Mutagenic DNA repair in Escherichia coli. VI. Gamma radiation mutagenesis in a tif-1 strain.

Summary In the non-filamenting tif-1 strain WP44_s NF trp a dramatic enhancement of both UV and gamma ray mutability to Trp⁺ was observed when irradiated bacteria were incubated on plates at 43°. This enhanced mutability was progressively suppressed when the initial plating density exceeded 10⁸ bacteria per plate and was not demonstrable in liquid media. Under optimal conditions more mutants were induced by gamma radiation than could reasonably be accounted for by the initial number of radiation-induced lesions in the DNA, implying the existence of some mechanism for amplifying the radiation effect. Moreover, the tif-enhanced mutation frequency could be obtained if incubation at restrictive temperature was delayed for up to 60 min in nutrient broth after irradiation, at a time when all known reparable DNA damage had been repaired and the number of viable bacteria had more than doubled. On plates the effect of high temperature was still fully demonstrable 120 min after irradiation. The results are hard to reconcile with the hypothesis that incubation of tif-1 bacteria at restrictive temperature causes the induction of a repair system acting on DNA damaged by gamma radiation. A more compatible interpretation would be that radiation causes a persisting physiological disturbance in the cell and that this enhances the spontaneous mutator effect occurring in tif-1 bacteria subjected to subsequent thermal shock.     

Ultraviolet Mutagenesis in Strains of <Emphasis Type="Italic">E. coli</Emphasis> deficient in DNA Polymerase

CERTAIN mutations in Escherichia coli which cause increased sensitivity to ultraviolet light (UV) drastically change the UV mutability of the sensitive strain. Strains lacking the ability to excise pyrimidine dimers, for example, exhibit greatly increased UV mutability, producing induced mutations at doses of UV far smaller than those required to induce mutations in wild type strains^{1, 2}. Mutants owing their UV sensitivity to reduced ability to perform genetic recombination, on the other hand, show reduced mutability in response to UV compared with the wild type and some (recA or exrA strains, for example) are stable to UV, producing no detectable induced mutations at any dose^3–5. Analysis of UV mutagenesis in such strains has led to the hypothesis that most UV-induced mutations in E. coli are errors in the recombinational repair of gaps in the daughter-strand which are located opposite unexcised pyrimidine dimers^{6, 7}.     

The dependence of postreplication repair on uvrB in a recF mutant of Escherichia coli K-12.

Summary Mutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated vir and wild type rates of excision gap closure in repairing UV damage to their own DNA. The same mutants showed reduced rates of postreplication repair strand joining. When uvrA ^- recF^- or uvrB ^- recF^- strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m². We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF may be similar. An intermediate in postreplication repair may accumulate in the uvr ^- recF^- strain.     

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.     

Influence of the yellow Locus on Sensitivity of Drosophila Germ Cells to Chemical Mutagens

The effect of theyellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the sex-linked recessive lethals (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin K; y; y ct v; and y mei-9 ^a) were treated by mutagenic agents and then crossed to Bascfemales. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that theyellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Bascmales were crossed to females of various genotypes including y and y mei-9 ^a ones. The crosses involving y females yielded F₁ progeny with high spontaneous lethality, whereas in F₂, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny F₁. The results obtained using the above two approaches suggest that theyellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.     

Studeis on the biochemical basis of mutation. IV. Effect of amino acid substitution on the enzymatic and biological properties of bacteriophage T4 DNA polymerase

The effects of substituting specific amino acids at specified loci in the bacterio-phage T4 DNA polymerase molecule have been studied. Gene 43 (DNA polymerase) amber mutants grown on suppressor strains which substitute serine, glutamine, or tyrosine at specific sites in the polymerase molecule, produce enzymes with substantially different physical, enzymatic and biological properties when compared to wild type. When amB22, a gene 43 mutant which makes a DNA polymerase fragment with only 3′-exonuclease activity, was grown in Escherichia coli B40(sup⁺1), -(sup⁺ 2) or -(sup⁺3), enzymes with different temperature sensitivities and nuclease to polymerase ratios were produced. Measurements of spontaneous mutation rates in these suppressed strains indicated that the two with higher than normal exonuclease activity were antimutators, and the one with a slightly lower exonuclease activity was a mutator. The substituted amino acids at the amB22 site perturbed the 3′-exonuclease activity creating either antimutator or mutator phenotypes. Thus, the B22 enzymes provide additional biochemical evidence to support the hypothesis that the exonuclease to polymerase ratio may influence the spontaneous mutation rate in phage T4.     

Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

The effect of bacteriophage T4 gene 59 mutations (DNA-arrested synthesis) on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. When Escherichia coli B was infected by a T4 gene 59 mutant, DNA synthesis proceeded to increase linearly after initiation, but started to decrease at 8 min and was completely arrested at 12 min at 37°C. At various incubation temperatures (20 to 42°C), the initial rates and times of arrest of DNA synthesis were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA molecules to other replicative intermediates (39). The abnormality in protein synthesis caused by gene 59 mutation is manifested by (i) a delayed shutoff in the expression of early proteins (gene 43, 46, 39, 52, 63, 42-45, and some unidentified proteins), (ii) a reduced rate of late gene expression (gene 34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence of cleavage of certain late proteins (23, 24, IPIII and 22 to 23^*, 24^*, IPIII^*, and small fragments). It appears that there was no effect on the expression of gene 33, 55, and 32 by a mutation in gene 59. Results obtained from an addition of rifampin at the prereplicative cycle after infection indicated that mRNA from genes 43, rIIA, 46, 39, 52, and 63 are more stable in T4amC5 (gene 59) than in wild-type-infected cells. mRNA remained functional longer in mutant-infected cells, and this may explain the prolonged synthesis of certain early proteins. The gene expression of other DNA arrested mutants—those in genes 46 and 47—showed a pattern of abnormal protein synthesis similar to that found in gene 59 mutant-infected cells, except more late proteins are synthesized. The gene expression in terms of phage DNA structure is discussed.     

Bacteriophage T4 DNA polymerase determines the amount and specificity of ultraviolet mutagenesis

Summary Ultraviolet mutagenesis in bacteriophage T4 proceeds via error-prone repair (EPR) and requires the functional integrity of the uvsWXY system which mediates genetic recombination, recombinational repair, and mutability by diverse DNA damaging agents. Current opinion holds that mutagens acting through EPR generate DNA damage which blocks the progress of the replication complex and that EPR consists of the facilitated bypass of such inaccurate, damaged templates. This notion predicts that the T4 DNA polymerase (encoded by gene 43) mediates EPR in UV irradiated phage T4. This prediction is verified by the discovery that gene 43 mutations often enhance or reduce UV mutagenesis (which is scored by the induction of r mutants) and sometimes change its specificity.     

The mutagenic effect of ultraviolet radiation and nitrous acid onMycobacterium phlei

Lethal and mutagenic effects of nitrous acid and UV radiation onMycobacterium phlei were studied Three auxotrophic strains of the PA strain ofMycobacterium phlei were obtained: ala^-, his^-, and gly^- (ser^-) INH^r Bods of the his^- strain grown in liquid media are longer to filamentous as compared with cells of the prototrophic PA strain grown in the same media, whereas cells of the gly^- (ser^-) INH^r mutant are shorter to coccobacillary. Cells of the ala^- strain are characterized by their various length from normal to coccobacillary. The auxotrophic strains obtained differ from each other by a frequency of spontaneous reversions to prototrophy. The his^- strain is the most stable, a frequency of spontaneous reversions to prototrophy being 10^-9. The gly^- (ser^-) INH^r strain reverts spontaneously to prototrophy with a frequency of 10^-8 to 10^-7. The ala^- strain spontaneously reverting with a frequency of 10^-5 is the most labile. The auxotrophic mutants obtained do not differ from the original prototrophic strain in the other properties studied. A change in a frequency of INH and STM-resistant mutants was also studied. It was found that under the influence of UV radiation a frequency of INH-resistant mutants increases 43 to 80 fold as compared with a frequency of spontaneous mutations, this latter being about 2.6 × 10^-6. No substantial increase in a frequency of STM-resistant mutants was found using UV irradiation under the given experimental conditions; their spontaneous frequency equals to 9.0 × 10^-9 to 2.0 X 10^-8.     

Influencing the spontaneous and chemically induced mutability of E. coli through changing the genetic background

Summary The influence of a second auxotrophic marker to the spontaneous and chemical-induced mutability to prototrophy of a first auxotrophic marker in 7 monoauxotrophs and 31 biauxotrophs of E. coli K 12 was studied by growth layer technics. No case of influence of a second auxotrophy to the mutagen-induced mutability (7 mutagens tested) of the first auxotrophy among 172 possibilities was found. An influence to the spontaneous mutability seemed to be present in 4 cases out of 31. But 3 of them were shown to be imitated by influences of components of the medium to the growth of mutants or parent type. In one case a mutator mutation is responsible for the about 8 times higher rate of the mutation met 1+ in strain met 1/his 7 than in strain met 1. But backmutation and crossing experiments showed that the mutator (mum ⁺) was separate from the auxotrophic marker his 7 (recombination frequency 5/16). This mutator did not increase remarkably the spontaneous mutability of other markers tested (resistence to phages T1 or T4 or to Streptomycin 3, 5, 10, or 100 /ml, or to Chloramphenicol 2 /ml). It is assumed that the spontaneous mutations in the wild-type are at least partially different in their nature from the mutator promoted ones.     

Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Li²⁺, NaN₃ and isopropanol was observed. The K_m and V_max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.Communicated by W.D. Grant     

On the role of the recipient cell during conjugation in Escherichia coli

To study the role of the E. coli recipient cell in conjugation recipient cell mutants deficient in conjugation (Con^-) were isolated. Mutants specific for F-type E. coli donor cells (ConF^-) and mutants specific deficient in conjugation with I-type donor cells (ConI^-) were isolated.Both ConF^- and ConI^- mutants were blocked in stable mating pair formation. Biochemical analysis of the mutants suggests that the outer membrane protein coded by the ompA gene and LPS are important for recipient activity in F-type conjugation while LPS is important for recipient activity in I-type conjugation.     

Detection of <Emphasis Type="Italic">Candida albicans</Emphasis> by Mass Spectrometric Fingerprinting

Candida albicans is one of the most frequent causes of fungal infections in humans. Significant correlation between candiduria and invasive candidiasis has previously been described. The existing diagnostic methods are often time-consuming, cost-intensive and lack in sensitivity and specificity. In this study, the profile of low-molecular weight volatile compounds in the headspace of C. albicans-urine suspensions of four different fungal cell concentrations compared to nutrient media and urine without C. albicans was determined using proton-transfer reaction mass spectrometry (PTR-MS). At fungal counts of ≥1.5 × 10⁵ colony forming units (CFU)/ml signals at 45, 47 and 73 atomic mass units (amu) highly significantly increased. At fungal counts of <1.5 × 10⁵ CFU/ml signals at 47 and 73 amu also increased, but only at 45 amu a statistically significant increase was seen. Time course alterations of signal intensities dependent on different cell concentrations and after addition of Sabouraud nutrient solution were analysed. Recommendations for measurement conditions are given. Our study is the first to describe headspace profiling of C. albicans-urine suspensions of different fungal cell concentrations. PTR-MS represents a promising approach to rapid, highly sensitive and non-invasive clinical diagnostics allowing qualitative and quantitative analysis.     

The Origin of Spontaneous Mutation in SACCHAROMYCES CEREVISIAE

Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.     

The recombinational analysis of aberrations and the position of the notch locus on the polytene chromosome of Drosophila

Summary The recombinational analysis of heterozygotes for a point-mutant N and a deficiency N suggests that the map region approximated by the interval fa to nd ² is at the right edge of salivary band 3C7 or in the interband to the right. The map region N ^55ell to fa can be anywhere between the left interband and the right edge of 3C7. We discovered that small inversions also can be used in the recombinational analysis, and the inversion data support the conclusions already described.The reactivation of latent mutability in a Notch inversion resulted in reinversion of the original aberration, followed by reversion of N to N ⁺. From the same Notch inversion, we isolated a spontaneous deficiency superimposed upon the original aberration, which supported our hypothesis that two of our w to N deficiencies probably originated as deficiencies superimposed upon inversions.     

Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

Summary We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42° C (relative to 30° C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30° C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42° C; and 4) that defects in excision repair or in other Uvr⁺-dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions.     

Syntheses of 14C-Labeled Pyrethrin I,Allethrin, Phthalthrin,and Dimethrin on a Submillimole Scale

Studies on the metabolic fate and degradation chemistry of pyrethroid insecticide chemicals are greatly facilitated by the use of compounds radiolabeled, in separate preparations, in the acid and alcohol moieties. Acid-labeled preparations were made by converting d-trans-chrysanthemic acid-1-¹⁴C (88 mg, 1.3 mCi/mm) into d-trans-d-pyrethrin-1-¹⁴C (68 mg, 1.3 mCi/mm), d-trans-d-allethrin-¹⁴C (43 mg, 1.3 mCi/mm), d-trans-dimethrin-¹⁴C (54 mg, 0.294 mCi/mm), and d-trans-phthalthrin-¹⁴C (47 mg, 0.294 mCi/mm), incorporating approximately 81% of the starting radiocarbon into the four pyrethroid preparations. Alcohol-labeled preparations were made by converting acetone-1,3-¹⁴C into d-trans-dl-ailethrin-¹⁴C (146 mg, 0.162 mCi/mm) and formaldehyde-¹⁴C into d-trans-phthalthrin-¹⁴C (299 mg, 0.276 mCi/mm). Each labeled compound had a high stereochemical purity and a radiochemical purity of greater than 99%. Detailed procedures were worked out for all conversions which took place in high yields except in one case: the synthesis of allethrin labeled in the alcohol moiety.     

Genetic instability at the opaque-2 locus of maize

Summary A case of genetic variegation discovered at the opaque-2 locus of maize that includes a two-element system with a receptor and regulatory element is described. The somatic mutability depends on the existence of two genetic factors: a responsive allele (with receptor element), o2m(r), and a regulatory element, Bg, that induces mutability of o2m(r). In the absence of Bg, o2m(r) is indistiguishable from the recessive alleles of the O2 locus; in the presence of the regulatory element, o2m(r) mutates giving rise to sectors of flint-like endosperm in an opaque back-ground. The regulatory element Bg may be located independently or at the controlled locus. The genetic properties of the new system, somatic mutability, transposition, existence of different patterns of mutability, are apparently similar to those previously described in maize for the classical systems of controlling elements. In addition, the recovery of the o2 mutability from crosses between spontaneous o2 alleles suggests that transposable genetic elements may be involved in the origin of natural mutability.     

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Modulation of allele leakiness and adaptive mutability inEscherichia coli

It is shown that partial phenotypic suppression of two ochre mutations (argE3 andlacZU118) and an amber mutation (inargE) by sublethal concentrations of streptomycin in anrpsL ⁺ (streptomycin-sensitive) derivative of theEscherichia coli strain AB1157 greatly enhances their adaptive mutability under selection. Streptomycin also increases adaptive mutability brought about by theppm mutation described earlier. Inactivation ofrecA affects neither phenotypic suppression by streptomycin nor replication-associated mutagenesis but abolishes adaptive mutagenesis. These results indicate a causal relationship between allele leakiness and adaptive mutability.