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We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.

Many metals have been shown to alter the function of a wide range of enzyme systems, including those involved in DNA repair and replication. To assess the impact in vivo of such metal actions a "Microtitre" fluctuation assay was used to examine the ability of Ni(II) to act as a comutagen with simple alkylating agents. In E. coli, Ni(II) chloride potentiated the mutagenicity of methyl methanesulfonate (MMS) in polymerase-proficient strains (WP2+ and WP2-), but not in polA- strains (WP6 and WP67) or in lexA- (CM561) or recA- (CM571) strains. The absence of UV excision repair (WP2- and WP67) had little, if any, effect. An extended lag phase was seen at 2-4 h in the polA- strains following treatment with Ni(II) chloride and MMS, but normal growth resumed thereafter. Results suggested that mutations induced by MMS were fixed during log phase growth and that more than 2 h of exposure were necessary for potentiation by Ni(II) to be observed. Thus, the extended lag phase probably cannot explain the lack of potentiation. RecA-dependence of the comutagenic effect was corroborated with S. typhimurium TA1535 and TA100. Only in the pKM101 containing strain, TA100, was potentiation of ethyl methanesulfonate (EMS) and MMS by Ni(II) chloride evident. The mucAB genes carried on pKM101 increase the sensitivity of TA100 to a variety of mutagens, providing there is a functional recA gene product. Taken together, the data suggest that Ni(II) acts indirectly, as a comutagen, in bacterial systems, possibly affecting processes involving recA- and/or polA-dependent function(s).

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.