Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.
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.
Xu
Z
Z
Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, USA.
Yu
Y
Y
Schwartz
J L
JL
Meltz
M L
ML
Hsie
A W
AW
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000201:enzymology*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005091:Exons; D016368:Frameshift Mutation; D017353:Gene Deletion; D005797:Genes, araC / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012326:RNA Splicing
D017931:DNA Primers; D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase