Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells.
The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.
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Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709.
Simpson
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Cochrane
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Liber
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Skopek
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TR
D001482:Base Composition; D001483:Base Sequence; D015139:Blotting, Southern; D002273:Carcinogens / Q000633:toxicity*; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005027:Ethylene Oxide / Q000031:analogs & derivatives* / Q000633:toxicity; D005091:Exons; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009154:Mutation; D016133:Polymerase Chain Reaction
D002273:Carcinogens; C033154:2-cyanoethylene oxide; D007041:Hypoxanthine Phosphoribosyltransferase; D005027:Ethylene Oxide