Genomic DNA sequencing of mRNA splicing mutants in the hprt gene of Chinese hamster ovary cells.
We have analyzed 41 mRNA-splicing mutants from the hypoxanthine-guanine phosphoribosyl-transferase (hprt) gene of Chinese hamster ovary (CHO) cells. Twenty-two of these mutants produced single cDNA PCR products with a partial or complete exon deletion; 19 mutants produced multiple cDNA PCR products, and most of these products contained one or more deleted exons. The affected exons and surrounding introns were amplified from genomic DNA and sequenced in order to identify mutations causing aberrant splicing. We found acceptor site mutations in 10 mutants, exonic mutations in 8 mutants, and no mutations in 5 mutants. Four mutants from solvent controls did not amplify the appropriate exons and were considered genomic deletion mutants. Our previous work [Manjanatha MG et al. (1994): Mutat Res 308;65-75] showed that nonsense mutants in the hprt gene of CHO cells are associated with multiple cDNA PCR products containing deleted exons and a low abundance of hprt mRNA if the mutation is found in an internal exon. The present results are consistent with these associations being facilitated by instability of mRNA after ribosome termination at nonsense codons.
Valentine
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Division of Genetic Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079-9502, USA.
Heflich
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D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects; D002911:Chrysenes / Q000633:toxicity; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005091:Exons; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity; D017354:Point Mutation; D011721:Pyrenes / Q000633:toxicity; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D017384:Sequence Deletion
D002911:Chrysenes; D017931:DNA Primers; D009153:Mutagens; D011721:Pyrenes; D012333:RNA, Messenger; C035986:6-nitrochrysene; D007041:Hypoxanthine Phosphoribosyltransferase; C032668:1-nitropyrene