Reversion of Frameshift Mutations Stimulated by Lesions in Early Function Genes of Bacteriophage T4

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.     

Adaptive Reversion of a Frameshift Mutation in Escherichia Coli

Mutation rates are generally thought not to be influenced by selective forces. This doctrine rests on the results of certain classical studies of the mutations that make bacteria resistant to phages and antibiotics. We have studied a strain of Escherichia coli which constitutively expresses a lacI-lacZ fusion containing a frameshift mutation that renders it Lac-. Reversion to Lac+ is a rare event during exponential growth but occurs in stationary cultures when lactose is the only source of energy. No revertants accumulate in the absence of lactose, or in the presence of lactose if there is another, unfulfilled requirement for growth. The mechanism for such mutation in stationary phase is not known, but it requires some function of RecA which is apparently not required for mutation during exponential growth.     

Temperature-Sensitive Nonsense Mutations in Essential Genes of Escherichia coli

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.     

Mutator Gene Studies in Escherichia coli

An Escherichia coli mutator gene, mutT, has been shown by P1-mediated crosses to map between the leucine and azide loci. The mutT1 and azi-r alleles cotransduce with a frequency of >92%. In mutT1/mutT⁺ merodiploids, the mutT1 phenotype is recessive; in mutT1/F′trp or mutT1/F′lac merodiploids, the mutT1 allele has a trans effect. The gene can mutate λ and T7 phage but not T1, T3, T4, T5, and S13.     

Mutator Gene of Escherichia coli B

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.     

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.     

Spontaneous and ICR191-A-induced Frameshift Mutations in the A Gene of Escherichia coli Tryptophan Synthetase

Thiaisoleucine-resistant mutants of Escherichia coli strain K-12 which exhibited reduced isoleucyl soluble ribonucleic acid synthetase activity were isolated. Resistance was apparently achieved by the selection of a synthetase with a 10-fold decrease in apparent affinity for thiaisoleucine. This mutation also resulted in a 2.5-fold decrease in apparent affinity for the natural substrate, l-isoleucine, and less activity than found in wild type. The mutants grew more slowly than wild type and were derepressed for three of the five enzymes in the pathways to isoleucine and valine.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Levels of the Vsr Endonuclease Do Not Regulate Stationary-Phase Reversion of a Lac− Frameshift Allele in Escherichia coli

Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac⁻ frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.     

Mutator specificity of Escherichia coli alkB117 allele

The Escherichia coli AlkB protein encoded by alkB gene was recently found to repair cytotoxic DNA lesions 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) by using a novel iron-catalysed oxidative demethylation mechanism that protects the cell from the toxic effects of methylating agents. Mutation in alkB results in increased sensitivity to MMS and elevated level of MMS-induced mutations. The aim of this study was to analyse the mutational specificity of alkB117 in a system developed by J.H. Miller involving two sets of E. coli lacZ mutants, CC101-106 allowing the identification of base pair substitutions, and CC107-CC111 indicating frameshift mutations. Of the six possible base substitutions, the presence of alkB117 allele led to an increased level of GC-->AT transitions and GC-->TA and AT-->TA transversions. After MMS treatment the level of GC-->AT transitions increased the most, 22-fold. Among frameshift mutations, the most numerous were -2CG, -1G, and -1A deletions and +1G insertion. MMS treatment appreciably increased all of the above types of frameshifts, with additional appearance of the +1A insertion.     

Mutagenesis by Mutator Gene mutH1 in Continuous Cultures of Escherichia coli

Mutability as a function of growth rate was examined in bacterial strains containing mutator gene mutH1. The rate of mutation to bacteriophage T5 resistance was found to be proportional to rate of growth in glucose-limited chemostat cultures. This finding supports the hypothesis that the mutator mutH1 gene product increases the error frequency during deoxyribonucleic acid replication.     

Isolation, Characterization, and Genetic Analysis of Mutator Genes in Escherichia coli B and K-12

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.     

Reversion of Ribosomal Helix Formation in Escherichia coli

After transfer into fresh medium, Escherichia coli cells containing ribosomal helices resume growth without a lag period. The helices disappear within 15 min after transfer, the number of 70S ribosomes decreases, and a steady-state ribosomal profile appears within one cell generation time. Subunits isolated from the helices support in vitro protein synthesis, but efficiency is optimal only when supplemented with an undetermined factor that is contained in the S-100 fraction of log-phase cells. The data suggest a possible role of helices as ribosomal reserve units.     

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs. The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations. The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed.     

Mutator effects in Escherichia coli caused by the expression of specific foreign genes

Certain genes from Lactococcus lactis and Pseudomonas aeruginosa, including the nfxB gene, generate a mutator phenotype in Escherichia coli. The results of this study, together with those of a previous study, support conservation of regulatory sequences in E. coli and P. aeruginosa and suggest that some efflux pumps prevent mutagenicity by exporting mutagenic products of metabolism.     

A Set of Lacz Mutations in Escherichia Coli That Allow Rapid Detection of Specific Frameshift Mutations

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.     

Reversion of a streptomycin-dependent strain of Escherichia coli

Summary A streptomycin dependent, spectinomycin resistant mutant ofEscherichia coli was used to select spontaneous phenotypic revertants to non-dependence on streptomycin. The ribosomes from one such revertant, which is inhibited by both streptomycin and spectinomycin, were analyzedin vitro. The altered protein responsible for the suppression of the streptomycin dependent phenotype was identified; this protein is 30S-10. The genetic locus for this mutation is a newly identified locus and it has been positioned close to thestr locus. The identification of the altered component responsible for the suppression of the spectinomycin resistant phenotype may be the same as that for the streptomycin dependent phenotype, but this is unproven.     

Reversion reactions of beta-galactosidase (Escherichia coli)

The reversion reactions of beta-galactosidase (Escherichia coli) produced beta-galactosyl-galactoses and beta-galactosyl-glucoses. About 10 beta-galactosyl-galactose and 10 beta-galactosyl-glucose gas-liquid chromatographic peaks were detected and it is thus very likely that every possible isomer of beta-galactosyl-galactose and beta-galactosyl-glucose was formed by the reversion reactions (taking into account both anomers for each isomer). The presence of lactose and allolactose among the beta-galactosyl-glucoses was confirmed with standards. An important finding relating to the role of allolactose as an inducer of the lac operon was that allolactose (beta-D-galactosyl-(1----6)-D-glucose) was the only disaccharide formed initially, and at equilibrium it was present in the largest amount (50%). Obviously the enzyme is specific in its ability to form allolactose, and allolactose is the most stable beta-galactosyl-glucose, both important inducer properties. The equilibrium constant (concentration of disaccharides divided by the concentration of reactants at equilibrium) of the reaction was about 9.5 mM-1. This is the first report of an equilibrium constant for the beta-galactosidase reaction. Of mechanistic significance is the fact that only three compounds were able to replace D-galactose as a reversion reactant. Two of these (L-arabinose and D-fucose) had alterations at carbon 6. The 6 position, therefore, is not essential for reactivity. The third compound was D-galactal. Any other sugars tested (even with very minor changes relative to D-galactose) did not react. Of special consequence is the 2 position. The results strongly suggest that there has to be either an equatorial hydroxyl at the 2 position of a sugar or a special reactivity (as with D-galactal) in order for the enzyme to catalyze the beta-galactosidase reaction.