A comparative study of the effects of UV irradiation upon diploid cultures of yeast defective at the rad 3 locus

Summary Therad 3 gene ofSaccharomyces cerevisiae appears to code for one of the enzymes involved in the repair of UV induced pyrimidine dimers. Haploid and diploid yeast cultures carrying different mutant alleles of therad 3 gene show considerable variation in their responses to both UV inactivation and post UV modifying treatments such as liquid holding in basal medium and photoreactivation. Positive liquid holding recovery was shown only by those diploid cultures containing alleles which conferred the highest levels of UV resistance. The results indicate that liquid holding recovery in yeast requires the activity of the excision-repair pathway for expression.     

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Summary Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (radl-1, 2, 3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11 and 22 show marked LHR after UV but no increase of survival after DEB treatment.     

Confluent holding leads to a transient enhancement in mutagenesis in UV-light-irradiated xeroderma pigmentosum, Gardner's syndrome and normal human diploid fibroblasts

The influence of confluent holding periods of 0-24 h of UV-light-induced mutagenesis has been investigated in several human cell strains including xeroderma pigmentosum complementation group A (XPA), Gardner's syndrome (GS) and normal human diploid fibroblasts (NHDF). These cells strains were chosen for the variety of their responses in cytotoxicity experiments. Confluent cultures of NHDF exposed to UV light exhibited a time-dependent increase in survival when subculture was delayed up to 24 h after irradiation. GS and XPA fibroblasts showed no such increase. In dose-response experiments, GS cells from 3 kindreds were moderately hypersensitive to cell killing by UV light whereas XPA cells were strongly hypersensitive. Confluent cultures of GS cells were slightly and XPA markedly hypermutable to 6-thioguanine resistance (6-TGR) when the cells were subcultured immediately after UV exposure. When allowed confluent holding periods of 1.5-24 h, GS, XPA and NHDF all exhibited a transient enhancement of mutagenesis such that a 5-10-fold increase in mutation frequency was observed in cells subcultured at 6-9 h after irradiation as compared to cells subcultured at 3-6 h. A decline in mutation frequency prior to the mutagenesis peak was observed in GS and normal cells but not in XPA. After 24 h of confluent holding, the mutation frequency in irradiated GS and NHDF had returned to near background levels although XPA mutation frequencies remain similar to those observed in immediately subcultured cells. A model to explain these overall results is discussed.     

The response to chemical mutagens of the individual haploid and homoallelic diploid UV-sensitive mutants of the rad 3 locus of Saccharomyces cerevisiae

Summary Mutant cultures of yeast defective at the generad 3 show increased sensitivity to the lethal effects of UV light. The order of UV sensitivity shown by haploid and homoallelic diploid cultures carrying the variousrad 3 alleles was duplicated by their sensitivity to the action of nitrous acid. In contrast, after treatment with the alkylating agents ethyl-methane sulphonate and methylmethane sulphonate therad 3 cultures showed only small differences in sensitivity compared with the wild-typeRAD culture. These small differences in sensitivity appear to result from variation in the metabolic condition of the cultures when treated with alkylating agents.The results indicate that the product of therad 3 gene in yeast is involved in the repair of UV induced pyrimidine dimers and deaminated bases produced by nitrous acid but does not participate in the repair of single strand DNA breaks produced by alkylating agents.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

Problems in the estimation of mutation rates and in the detection of induced mutations in man

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stiationary phase haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the “petite” recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of “petites” in stationary phase cells (increase of the frequency of “petites” during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.     

UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.     

Formation of the heterozygous tandem duplication in the process of conjugation recombination in Escherichia coli: study of the effect of mutations for the recQ, uvrD, and recJ genes]

Stable tandem duplications were shown to originate from conjugational recombination between Escherichia coli HfrH strains carrying mutations for the deo operon. The duplications deoC deoD/deoA deoB::Tn5 usually constitute approximately 5% of the Deo+ offspring. The effect of mutations for the recQ, uvrD, and recJ genes on the frequency of duplications was studied. The CM1563 strain carrying the recQ mutation was shown to give, as a recipient, 20% of duplications in the Deo+ offspring. However, this property of CM1563 seems to depend on the presence of a spontaneous mutation of unknown nature, which also increased UV sensitivity of bacteria. The recQ mutation itself increased the frequency of duplications by less than 50%. The recJ mutation did not virtually affect the frequency of duplications. The uvrD mutation possessing the recombinogenic effect was shown to increase the frequency of deo+ recombinants and simultaneously decrease the frequency of duplications. Tandem duplications are assumed to be normal intermediates of multi-stage conjugational recombination initiated by the integration of the proximal region of the Hfr chromosome into different nonhomologous regions of the recipient chromosome.     

Dimeric circular duplex DNA of bacteriophage phiX174 and recombination

Summary Bacteriophage X174 replicative from DNA (RF DNA) was formed in the presence of chloramphenicol at a concentration of 40 g per ml and isolated at 12 and at 55 min. after infection. The component I RF DNA (double stranded covalently closed and twisted form) was separated and divided into a monomer and multimer (dimer) fraction.The frequency of recombinants found after phage formation in the chloramphenicol treated cells and that found after spheroplast infection with the monomer molecules both increase with the time of RF formation. However, the frequency of recombinant molecules among the dimers remained constant. This finding is explained by the hypothesis that two separate mechanisms act in X174 recombination, one of which is restricted to the formation of dimers.Irradiation with UV of phage prior to infection showed that the frequency of recombinants in monomers increased, as the recombination frequency of phage after (a single) growth (step) did, but that neither the frequency of recombinant molecules in dimers is raised, nor the frequency of dimers. Using a recombination negative host the frequency of recombinant dimer molecules was three to fourfold decreased, whereas the frequency of dimers was only slightly lower (relative to the normal host). These results support the hypothesis mentioned above and moreover lend support to the view that the greater part of the dimers is not formed by recombination events.     

Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.

The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair. Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration. Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light. The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC. The products of these genes have been linked with the initiation of recombination. We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme. The strains constructed were also tested for sensitivity to UV light. Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains. The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.     

Genetic Control of Resistance to the Piperidine Fungicide Fenpropidin in Ustilago maydis

Mutants of Ustilago maydis (DC.) Corda, resistant to the piperidine fungicide fenpropidin, were isolated in a mutation frequency of 3.2 × 10^–5, after UV‐irradiation and selection on media containing 75 μg/ml fenpropidin. Genetic analysis with 15 such mutant isolates resulted in the identification of two unlinked chromosomal loci, U/fpd‐1 and U/fpd‐2. The U/fpd mutations are responsible for moderate resistance levels to fenpropidin (Rf: 42–56 or 15 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively). Haploid strains carrying both U/fpd mutations do not exhibit higher levels of resistance to fenpropidin, indicating no additivity of gene effect between non‐allelic genes. Cross‐resistance studies with other Sterol Biosynthesis inhibitors (SBIs) showed that the U/fpd‐mutant isolates exhibited a positive cross‐resistance to the piperidine piperalin and to the related morpholine fungicides fenpropimorph and tridemorph, but not to the inhibitors of C‐14 demethylase and squalene epoxidase. Crosses between mutants carrying the U/fpd‐genes with compatible isolates carrying the U/fpm or U/tdm mutations, which have been identified in previous genetic studies for resistance to morpholine fungicides fenpropimorph and tridemorph, yielded, with the exception of U/fpd‐2 × U/fpm‐2 crosses, a large number of recombinants with wild‐type sensitivity, indicating that the mutant genes involved were not allelic. Analysis of progeny from crosses between U/fpd‐2 and U/fpm‐2 mutants yielded no recombinants with wild‐type sensitivity, but a 1 : 1 progeny segregation was observed at the MIC for the U/fpd‐2 isolates, indicating that these genes are alleles of the same locus. A study of the fitness of fenpropidin‐resistant isolates showed that the U/fpd mutations do not affect the phytopathogenic fitness‐determining characteristics such as growth in liquid culture and pathogenicity on young corn plants.     

Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG.

ruvC mutants of Escherichia coli appear to lack an activity that resolves Holliday intermediates into recombinant products. Yet, these strains produce close to normal numbers of recombinants in genetic crosses. This recombination proficiency was found to be a function of recG. A "mini-kan" insertion in recG was introduced into ruvA, ruvB, and ruvC strains. Conjugational recombination was reduced by more than 100-fold in recG ruvA::Tn10, recG ruvB, and recG ruvC strains and by about 30-fold in a recG ruvA strain carrying a ruvA mutation that is not polar on ruvB. The double mutants also proved very deficient in P1 transduction and are much more sensitive to UV light than ruv single mutants. Since mutation of recG alone has very modest effects on recombination and sensitivity to UV, it is concluded that there is a functional overlap between the RecG and Ruv proteins. However, this overlap does not extend to circular plasmid recombination. The possibility that RecG provides a second resolvase that can substitute for Ruv is discussed in light of these findings.     

pH induced damage and repair in E. coli

Escherichia coli lost its colony-forming ability when suspended in Tris/NaOH or Tris/Mg2+ buffers of pH 10.0 and 4.0, respectively. A significant decrease in the survival of radiation-sensitive mutants recA, polA, res, rer and lexA was observed as compared to their wild-type counterpart under these conditions. The alkali-injured cells were found to recover when incubated at 37 degrees C for 2 h in 0.05 M phosphate buffer of pH 8.0, whereas no such liquid holding recovery was observed in recA and lexA mutants. Recovery in phosphate buffer was not affected by metabolic inhibitors. As a result of alkali treatment, the sensitivity of bacteria to ultraviolet light (UV) was enhanced. However, on incubation for 2 h in recovery buffer at 37 degrees C, the bacteria regained partial UV resistance. Bacteria exposed to alkaline environment exhibited an enhanced level of mutagenesis. Contrary to the treated wild-type, the mutants recA and lexA did not exhibit any increase in the mutation frequency. Alkali treatment to GC----AT transition mutants of Salmonella typhimurium, TA102 and TA104 resulted in the highest number of revertants per plate.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.     

Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair

The recD, recJ and recN genes of Escherichia coli K-12 have been shown to be involved in genetic recombination and DNA repair in this organism. Yet, mutation of any one of these genes does not seem to interfere much with the recovery of recombinants from conjugational crosses. Strains carrying all possible combinations of mutations inactivating these genes were constructed and examined for their recombination proficiency and sensitivity to UV light. The recD recJ and recJ recN double mutants are moderately sensitive to UV light and slightly deficient in recombination. A combination of mutations in all 3 genes produced strains that are very deficient in recombination (50- to 100-fold reduction) and strikingly sensitive to UV light. We conclude that these genes provide overlapping activities that compensate for one another in the single mutants. On the basis of these and other data, recombination genes are classified into 3 epistatic groups that define activities which function pre-synaptically or post-synaptically to promote genetic exchanges catalysed by RecA.     

Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.     

Recombination-induced suppression of cell division following P1-mediated generalized transduction in Klebsiella aerogenes

Klebsiella aerogenes recombinants resulting from bacteriophage P1-mediated generalized transduction failed to increase in number for approximately six generations after transduction. Nevertheless these recombinants continued to grow and became sensitive to penicillin after a transient resistance, suggesting that the cells were growing as long, non-dividing filaments. When filamentous cells were isolated from transduced cultures by gradient centrifugation, recombinants were 1000-fold more frequent among the filaments than among the normal-sized cells. The suppression of cell-division lasted for six generations whether markers near the origin (gln, ilv) or terminus (his, trp) of chromosome replication were used, despite a 50-fold difference in transduction frequencies for these markers. The suppression of cell division was a host response to recombination rather than to P1 invasion since cells lysogenized by P1 in these same experiments showed only a short (two generation) suppression of cell division. We speculate that the suppression of cell-division is an SOS response triggered by the degraded DNA not incorporated in the final recombinant. We demonstrate that both the filamentation and the transient penicillin resistance of recombinant cells can be exploited to enrich greatly for recombinants, raising transduction frequencies to as high as 10(-3).     

Screening of multi-copy mannanase recombinants of Pichia pastoris based on colony size

Pichia pastoris pGAP (glyceraldehyde dehydrogenase promoter) expression system was widely used for the expression and production of heterologous proteins. Screening multi-copy recombinants was an effective strategy to improve the heterologous protein production in P. pastoris. Because multiple gene insertion events occurred with a low frequency, hundreds to thousands of antibiotic-resistance recombinants need to be screened. The common way of improving screening efficiency was to increase antibiotic concentration in screening plates. Here we developed a screening method by selecting small colonies from low-concentration antibiotic screening plates. This strategy greatly improved the probability of obtaining multi-copy mannanase gene (man) recombinants and it could replace the common strategy by increasing antibiotic concentration in screening plates. The further study in liquid shake flask cultures revealed that cell concentrations, growth rates and substrate consumption rates of recombinants gradually decreased with the increase in man copy number. This indicated that such a screening strategy was effective to screen multi-copy recombinants based on colony size.