Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer
01 Dec 1988-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 85, Iss: 23, pp 8998-9002
TL;DR: The efficacy of this cDNA cloning strategy was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases.
Abstract: We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.
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TL;DR: The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.
Abstract: The mechanisms that balance food intake and energy expenditure determine who will be obese and who will be lean. One of the molecules that regulates energy balance in the mouse is the obese (ob) gene. Mutation of ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.
12,394 citations
TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients
6,731 citations
TL;DR: In this paper, a gene was identified that directed the expression of antigen MZ2-E on a human melanoma cell line, which belongs to a family of at least three genes.
Abstract: Many human melanoma tumors express antigens that are recognized in vitro by cytolytic T lymphocytes (CTLs) derived from the tumor-bearing patient. A gene was identified that directed the expression of antigen MZ2-E on a human melanoma cell line. This gene shows no similarity to known sequences and belongs to a family of at least three genes. It is expressed by the original melanoma cells, other melanoma cell lines, and by some tumor cells of other histological types. No expression was observed in a panel of normal tissues. Antigen MZ2-E appears to be presented by HLA-A1; anti-MZ2-E CTLs of the original patient recognized two melanoma cell lines of other HLA-A1 patients that expressed the gene. Thus, precisely targeted immunotherapy directed against antigen MZ2-E could be provided to individuals identified by HLA typing and analysis of the RNA of a small tumor sample.
3,497 citations
TL;DR: A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene, termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-d determining gene, TDF.
Abstract: A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene. This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript. It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2. This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.
3,019 citations
Johns Hopkins University1, University of Helsinki2, National Institutes of Health3, University of Denver4, Los Alamos National Laboratory5, Fred Hutchinson Cancer Research Center6, University of Texas Health Science Center at San Antonio7, Centre national de la recherche scientifique8, Memorial University of Newfoundland9, University of Auckland10, Creighton University11
TL;DR: Somatic as well as germline mutations of the gene were identified in RER+ tumor cells, and this mutS homolog is likely to be responsible for HNPCC.
2,297 citations
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