Mutation frequency decline following chemical mutagenesis of Salmonella typhimurium

The occurrence is reported of a mutation frequency decline process (MFD) following treatment of Salmonella typhimurium strain trpC₃ with two chemical mutagens which give rise predominantly to suppressor revertants. With the carcinogen 4-nitroquinoline-N-oxide (4NQO) the results are analogous to those obtained for UV-mutagenesis. In the case of methoxynamine, the process is due to specific excision of premutational lesions, since lethality is low and lethal lesions are non-excisable. Mutants are described which cannot perform MFD of lesions induced by one or both of the chemical mutagens, indicating that the loss of revertants is in each case due to a bacteial repair system rather than to spontaneous degradation of the induced lesion. The mutants, however, were isolated because of an altered response to UV mutagenesis, viz., their ability to express UV-induced mutants in the absence of amino acids to stimulate active post-irradiation protein synthesis. In all other respects tested, their response to UV is identical with that of the parent strain. The hypothesis is discussed that the total absence of UV-induced revertants of the strain S. typhimurium trpC₃ when active protein synthesis is inhibited is due to two processes, first, rapid MFD due to the specific excision of pyrimidine dimers (the predominant UV-lesion) and secondly, the slow excision of other premutational damage which may be other photoproducts or secondary distortions caused by close juxtaposition of several pyrimidine dimers.     

Ultraviolet light-induced responses of an mfd mutant of escherichia coli B/r having a slow rate of dimer excision

A mutant of Eschirichia coli B/r designated mfd has drastically reduced ability to exhibit “mutation frequency decline” (MFD) the irreversible loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with U.V. We have found that the initial rate of thymine dimer excision in the mfd mutant is only about one-third that of its mfd⁺ parent strain after a UV dose of 400 erg/mm². The yield of UV-induced Tyr⁺ revertants is 4–10 times higher in the mfd strain than in the mfd⁺ strain. This is comparable to the level of UV-mutability in the mfd⁺ strain in the presence of caffeine, an inhibitor of dimer excision. UV-mutability, prophage induction and Weigle reactivation of irradiated λ phage occur to a greater extent at low UV doses (10–50 erg/mm²) in the mfd strain compared to the mfd⁺ strain. We propose that the slow excision repair in the mfd mutant results in a shift in the induction threshold for these UV-inducible functions toward lower UV doses.     

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.     

The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D)C proteins. The frequency of argE(ochre)Arg⁺ mutations (which occur predominantly by ATTA transversions) and Rif^SRif^R mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD₂C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.     

Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation

Protein synthesis was studied comparatively in a wild-type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant differences between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two-dimensional gel electrophoresis showed that histidine-starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT+ cells.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.     

The isolation and mapping of EF-Tu mutations in Salmonella typhimurium

Summary The first isolation of EF-Tu mutations in Salmonella typhimurium is reported. The mutations were isolated by selecting for resistance to the antibiotic mocimycin (= kirromycin). The mocimycin resistant phenotype is the result of mutations in each of two genes, tufA and tufB. Strains mutant in only one of the two tuf genes are sensitive to mocimycin. The spontaneous mutation rate of each of the two tuf genes to a mocimycin resistant phenotype differs by an order of magnitude. tufA maps at minute 71–72, closely linked to rpsL. tufB maps at minute 88–89, closely linked to rpoB. These map positions correspond to the locations of tufA and tufB in E. coli.Abbreviations EF-Tu protein elongation factor Tu - MOC mocimycin     

Effect of nitrosourea in small doses on the frequency of mutation in Salmonella typhimurium

The effect of N-nitroso-N-methylurea (NMU), N-nitroso-N,N'-dimethylurea (NDMU) and N-nitroso-N-ethylurea (NEU) at doses less than 100 mkg/ml on mutability of Salmonella typhimurium strains of Ames' system (G-46, TA-1950, TA-1535, TA-100, TA-1538) has been studied. NMU and NEU at doses of 5-10 mkg/ml have been found to increase the survival and decrease the number of reversions from auxotrophity in histidine to prototrophity. The effect of given doses of NMU and NEU on bacteria repair activity has been shown. The role of pk M101 plasmide in this process is being discussed. NDMU in contrast to NMU and NEU induces read frome shift mutations and exhibits high mutagenous activity at all doses examined.     

The isolation and characterization of a T4 mutant partially defective in recombination

Summary Starting with an rII diploid isolate of T4 a procedure is reported for isolating diploid variants which generate segregants at a reduced frequency. The properties of one such variant, which exhibits a four-fold reduction in segregation frequency, is discussed in detail. It is shown: 1) this variant has acquired at least two extra mutations outside the rII region 2) these mutations confer a four to seven-fold reduction in recombination frequency as measured in standard phage crosses 3) the mutations also cause a 20% increase in UV-sensitivity and a marked effect both on multiplicity-reactivation and on the intracellular development of radiation-resistance.     

Induction of mutations in bacteriophage T7 by gamma-rays: no influence of the SOS repair pathway

Under conditions where the reversion of an amber mutant of bacteriophage lambda by gamma-rays is enhanced by subjecting the irradiated phage to SOS repair, gamma-ray-induced reversion of two T7 ambers is not influenced by this error-prone bacterial repair system. The survival of T7 gamma-irradiated under anoxic conditions is somewhat enhanced by SOS repair, whereas the survival of phage irradiated under oxygen is not affected.