DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.
DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.
复制标题
N-乙酰氧基-2-乙酰氨基芴诱导的中国仓鼠卵巢细胞二氢叶酸还原酶突变体的DNA碱基变化和RNA水平。
DOI：
10.1016/0022-2836(89)90506-8
复制
发表时间：
1989
影响因子：
5.6
通讯作者：
Grunberger,D
中科院分区：
文献类型：
--
作者：
Carothers,AM;Steigerwalt,RW;Urlaub,G;Chasin,LA;Grunberger,D
Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced byN-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 basedhfrgene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage λ vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G · C to T · A transversions in either thedhfrcoding sequence or at splice sites; transition G · C to A · T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of a G · C pair and the other was an insertion of an A · T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels ofdhfrmRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels ofdhfrmRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the lowdhfrmRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of thedhfrgene.
