Salmonella tester strain TA102 carries the hisG428 ochre mutation on the multicopy plasmid pAQ1. DNA sequence analysis of 45 spontaneous revertants of hisG428 on the chromosome in the presence of pKM101 (strain TA103) indicates that hisG428 revertants fall into three major categories: (t) small, in-frame deletions (3 or 6 base pairs) that remove part or all of the ochre triplet; (it) base substitution mutations at the ochre site; (iii) extragenic ochre suppressors. Deletion revertants are identified in a simple phenotypic screen by their resistance to the inhibitory histidine analog thiazolealanine, which feedback inhibits the wild-type hisG enzyme but not the enzyme resulting from the deletions. The effect of various genetic backgrounds on the generation of spontaneous deletion revertants was examined. The error-prone repair system encoded in the pKM101 plasmid markedly increased the frequency of total spontaneous reversion events in all genetic backgrounds except recA but did not affect the frequency of spontaneous deletion revertants in any background except polA. The presence of a polA mutation increased the frequency of spontaneous deletion revertants by 2-fold in the absence of pKM101 and by 20-fold with pKM101. The presence of a uvrB mutation or a recA mutation suppressed the generation of spontaneous deletion revertants to approximately 1/2.5. When hisG428 was in multiple copies on pAQ1, the frequency of spontaneous deletion revertants increased by 40-fold, which is the approximate copy number of pAQ1. Mutagenic agents that induce single-strand breaks in DNA (e.g., x-rays, bleomycin, and nalidixic acid) induced deletion revertants in TA102. These agents induced deletion revertants only in hisG428 on pAQ1 and only in the presence of pKM101. Deletion revertants were not induced by frameshift mutagens (i.e., ICk-191 and 9-aminoacridine). These results indicate that different pathways exist for the generation of spontaneous and mutagen-induced deletion revertants of hisG428.Genetic rearrangements such as deletions and insertions constitute an important class of mutations. Although it has been suggested that they are important as initiating events in tumor production (1, 2), their study has been largely neglected. Several systems have been described for detecting large deletions (0.7-40 kilobases) in bacteria (3-6). Large deletions are induced by the mutagens mitomycin C, 4-nitroquinoline 1-oxide, nitrous acid, nitrogen mustard, x-rays, and UV light (4-6), although there is variation among different systems.In this study we present a simple system for detecting a class of small deletions [3 or 6 base pairs (bp)] in the hisG gene of Salmonella, using the recently described tester strains TA102 and TA104 (7,8). The occurrence of spontaneous and mutagen-induced deletions was studied in a variety of DNA repair backgrounds.
MATERIALS AND METHODSMaterials. N-(2-Thiazolyl)-DL-alanine, methyl methanesulfonate, nitrogen mustard, and 2,7-dinitro-9-fluorenone were from Aldrich; bleomycin sulfate, nalidixic acid,...
