title: G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E. coli host designed for assaying pZ189 supF mutational specificity.
abstract: Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 x 10(-7) per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.
authors: Division of Industrial Health, Osaka Prefectural Institute of Public Health, Japan.
authors: S
authors: S
authors: Akasaka
authors: K
authors: K
authors: Takimoto
authors: K
authors: K
authors: Yamamoto
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009268:Nalidixic Acid / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012343:RNA, Transfer / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D009838:Oligodeoxyribonucleotides; D009268:Nalidixic Acid; D004247:DNA; D012343:RNA, Transfer; D001616:beta-Galactosidase