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.     

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.     

Reversion of DNA polymerase-deficient Escherichia coli

Summary When a DNA polymerase-deficient strain of E. coli (pol A^- mutant; amber nonsense) was exposed to methyl methanesulfonate or to nitrosomethylurethan, drug-resistant mutants which had recovered the pol A⁺ character were isolated. On the other hand, exposure of pol A^- bacteria to nalidixic acid or to streptozotocin resulted in the recovery of mutants with specific resistance only to nalidixic acid or streptozotocin. These mutants retained the pol A^- character.     

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.     

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.     

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.     

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.     

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.     

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 Instability in the Galactose operon of Escherichia coli

The gal-3 mutation, which had been shown previously to produce unstable revertants, was combined in three instances with recA, a mutation which suppresses recombination. Unstable revertants were produced in the gal-3 recA recombinants qualitatively as frequently as in the absence of the recA gene, and it is concluded that a recombination mechanism is not the basis of the instability observed.     

Ultraviolet-Sensitive Mutator mutU4 of Escherichia coli Inviable with polA

Attempts to transduce the ultraviolet-sensitive mutator lesion mutU4 into strains deficient in deoxyribonucleic acid polymerase I (polA) were unsuccessful. Mutator recombinants were found when the polA recipient had first been reverted to Pol(+) by selection for resistance to methyl methanesulfonate. The inviability of the mutU4 polA double mutant was demonstrated by a reduction in the absolute number of transductants when the recipient was polA as compared with Pol(+), and selection was made for markers very close to mutU4. Double mutants containing mutU4 and polA4, which determines a cold-sensitive polymerase, were unable to grow at 24 C, the nonpermissive temperature.     

Reversion of the Frameshift hisD3018 of Salmonella typhimurium

Previous studies on histidinol dehydrogenase from His(+) revertants have shown that the frameshift mutation hisD3018 is a +1 type, resulting from inclusion of an extra cytidylate residue in messenger ribonucleic acid. Histidinol dehydrogenase from newly isolated spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced intragenic revertants has been examined for amino acid replacements. The results provide additional evidence that NG can delete guanine plus cytosine base pairs from deoxyribonucleic acid. One spontaneous revertant was found to result from a +2 addition of approximately 16 nucleotide residues before the +1 parent frameshift, and another by a -4 deletion about six residues before the same. Circumstantial evidence suggests the in vivo codon assignment GAG for glutamic acid. A region of histidinol dehydrogenase highly permissive of amino acid changes encoded in the minus (-) phase is now apparent.