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Genomic DNA sequencing of mRNA splicing mutants in the hprt gene of chinese hamster ovary cells
Author(s) -
Valentine Carrie R.,
Heflich Robert H.
Publication year - 1995
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850250202
Subject(s) - chinese hamster ovary cell , exon , mutant , microbiology and biotechnology , nonsense mutation , biology , genomic dna , gene , genetics , complementary dna , hypoxanthine guanine phosphoribosyltransferase , intron , mutation , rna splicing , rna , missense mutation , cell culture
We have analyzed 41 mRNA‐splicing mutants from the hypoxanthine‐guanine phosphoribo‐syl‐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 in‐trons were amplified from genomic DNA and sequenced in order to identify mutations causing aberrant splicing. We found acceptor site mutations in 14 mutants, donor site mutations in 10 mutan's, 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. © 1995 Wiley‐Liss, Inc.