A new shuttle vector system for the identification of spontaneous and radiation-induced mutations in the fission yeast Schizosaccharomyces pombe.
A shuttle vector, pCRR1, has been constructed for the detection of spontaneous and radiation-induced mutations in the fission yeast Schizosaccharomyces pombe. This vector contains an Escherichia coli supF suppressor tRNA gene as the target for mutagenesis and bacterial pMB1 and yeast ars1 replication origins, which can be used to propagate the plasmid in bacterial and fission yeast cells, respectively. supF mutations can be detected after plasmid transformation into S. pombe and recovery in a bacterial indicator system, KS40/pKY241, by selecting for nalidixic acid resistance and/or by screening for lacZ- cells. We found that UV light or gamma-rays induced mutations in a dose-dependent manner in this system. Treatment of ultraviolet light (UV)-irradiated DNA with E. coli photolyase, which monomerizes cyclobutane pyrimidine dimers, before introduction into S. pombe reduced mutation frequencies to nearly background levels, indicating that this type of lesion is the major source of mutations. Comparison of spontaneous and UV-induced mutation frequencies in rad+, rad8-190 and rad13-A cells revealed no significant difference in background levels or induced levels after exposure to 100 J/m2 of UV. However, when plasmid DNA was UV-irradiated with 500 J/m2, the rad8-190 cells generated only 38% as many induced supF mutations as the rad+ strain, whereas the rad13-A cells produced more than a 6-fold increase in mutability relative to the level observed for the wild-type strain. These mutability patterns are consistent with previous studies that characterized rad8-190 cells as hypomutable and rad13-A cells as hypermutable by UV light at chromosomal loci. Thus, this shuttle vector system provides a useful and sensitive tool to assess mutability in S. pombe.
Zhao
Y
Y
Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Goriparthi
L
L
Lieberman
H B
HB
D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005720:Gamma Rays; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011740:Pyrimidine Dimers; D012568:Schizosaccharomyces / Q000235:genetics / Q000528:radiation effects*; D014466:Ultraviolet Rays
D017931:DNA Primers; D011740:Pyrimidine Dimers