The mutation spectra of 4-nitroquinoline N-oxide-induced frameshift reversion ofhis4-38 in REV1 and rev1-1 strains of Saccharomyces cerevisiae

The purpose of this study was to recover DNA sequence information from the HIS4A region of chromosome III in a large number of independently arising, 4-nitroquinoline N-oxide (4NQO)-induced His$\sp+$ revertants of the +1 frameshift mutation his4-38 in Saccharomyces cerevisiae strains XY729 (REV1) and XY760 (rev1-1). These two strains differ in their ability to repair DNA. The REV1 gene is involved in error-prone repair of DNA. The two strains differed in their sensitivity to the lethal effects of 4NQO. To control for this difference both strains were treated with a concentration of 4NQO that led to 37% survival (one lethal hit). To obtain the DNA sequence information, methods for DNA amplification by the polymerase chain reaction (PCR) and subsequent dideoxy sequencing of the double-stranded PCR product were developed. PCR was optimized to generate a high yield of the amplified DNA, and to increase the stringency of the reaction to prevent the formation of artifacts. Double-stranded, dideoxy sequencing with $\sp{35}$S-dATP of the PCR-amplified DNA was developed and optimized to produce clear, readable sequence autoradiographs. One hundred His$\sp+$ revertants were sequenced from each strain. Of these 86 of the XY729 revertants and 94 of the XY760 revertants were due to $-$1 frameshift events, with the rest of the reversion events being due to complex mutations. The distribution of XY729 $-$1 frameshift events was different from the spontaneous control distribution (Kalinowski, 1991; Kalinowski et al., 1991). The distribution of the XY760 $-$1 frameshift events was different from both the spontaneous controls and the XY729 distribution. The XY729 distribution exhibited a hot spot for mutation at the original site of the his4-38 mutation. This hot spot was absent from the XY760 distribution. The types of 4NQO-induced complex mutations observed also differed from the spontaneous controls and between the two strains, with several types observed in XY729 and only one type observed in XY760. 4NQO's mutagenic effects occurred primarily at guanine residues in both strains. The REV1 gene product appeared to play a role in the specificity of 4NQO-induced mutation.U of I OnlyETDs are only available to UIUC Users without author permissio