Restoration of mutability in non-mutable Escherichia coli carrying different plasmids.

Summary N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA ⁺ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA ^- strains (class I) and those leaving lexA ^- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while class II plasmids cause far fewer MMS revertants in this strain than in lexA ⁺. A pair of class I and II plasmids in lexA ^- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

Reversions of the two missense and one nonsense trp-mutations induced by UV-light or methyl methanesulfonate in E. coli wild type and its polAi and uvrE502 mutants.

Two missense mutations, trpA58 and trpA78, and one nonsense mutation-trp-ochre, were used to determine the types of base-pair substitution caused by ultra, violet irradiation and methyl methanesulfonate (MMS) in Escherichia coli. UV irradiation of the wild-type bacteria led to the formation of revertants mainly arising as a result of GC yields AT transitions (suppressor revertants of the trpA58 mutant). True revertants of the trp- mutant (arising via transitions of AT pairs) and 5-methyl tryptophan-sensitive (MT-s) Trp+ of the trpA78 mutant (arising via unidentified transversions) occurred at a lower frequency. The polAI mutation did not change the frequency of the UV-induced transitions GC yields AT or that of the substitutions of the AT pairs. The uvrE502 mutation significantly increased the frequency of the UV-induced revertants arising via the transition GC yields AT. Treatment of the wild-type bacteria with MMS resulted in the formation of revertants mainly due to the GC yields AT substitution, and with a lower frequency to the AT yields GC transitions. MMS also induced, with a low frequency, some transversions. The frequency of the MMS-induced GC yields AT transitions was enhanced in the uvrE502 mutant. On the other hand, the uvrE502 mutation eliminated or significantly lowered MMS-induced revertants arising as a result of AT yields GC transitions or transversions.     

Isolation and characterization of cAMP suppressor mutants of Escherichia coli K12

Summary We have isolated spontaneous and chemically induced revertants of cya mutant strains of Escherichia coli. Three different classes of revertants were obtained. One class consisted of primary site revertants; a second class was pseudorevertants that had phenotypically reverted to wild type but retaining the original cya mutation and the third class of revertants, designated csm, were pseudorevertants hypersensitive to exogenous cAMP. Transductional analysis of the csm mutation indicated the mechanism of suppression in these strains was intergenic. The csm mutation and hypersensitivity to cAMP map in or near the crp gene. Growth of the csm strains of PTS (phosphoenolpyruvate phosphotransferase system) and non-PTS substrates was inhibited by 5 mM cAMP. The csm strains were found to accumulate toxic levels of methylglyocal when grown on non-PTS substrates in the presence of exogenous cAMP. All csm strains were sensitive to catabolite repression mediated by -methylglucoside. Revertants selected as resistant to cAMP fell into four major classes that could be distinguished by their fermentation patterns in the presence and absence of cAMP as well as by their growth response to streptomycin in the presence of cAMP.Paper No. 6623 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650, USA     

Ammonium assimilation in Rhizobium meliloti

We have characterized a mutant of Rhizobium meliloti strain 2011 which cannot use ammonium as a nitrogen source. This mutant, RTm2620, was found to have significantly altered glutamate synthase activity. Both the mutant and the wild-type strains had glutamate dehydrogenase activity, which, although stimulated in the presence of glutamate and ammonium, was apparently insufficient to allow ammonium assimmilation. We conclude that the glutamine synthetase-glutamate synthase pathway may be the normal mode of ammonium assimilation by this strain in the free-living state. Independent revertants of Rm2620 were isolated and fell into two classes. Class I revertants regained partial glutamate synthase activity and had the same levels of glutamate dehydrogenase activity as Rm2620. Class II revertants retained the altered glutamate synthase activity but acquired a very high level of assimilatory glutamate dehydrogenase activity. Both classes were found to be altered in their symbiotic properties, although the original Rm2620 mutant was normal in this regard.     

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.     

Suppressor system in Bacillus subtilis 168

Multiple auxotrophic strains of Bacillus subtilis 168 were tested for joint one-step reversion of two or more auxotrophic markers to the wild-type phenotype. Mu8u5u5, a strain requiring leucine, methionine, and threonine, yielded revertants that grew without added methionine or threonine and proved to have a suppressor gene. When transferred by transformation with deoxyribonucleic acid, this suppressor gene also suppressed the adenine mutation in another strain, Mu8u5u6. The one-step double revertants fell into two distinct classes: strains of class su(+) (I) grow well in broth; strains of class su(+) (II) grow poorly. Strains su(+) (II) tend to revert frequently to the su(+) (I) or su(-) state. Conditional lethal mutants of phage phie were isolated which can grow on the su(+) and not on the su(-) strains.     

Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements

Mouse expanded simple tandem repeats (ESTRs) provide highly informative loci for analyzing spontaneous and induced germline mutation. We have conducted an extensive sequence database search and identified 17 new members of the highly unstable rodent-specific ESTR family called MMS10. This family has arisen by independent expansions of a common GGCAGA repeat unit from within a subset of both ancestral and modern SINE B1 elements during the course of mouse evolution. Analysis of the interspersion patterns of variant repeats along alleles of 20 of these MMS10 loci revealed two distinct classes of tandem arrays: one composed of uninterrupted GGCAGA repeats and the second with generally larger arrays interrupted by variant units. Surveys of allelic diversity at 11 representative members of these two classes of loci in various laboratory strains and BXD recombinant inbred lines revealed that the level of repeat instability was positively correlated with the length of uninterrupted repeats. Turnover processes at MMS10 loci, therefore, appear similar to the type of mechanism observed at human microsatellites. The MMS10 family thus provides a potentially useful murine model for studying dynamic mutation at simple tandem repeats.     

Characterization of multiple changes of antibiotic resistance characters in Streptomyces coelicolor A3(2)

Among mutants of Streptomyces coelicolor A3(2) studied which were sensitive to chloramphenicol (Cmls), strains sensitive to a number of antibiotics (ristomycin, tetracycline, polymyxin, lincomycin) amount of 46%. Antibiotic-sensitive mutants are capable to form different classes of resistant revertants with frequency varying from 10(-2) to 10(-6) in independent strains. Ristomycin-sensitive clones (Rims) have been found to occur with high frequency in Cmls strains and Cmlr revertants. Mutations mediating the Rims phenotype are mapped in a locus linked to the gene for resistance to chloramphenicol. The results obtained are discussed, in accordance with the notion about possible role of cml mutation in induction of secondary mutational changes in the genome of S. coelicolor A3(2).     

Genetic events associated with an insertion mutation in yeast

The his4-912 mutation shares similar genetic properties with mutations promoted by procaryotic insertion elements. This mutation lacks all three his4 functions. Many different classes of His+ revertants have been obtained from his4-912. The most frequent class of His+ revertants results from a site mutation which confers a cold-sensitive His- phenotype. Other classes of revertants contain translocations (one between chromosomes I and III and the other between chromosomes III and XII), a transposition of the his4 region to chromosome VIII, and an inversion of most of the left arm of chromosome III. Another class contains deletions which extend from his4-912 into the his4 region. In each of these classes of revertants, the his4 region is closely linked to the chromosomal aberration. Many of these revertants contain additional changes in chromosome structure (duplication, deletion and aneuploidy) that are unrelated to the reversion of his4-912 to His4+.     

A system to determine basepair substitutions at the molecular level, based on restriction enzyme analysis; influence of the muc genes of pKM101 on the specificity of mutation induction in E. coli

A system has been developed for the analysis of basepair substitutions that are involved in the reversion of a specific missense mutation. The method is based on the ability of restriction enzymes to recognize and cut specific DNA sequences. Wild-type revertants arising from AT----GC transitions, pseudo wild-type revertants arising from AT-transversions and second site revertants can be distinguished. 4 mutagenic agents have been used, 2,6-diaminopurine, MMS, EMS and ENU, which differ in the types of damage they cause in DNA and in the susceptibility of the damage to repair. All 4 mutagens effectively enhanced the reversion of the mutation studied, trpA223, particularly by increasing the fraction of AT----GC transitions. In this system the influence of the muc genes of plasmid pKM101 was investigated. The presence of these genes reduced the fraction of AT----GC transitions and enhanced the fraction of AT-transversions as well as the fraction of second-site mutations. This change in mutation specificity is found irrespective whether mutation induction occurs mainly via SOS repair (MMS, ENU) or via mainly misreplication (2,6-diAP, EMS). These data suggest that the muc genes are involved in the induction of mutations not only during SOS repair, but also during misreplication. The change in mutation specificity may be caused by a change in the selection and insertion of nucleotides by the DNA-polymerising complex, or by interference with the repair of mismatched bases.     

Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli

In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E. coli dam- cells are able to mutate following MMS treatment. Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E. coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains. The mutagenicity of MMS was also tested in several of these strains. The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system. The role of strand breaks in repair of mismatches induced by alkylating agents is discussed.     

Isolation and Characterization of Revertant Cell Lines VI. Susceptibility of Revertants to Retransformation by Simian Virus 40 and Murine Sarcoma Virus

The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied. Both serum-sensitive and density-sensitive revertants are not retransformable by SV40. MSV can transform both types of revertants. The MSV-transformed revertants grow to high cell densities and form colonies when suspended in semi-solid methylcellulose medium, but are unable to grow in 1% calf serum. The MSV-transformed revertants produce infectious MSV and murine leukemia virus and possess the same number of chromosomes as the untransformed revertants.     

Isolation and characterization of T antigen-negative revertants from a line of transformed rat cells containing one copy of the SV40 genome.

Negative selection with FUdR produced revertants from the transformed rat line 14B, which contains one insertion of the SV40 viral genome (Botchan, Topp and Sambrook, 1976). 14B contains nuclear T antigen, grows to a high density, grows in low serum and is anchorage-independent. The revertants fall into three classes with regard to viral DNA sequences: the SV40 DNA is retained; the SV40 DNA is retained but has undergone a deletion; and the SV40 DNA is lost, generating a cured cell. This heterogeneity is not a result of long-term passage. The revertants arise with a frequency of one in 8.4 X 10(5) cells after as few as 12 passages. All three classes of revertants are T antigen-negative, density-sensitive, more serum sensitive than 14B and anchorage-dependent. These data argue for a direct role of the functioning viral genome in the maintenance of the transformed state, and that with 14B, the phenotypes of transformation are not virus gene dosage-dependent.     

UV-mutagenesis in Bacillus subtilis. IV. Analysis of revertants to methionine prototrophy]

UV light induces in Bacillus subtilis met5 ade6 two classes of revertants to prototrophy to methionine which can be easily distinguished by their phenotype: double (Met+Ade+) and solitary (Met+) revertants. Crosses of revertants with the wild type, carried out in transformational experiments, showed that original (direct) mutation met5 is presented in chromosome of double revertants. Consequently they are extragenic suppressor revertants. In the chromosome of solitary revertants Met+ an extragenic suppressor was not detected; reversions Met+ seem to be of an intragenic nature. It is possible to use reversions to prototrophy to methionine as a model to study UV-mutagenesis in suppressor and non-suppressor genes.     

Antimutagenic activity of methanolic extracts of four ayurvedic medicinal plants

Methanolic extracts of Acorus calamus (Rhizome), Hemidesmus indicus (Stem), Holarrhena antidysenterica (Bark) and Plumbago zeylanica (Root), were tested for their antimutagenic potential. These extracts, at tested concentrations, showed no sign of mutagenicity to Salmonella typhimurium tester strains. The extracts of the plants exhibited varying level of antimutagenicity. At a dose of 100 microg/plate, the extracts exhibited the inhibition of His+ revertants from 18.51% to 82.66% against direct acting mutagens, methyl methanesulphonate (MMS) and sodium azide (NaN3) induced mutagenicity in Salmonella tester strains TA 97a, TA 100, TA 102 and TA 104. However, at lower concentrations (25 and 50 mcirog/plate) of the plant extracts, a decrease in antimutagenic activity was recorded. Dose dependent antimutagenic activity of the extracts is also evident from linear regression analysis of the data. The over all antimutagenic potential of above four extracts was found to be in order of A. calamus > H. indicus > H. antidysenterica > P. zeylanica. Further, total phenolic content of these extracts did not correlate with its antimutagenic activity in A. calamus and P. zeylanica.