Molecular nature of spontaneous mutations at the hypoxanthine‐guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

Abstract

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

Comparative analysis of the mutagenic activity of oxaliplatin and cisplatin in the Hprt gene of CHO cells

“…The spontaneous mutations that were identified were predominantly base substitutions, consisting mostly of G:C?T:A, followed by G:C?C:G and A:T?G:C substitutions. These observations are consistent with spontaneous Hprt mutation spectra previously reported in CHO [Xu et al, 1995] and in V79 cells [Zhang et al, 1992], in which base substitutions were also the predominant change, with transversions being more common than transitions. Although there was no clear shift in the overall distribution of mutations between the spontaneous and the cisplatin-and oxaliplatin-induced mutant sets, there were specificities in the type of mutations and also in the sequence context that discriminated between the spectra.…”

Lead acetate mutagenicity and mutational spectrum in the hypoxanthine guanine phosphoribosyltransferase gene of Chinese hamster ovary K1 cells

“…Sequencing analyses showed lead-induced single-base substitutions (primarily at G C base pairs), small sequence alterations, splicing defects, and gene deletions. Furthermore, the lead-induced mutational specificity in CHO K 1 cells differed from that occurring spontaneously [23] and from that induced by the metal carcinogen chromium(V1) oxide [24].…”

Chemical and toxicological characterization of slurry reactor biotreatment of explosives-contaminated soils

“…This is an established assay that has been used to evaluate compounds either for their direct mutagenic activity or after "activation" with liver enzyme extracts -so called "S9 preparations." This system is used particularly to identify the types of mutations caused by specific agents (20,21). A wealth of data on the activity of a variety of compounds in this assay is available for comparison (20,(22)(23)(24)(25)(26).…”