Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-04-15
2002-10-22
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S325000, C530S350000, C536S023500, C536S024100
Reexamination Certificate
active
06468766
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to gastrointestinal abnormalities.
Gastroschisis is a life-threatening abdominal wall defect that occurs in approximately 1-7 of every 10,000 human births. The defect is thought to originate on the right side of the umbilical cord and may involve the formation of the omphalomesenteric artery. Infants with gastroschisis can be born with abdominal organs outside the body cavity, i.e., protruding through the defect. Factors associated with an increased risk for gastroschisis include a maternal age below 20 years, ingestion of aspirin, and ingestion of pseudoephedrine. The cause of gastroschisis has not been identified.
SUMMARY OF THE INVENTION
A novel human gene encoding aortic carboxypeptidase-like polypeptide (ACLP) has been discovered. A mutation in an ACLP gene has now been shown to be associated with the development of gastroschisis. Thus, a mutation in an ACLP gene is indicative of gastroschisis or a predisposition to develop the condition. Accordingly, the invention provides an isolated nucleic acid (e.g., genomic DNA, cDNA, or synthetic DNA) encoding an ACLP. By the term “human ACLP” is meant a polypeptide having the amino acid sequence of a naturally-occurring human ACLP. For example, the invention encompasses an ACLP with the amino acid sequence of SEQ ID NO:2 as well as naturally-occurring variants thereof such as mutant forms associated with gastroschisis or isoforms resulting from alternative splicing of exons of the ACLP gene.
The invention includes a nucleic acid molecule which contains the nucleotide sequence of human ACLP cDNA (SEQ ID NO:1). A nucleic acid molecule which contains nucleotides 140-3613 (ACLP coding sequence), inclusive, of SEQ ID NO:1 or a degenerate variant thereof, is also within the invention. Nucleotides 214-3613 encode an ACLP which lacks the first 25 residues (a putative signal peptide). Preferably, the nucleic acid molecule contains a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 87% identical to the sequence of SEQ ID NO:2. More preferably, the sequence is at least 90% identical to SEQ ID NO:2, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably, the nucleotide sequence encodes a polypeptide the amino acid sequence of which is SEQ ID NO:2.
An isolated nucleic acid molecule containing a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, or the complement thereof is also within the invention. The nucleic acid molecule may be a primer useful to amplify ACLP DNA in a polymerase chain reaction (PCR). For example, the nucleic acid is at least 5 nucleotides but less than 50 nucleotides in length. Alternatively, the nucleic acid molecule may encompass the entire coding sequence of ACLP CDNA, i.e., nucleotides 140-3613, inclusive, of SEQ ID NO:1. Preferably, the nucleic acid molecule spans a gastroschisis-associated mutation in an ACLP gene. Such a molecule is useful as a hybridization probe to identify a genetic alteration, e.g., a deletion, duplication, point mutation, or translocation, that indicates that an individual has gastroschisis, is predisposed to developing gastroschisis, or is a heterozygous carrier of a genetic alteration associated with gastroschisis.
By “isolated nucleic acid molecule” is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank an ACLP gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a procaryote or eucaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain an ACLP gene flanked by one or more other genes which naturally flank it in a naturally-occurring genome.
Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Hybridization is carried out using standard techniques such as those described in Ausubel et al.,
Current Protocols in Molecular Biology,
John Wiley & Sons, (1989). “High stringency” refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., hybridization and wash conditions of 65° C. at a salt concentration of 0.1×SSC. “Low” to “moderate” stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60° C. at a salt concentration of at least 1.0×SSC. For example, high stringency conditions may include hybridization at 42° C. in a solution containing 50% formamide; a first wash at 65° C. using a solution of 2×SSC and 1% SDS; followed by a second wash at 65° C. using a solution of 0.1%×SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an ACLP gene are detected by, for example, hybridization at 42° C. in the absence of formamide; a first wash at 2° C. in a solution of 6×SSC and 1% SDS; and a second wash at 50° C. in a solution of 6×SSC and 1% SDS.
TABLE 1
Human ACLP cDNA
1
tccctcgctc accccatcct ctctcccgcc ccttcctgga ttccctcacc cgtctcgatc
61
ccctctccgc cctttcccag agacccagaq cccctgaccc cccgcgccct ccccggagcc
121
ccccgcgcgt gccgcggcca tggcggccgt gcgcggggcg cccctgctca gctgcctcct
181
ggcgttgctg gccctgtgcc ctggagggcg cccgcagacg gtgctgaccg acgacgagat
241
cgaggagttc ctcgagggct tcctgtcaya gctagaacct gagccccggg aggacgacgt
301
ggaggccccg ccgcctcccg agcccacccc gcgggtccga aaagcccagg cggygggcaa
361
gccagggaag cggccaggga cggccycaga agtgcctccg gaaaagacca aagacaaagg
421
gaagaaaggc aagaaagaca aaggccccaa ggtgcccaag gagtccttgg aggggtcccc
481
caggccgccc aagaagggga aggagaagcc acccaaggcc accaagaagc ccaaggagaa
541
gccacctaag yccaccaaga aycccaagga ggagccaccc aaggccacca agaagcccaa
601
agagaagcca cccaaggcca ccaagaagcc cccgtcaggg aagaggcccc ccattctggc
661
tccctcagaa accctggagt ggccactgcc cccacccccc agccctggcc ccgaggagct
721
accccaggag ggaggggcgc ccctctcaaa taactggcay aatccaggag aggagaccca
781
tgtggaggca caggagcacc agcctgagcc ggaggaggag accgagcaac ccacactgga
841
ctacaatgac cagatcgaga gggaggacta tgaggacttt gagtacattc ggcgccagaa
901
gcaacccagg ccacccccaa gcagaaggag gaggcccgag cgggtctggc cagagccccc
961
tgaggagaag gccccggccc cagccccgga ggagaggatt gagcctcctg tyaagcctct
1021
gctgcccccg ctgccccctg actatggtga tggttacgtg atccccaact acgatgacat
1081
ggactattac tttgggcctc ctccgcccca gaagcccgat gctgagcgcc agacggacga
1141
agagaaggag gagctgaaga aacccaaaaa ggaggacagc agccccaagg aygagaccga
1201
caagtgggca gtggagaagg gcaaggacca caaagagccc cgaaaggycg aggagttgga
1261
ggaggagtgg acgcctacgg agaaagtcaa gtgtcccccc attgggatgg aytcacaccg
1321
tattgaggac aaccagatcc gagcctcctc catgctgcgc cacygcctgg gggcacagcg
1381
cggccggctc aacatgcaga ccggtgccac tgaggacgac tactatgatg gtgcgtggtg
1441
tgccgaggac gatgccagga cccagtggat agaggtggac accaggagga ctacccggtt
1501
cacaggcgtc atcacccagg gcagagactc cagcatccat
Layne Matthew D.
Lee Mu-En
Yet Shaw-Fang
Beattie Ingrid A.
Mintz Levin Cohn Ferris Glovsky & Popeo
President and Fellows of Harvard College
Romeo David S.
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