A direct confirmation of the specificity of mutation frequency decline for suppressor mutations

Summary In a phage T7-resistant and galactose-sensitive derivative of E. coli B/r trp^- it has been possible to show that MFD for UV-induced mutations is specific for Trp⁺ reversions (mainly of an ochre suppressor-containing type) but is without effect on galactose-resistant or D-fucose-resistant (ara C_c) forward mutations.     

Role of post-replication and excision repair mechanism in the induction ofTrp + revenants of UV-irradiatedEscherichia coli

Both the post-replication and the excision repair mechanism participate in the induction ofTrp ⁺ revertants inEscherichia coli B/rHer ⁺ thy trp after a UV-irradiation. At low radiation doses (surviving cell fraction > 10^?) mostTrp ⁺ reversions are due to post-replication repair mechanism while at high doses (surviving cell fraction « 10^?1) theTrp ⁺ reversions arise probably as the result of an inaccurate excision repair. The absolute accuracy of repair processes decreases with increasing radiation dose.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

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.     

Mutagenic DNA repair in Escherichia coli. VII. Constitutive and inducible manifestations.

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp⁺ mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating “fixation”) during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair.In contrast the frequency of UV-induced mutations to Trp⁺ (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. We conclude that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system.     

Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr⁺ or uvr^? strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His⁺, ColE^R and Spc^R, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N′-nitro-N-nitrosoguanidine-induced His⁺ mutations. In contrast, only 1 out of 13 trp^? frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC⁺-dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp⁺ mutations in most of the strains regardless of the umu⁺ or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC⁺-dependent pathway of error-prone repair.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.     

Development of papillae on colonies of two isopoly-auxotrophic strains ofSaccharomyces cerevisiae allelic inRAD6 during adenine starvation

Papilla formation on colonies of two isopolyauxotrophic strains (ade 2 his3 leu2 trp1 ura3) allelic inRAD6 was compared in order to find proper conditions for selecting mutants ofSaccharomyces cerevisiae with altered starvation-induced mutability. The most promising for this purpose appeared to be culturing low numbers of colonies on suboptimal plates with a growth-limiting amount of adenine at 28 °C for 20 d. Inactivation of theRAD6 gene which suppresses the level of starvation-associated mutagenesis markedly enhanced papilla formation under these conditions. Formation of almost all papillae on 20-d-old colonies of BJC3 was caused by mutation. Most of the papillae (75%) were white Ade⁺ revertants. Three groups of these papillae were distinguished (Ade⁺, Ade⁺ Rad6⁺ and Ade⁺ Trp⁺). Both, Ade⁺ Rad6⁺ and Ade⁺ Trp⁺ double reversions were very probably caused by a suppressor mutation. The less frequent red papillae had the same auxotrophic markers and UV sensitivity as BJC3 but their outgrowth in liquid media was greater. It appears that creation of these papillae is caused by mutation affecting the cell response to growth limitation by low concenttations of adenine.     

The dependence of UV-induced reversions to prototrophy on the streptomycin resistance allele in Escherichia coli

UV-induced mutability to prototrophy depended on the alleles of the strA locus. The yield of UV-induced suppressor mutations was decreased ten-fold in a Str^r strain compared with an isogenic Str^s strain. This decrease was due to the inability of the majority of suppressors to be expressed phenotypically in the Str^r strain in the course of selection. The addition of streptomycin to the selective media raised the number of selected suppressor revertants, by making such expression possible. The probable relation of streptomycin resistance and UV-induced suppressors is discussed.     

Revertants of a streptomycin-resistant, oligosporogenous mutant ofBacillus subtilis

Summary Revertants of a streptomycin-resistant (Str^R), oligosporogenous (Spo^-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: Str^S Spo⁺ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Str^R Spo⁺, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in Str^R Spo⁺ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Str^R revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Str^R parental strain. The Str^R parent grew slowly and sporulated at approximately 1% of the wild type level. The Str^S revertants closely resembled the wild type strain with regard to growth and sporulation. The Str^R revertants grew at rates intermediate between those of the Str^R parent and wild type, and sporulated at wild type levels.     

Streptomycin-resistant mutant production in a continuous-flow UV mutation device

Summary A continuous-flow UV-induced mutation device which incorporates starting strain cultivation, UV irradiation and mutant reproduction was conceptualized and tested in this study using streptomycin resistance as an indicator of mutant production. For the experimental conditions employed and populations used, the mutation frequency for streptomycin resistance ranged from 10^–4 to 10^–5 cfu/ml. These mutation frequencies are comparable with conventional batch UV mutation methods and represent a gain of 3 orders of magnitude over the spontaneous mutation frequency.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

The effect of extracellular metabolites on the frequency of thy+ revertants in Salmonella typhimurium populations

There is convincing evidence that adaptation and survival processes in bacterial populations depend on cell-to-cell interactions. Our studies showed that the frequency of stress-induced His⁺ reversions in an amino-acid-starved Salmonella typhimurium culture is inversely proportional to cell density in this culture. The effects of cell density and of different culture liquids prepared from cultures starved for histidine on the frequency of Thy⁺ revertants were also studied. It was found that the frequency of Thy⁺ revertants is inversely proportional (r = ?0.74) to the density of the bacterial culture starved of thymine. The culture liquid prepared from the culture starved of histidine exerted an inhibitory effect on the frequency of Thy⁺ reversions, indicating that mutations induced by different types of stress have a common mechanism. The study of the effect of the culture liquid prepared from a histidine-starved culture on the frequency of ethyl-methanesulfonate-induced His⁺ revertants showed that this liquid prevented the induction of His⁺ reversions.     

Mutagenic repair in Escherichia coli: VIII. Effect of gyrB mutations on ultravioet light mutagenesis

Introduction into Escherichia coli WP2 bacteria of a mutation in the gyrB locus previously shown to reduce the degreee of chromosomal superhelicity caused a small decrease in the frequency of UV-induced mutations to streptomycin resistance (but not significantly) and to tryptophan independence (mostly ochre suppressors) in excision repair-proficient bacteria. It did not influence the ‘broth effect’ or the rate or extent of ‘mutation frequency decline’ of suppressor mutations. In an excision-deficient (uvrA 155) background the yield of UV-induced streptomycin-resistant mutations was lower in gyrB bacteria at all doses; the yield of tryptophan-independent mutations was slightly lower at low doses and slightly higher at high doses. In both excision-proficient and -deficient the yield of UV-induced mutations to rifampicin resistance was apparently in gyrB mutants but this could be due at least in part to a hypersensitivity of some Rif^r gyrB bacteria to UV.The number of spontaneous tryptophan-independent mutations was lower in gyrB bacteria but this was almost certainly due to their poorer viability on trypophan-limiting plates and not to a lower spontaneous mutation rate. In a temperature-sensitive presumed gyrase-deficient strain a small decrease in mutant yield at low doses was observed following incubation at restrictive temperature befor UV. This was ascribed to an enhancement of excision repair.Our failure to find any significant effect of gyrB mutation does not support the hypothesis that hairpin formation (which should be dependent on a high degreee of superhelicity) is involved in determining the ‘broth effect’, ‘mutatiion frequency decline’ or the probability that a mutation will occur spontaneously. Dramatic effects of superhelicity on UV mutagenesis also seem to be unlikely.     

The antimutagenic effect of a truncated ε subunit of DNA polymerase III inEscherichia coli cells irradiated with UV light

It has previously been suggested that inhibition of the proofreading 3′-5′ exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations inEscherichia coli. Our previous work showing that overproduction of ε, the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response ofE. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of ε we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of ε — Exol and ExoII; the third segment, ExoIII, which is essential for 3′–5′ exonuclease activity, is deleted. We show that at 40°C, over-production of the truncated e subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in atrpE65 strain growing at 40°C causes a 10-fold decrease in the frequency of UV-induced Trp⁺ mutations. This antimutagenic effect of the truncated s is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains thednaQ49 allele encoding an ε subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in thetrpE65 strain. We propose that the DNA binding ability of free ε, rather than its 3′–5′ exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD′-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis.     

Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE

Reverse and forward mutation, induced by photoaddition of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CPs) or ultraviolet light (UV), are reduced in three pso mutants of Saccharomyces cerevisiae. The pso1–1 strain exhibits a lower frequency of spontaneous reversion (antimutator) and is almost entirely unaffected by the three agents in both the haploid and diploid states. The pso2–1 strain demonstrates very reduced frequencies of 8-MOP and 3-CPs plus 365 nm radiation-induced mutations in happloid and diploid cells. UV-induced mutations are slightly reduced, whereas survival is almost normal. The pso3–1 strain is mutable by 8-MOP and 3-CPs photoaddition only in the low-dose range. After UV treatment, survival of pso3–1 is nearly normal, whereas the frequencies of induced mutants are diminished as compared to the normal PSO⁺. An analogue of adenine, 6-N-hydroxyaminopurine, is capable of inducing reversions in wild type, as well as in pso and rad6–1 mutant strains, indicating that this drug may act as a direct mutagen in yeast. The comparison of photoaddition of the bifunctional agent (8-MOP) to that of the monofunctional one (3-CPs) confirms that cross-links, as well as monoadditions, are mutagenic in S. cerevisiae. Repair, of the recombinational type, taking place in diploid cells or in haploid cells in G2 phase leads to higher survival, but appears to be error-free.     

Bisulfite (sulfur dioxide) is a comutagen in E. coli and in Chinese hamster cells

The mutagenic and comutagenic effects of bisulfite were investigated. Under moderate exposure conditions (high survival) it was found that bisulfite is not mutagenic to either eukaryotic cells (Chinese hamster V79), or prokaryotic cells (Escherichia coli). However, bisulfite does act as a comutagen with UV irradiation. Bisulfite approximately doubles the mutation frequency in UV-irradiated Chinese hamster V79 cells, and it causes a greater than 8-fold increase in Trp⁺ revertants in UV-irradiated E. coli. The comutagenic effect occurs whether cells are exposed to bisulfite during or immediately after UV irradiation. Kinetic studies of the comutagenic effect in E. coli shows that it decays in a biphasic manner, with an apparent half-life of 15 min and a persistence of the comutagenic effect for up to 120 min after UV irradiation.Experiments with several strains of E. coli of varying DNA-repair capacities indicate that excision repair is necessary for a comutagenic effect by bisulfite. It is thought that bisulfite acts to inhibit excision repair, perhaps by effects on DNA polymerase I, or DNA ligase.     

Photoreactivation, Excision, and Strand-Rejoining Repair in R Factor-Containing Minicells of Escherchia coli K-12

Chromosomeless “minicells” are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K-12 mutant strain. Resistance (R)-factor deoxyribonucleic acid (DNA) can be introduced into minicells by segregation from an R⁺ (R64-11) derivative of the original mutant. We have assessed the ability of R⁺ minicells to correct defects produced in their plasmid DNA by ultraviolet (UV) and gamma radiations. Minicells harboring plasmid DNA, in comparison with their repair-proficient minicell-producing parents, possess (i) an equal competence to rejoin single-strand breaks induced in DNA by gamma rays, (ii) a reduced capacity for the photoenzymatic repair of UV-induced pyrimidine dimers, and (iii) a total inability to excise dimers, apparently owing to a deficiency in UV-specific endonuclease activity responsible for mediating the initial incision step in excision repair. Assuming that the DNA repair properties of R⁺ minicells reflect the concentration of repair enzymes located in the plasmid-containing polar caps of entire cells, these findings suggest that: (i) the enzymes responsible for rejoining single-strand breaks are distributed throughout the cell; (ii) photoreactivating enzyme molecules tend to be concentrated near bacterial DNA and to a lesser extent near plasmid DNA; and (iii) UV-specific endonuclease molecules are primarily confined to the central region of the E. coli cell and, thus, seldom segregate with R-factor DNA into minicells.