembled into a contig using the program Sequencher (Perkin Elmer, Norwalk, CT), and a consensus sequence derived from three
  • ESTs (AA318352, AA307119, and AA205892) was re-analyzed by BLAST.
  • EST AA317489 was included in the consensus cDNA sequence.
  • the consensus sequence derived from the four ESTs was compared with genomic sequences obtained from pCA759J12-2, PAC 466A11, and shotgun PAC 759J12 subclones using the programs Crossmatch and AceDB. This analysis verified the sequence and corrected sequencing errors that were found in AA318352, AA307119, AA205892, and AA317489. Comparison of cDNA and genomic sequences revealed a total of 7 exons.
  • Bioinformatic analysis did not allow the prediction of boudaries between exons 2 and 4, exons 6 and 7, and exons 7 and 8. In addition, there was no overlap between ESTs represented in exons 1 and 2 from one side and exons 4, 5, 6, 7, 8, 9, 10, and 11 from another. There was the possibility of the presence of additional exons in the CGICE gene that were not represented in the GenBank EST database.
  • CTAGTCGCCAGACCTTCTGTG (SEQ.ID.NO.:9) was paired with a reverse primer from ex4 (GR: CTTGTAGACTGCGGTGCTGA) (SEQ.ID.NO.:10), forward primer from ex4 (GF:
  • GAAAGCAAGGACGAGCAAAG (SEQ.ID.NO.:ll) was paired with a reverse primer from ex6 (ER: AATCCAGTCGTAGGCATACAGG )
  • PCR reaction was performed using the Taq Gold DNA polymerase (Perkin Elmer, Norwalk, CT) in the reaction buffer supplied by the manufacturer with the addition of dNTPs, primers, and approximately 0.5 ng of human retina cDNA.
  • PCR products were electrophoresed on a 2% agarose gel and DNA bands were excised, purified and subjected to sequence analysis with the same primers that were used for PCR amplification. The assembly of the DNA sequence results of these PCR products revealed that:
  • exons 1 and 2 from one side and exons 4, 5, 6, 7, 8, 9, 10, and 11 indeed represent fragments of the same gene
  • exon 7 (Hypothetical) predicted by the BLASTX analysis is present in the CGICE cDNA fragment amplified by EF/AR primers.
  • Two splicing variants of exon 7 were detected upon sequence analysis of RT-PCR products amplified from human retina cDNA with the primer pair EF/AR. Two variants utilize alternative splice donor sites separated from each other by 203 bp. Both splicing sites conform to the published consensus sequence.
  • RACE is an established protocol for the analysis of cDNA ends. This procedure was performed using the Marathon RACE template from human retina, purchased from Clontech (Palo Alto, CA). cDNA primers KR (CTAAGCGGGCATTAGCCACT) (SEQ.ID.NO.:19) and LR(TGGGGTTCCAGGTGGGTCCGAT) (SEQ.ID.NO.:20) in combination with a cDNA adaptor primer API
  • Genomic DNA from BMD patients from two Swedish pedigrees having Best's macular dystrophy was amplified by PCR using the following primer pair: exGJeft AAAGCTGGAGGAGCCGAG (SEQ.ID.NO.:23) exG_right CTCCACCCATCTTCCGTTC (SEQ.ID.NO.:24) This primer pair amplifies a genomic fragment that is 412 bp long and contains exon4 and adjacent intronic regions.
  • the patients were: Family SI:
  • Sl-3 a normal individual, i.e., not having BMD
  • sister of Sl-4 Sl-4 an individual heterozygous for BMD
  • Sl-5 an individual homozygous for BMD.
  • SL76-3 an individual heterozygous for BMD; mother of SL76-2 SL76-2, an individual heterozygous for BMD, son of SL-3.
  • PCR products produced using the primer sets mentioned above were amplified in 50 ⁇ l reactions consisting of Perkin-Elmer 10 x PCR Buffer, 200 mM dNTP's, 0.5 ul of Taq Gold (Perkin-Elmer Corp., Foster City, CA), 50 ng of patient DNA and 0.2 EM of forward and reverse primers. Cycling conditions were as follows:
  • Products obtained from this PCR amplification were analyzed on 2% agarose gels and excised fragments from the gels were purified using Qiagen QIAquick spin columns and sequenced using ABI dye-terminator sequenring kits. The products were analyzed on ABI 377 sequencers according to standard protocols.
  • Figure 6 shows a chromatogram from sequencing runs on the PCR fragments from patients Sl-3, Sl-4, and Sl-5. The six readings represent sequencing of both strands of the PCR fragments from the patients.
  • Both copies of the CGICE gene are mutated in homozygous affected Sl-5, while heterozygous affected Sl-4 contains both normal and mutated copies of the CGICE gene.
  • This mutation changes the codon that encodes the amino acid at position 93 of SEQ.ID.NO.:3 from TGG (encoding tryptophan) to TGC (encoding cysteine).
  • Patient Sl-3 a normal individual, has the wild-type sequence, TGG, at this codon.
  • This disease mutation that changes this TGG codon to a TGC codon was not found upon sequencing of 50 normal unrelated individulas (100 chromosomes) of North American descent.
  • Phenylalanine and tyrosine both being aromatic amino acids, are highly similar.
  • RT-PCR experiments were performed on "quick- clone" human cDNA samples available from Clontech, Palo Alto, CA. cDNA samples from heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and retina were amplified with primers AF (CCCTTTGGAGAGGATGATGA) (SEQ.ID.NO.:15) and CR (CTCTGGCATATCCGTCAGGT) (SEQ.ID.NO.:16) in the following PCR conditions:
  • Northern blot analysis Northern blots containing poly(A+)- RNA from different human tissues were purchased from Clontech, Palo Alto, CA. Blot #1 contained human heart, brain placenta, lung, liver, skeletal muscle, kidney, and pancreas poly(A+)-RNA. Blot #2 contained stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow poly(A+)-RNA.
  • the blots were washed by two 15 min incubations in 2X SSC, 0.1% SDS (prepared from 20X SSC and 20 % SDS stock solutions, Fisher, Pittsburgh, PA) at room temperature, followed by two 15 min incubations in IX SSC, 0.1% SDS at room temperature, and two 30 min incubations in 0.1X SSC, 0.1% SDS at 60°C. Autoradiography of the blots was done to visualize the bands that specifically hybridized to the radiolabeled probe.
  • the probe hybridized to an mRNA transcript that is uniquely expressed in brain and spinal cord.
  • Mouse probe for the murine ortholog of the GC1CE gene was generated based on the sequence of an EST with GenBank accession number AA497726.
  • the 246 bp probe was amplified from mouse heart cDNA (Clontech, Palo Alto, CA) using the primers mouseCGlCE_L (ACACAACACATTCTGGGTGC) (SEQ.ID.NO.:26) and mouseCGlCE_R (TTCAGAAACTGCTTCCCGAT) (SEQ.ID.NO.:27). Due to an extremely low expression level of the CGICE gene in mouse heart, repetitive amplification steps were used to generate this probe. The authenticity of this probe was verified by sequence analysis of the gel purified DNA band.
  • Northern blot containing poly(A+)-RNA from several rat tissues was purchase from Clontech, Palo Alto, CA.
  • the probe hybridized to an mRNA transcript that is expressed in testis only.

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Abstract

Nouvelles séquences d'ADN humain et de souris codant le gène CG1CE qui, lorsqu'il est muté, est responsable de la dystrophie cornéenne de Best. La présente invention concerne en outre de l'ADN génomique CG1CE ainsi que de l'ADNc qui code la protéine CG1CE, la protéine CG1CE codée par les nouvelles séquences d'ADN, des procédés d'expression de la protéine CG1CE dans des systèmes de recombinaison et des méthodes diagnostiques permettant d'identifier les patients possédant des gènes CG1CE mutés.
EP99908345A 1998-02-25 1999-02-22 Gene de la dystrophie corneenne de best Withdrawn EP1060184A4 (fr)

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US7594198P 1998-02-25 1998-02-25
US75941P 1998-02-25
US11292698P 1998-12-18 1998-12-18
US112926P 1998-12-18
PCT/US1999/003790 WO1999043695A1 (fr) 1998-02-25 1999-02-22 Gene de la dystrophie corneenne de best

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KR101154538B1 (ko) * 2009-08-24 2012-06-13 한국과학기술연구원 소뇌에서의 ｇａｂａ 방출 조절제

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US5705380A (en) * 1996-09-04 1998-01-06 Sequana Theraputics, Inc. Identification of a gene encoding TULP2, a retina specific protein
US5686598A (en) * 1996-08-22 1997-11-11 The Jackson Laboratory Genes associated with retinal dystrophies

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EP1060184A4 (fr) 2002-09-25
WO1999043695A1 (fr) 1999-09-02
CA2321129A1 (fr) 1999-09-02
JP2002504559A (ja) 2002-02-12

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