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 μM × 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 exon 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. © 1990.