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.     

Accumulation of Ade+ reversions in isoauxotrophic strains ofSaccharomyces cerevisiœ allelic inRAD6 during adenine starvation

A comparative method based on an analysis of accumulation of starvation-induced Ade⁺ reversions and cell death during adenine starvation was developed and exploited for estimating the role ofRAD6 in the starvation-induced reversions. It was shown that inactivation ofRAD6 function inSaccharomyces cerevisiœ markedly enhances the accumulation of Ade⁺ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells. This work was supported by a grant 204-1080-1993 from GACR to the last author andCharles University grant 274/1996 to the first author.     

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.     

Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae

Adaptive or selection-induced mutations are defined as mutations that occur in non-dividing cells as a response to prolonged non-lethal selective pressure such as starvation for an essential amino acid. In the absence of DNA replication, the processing of endogenous DNA lesions by repair enzymes probably acts as a source of mutations. We are studying selection-induced reversions of frameshift alleles in the eukaryote Saccharomyces cerevisiae. Here we show that respiration-deficient strains, totally devoid of mitochondrial DNA, yield selection-induced mutants at slightly elevated frequencies compared to isonucleic respiration-competent strains. Therefore factors of mitochondrial origin such as reactive oxygen species or hypothetical recombinogenic DNA fragments are unlikely to be mediators of selection-induced nuclear frameshift mutation in yeast. Furthermore we compared sequence spectra of reversions of the +1 hom3-10 frameshift allele and found a strong preference for −1 deletions in mononucleotide repeats in selection-induced and replication-dependent revertants, indicating slippage errors during DNA repair synthesis as well as during DNA replication. Remarkably, a higher degree of variation in the site of the reverting frameshift and accompanying base substitutions was found among selection-induced revertants. Received: 25 May 1998 / Accepted: 20 August 1998     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997     

Mutation induction by thymine deprivation in Escherichia coli B/R : II. Mutation in the repressed and derpressed tryptophan operon

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Simultaneous transposition of different mobile elements: Relation to multiple mutagenesis in Drosophila melanogaster

Summary Several different transposition events occur simultaneously in one and the same germ cell, as we have found by analyzing different genetic systems in Drosophila melanogaster. (i) In unstable ct ^MR2 strains, stable reversions to ct ⁺ and changes in the type of ct mutation, which depend on an excision or transposition of the mobile element mdg4 (Gerasimova 1981; Gerasimova et al. 1984), are frequently accompanied by the appearance of novel mutations in different loci of the X chromosome. Some of these (sn, w, g) seem to be induced by the P-element and copia. (ii) A stable ct ^MR2 reversion to the wild type frequently coexists with an insertion of one to five copies of the P-element in the X-chromosome. Thus, the number of independent transposition events registered by genetic analysis and in situ hybridization may be as great as six. (iii) In two strains with double unstable mutations (cm, ct, and ct, r), double reversions to the wild type occurred at a high rate (80%–97% of total revertants). They frequently coexisted with novel strain-specific mutations. (iv) The stable strain ct ⁶ g²is destabilized by crossing with the MRh12/Cy strain (which contains a number of P-element copies). Both mutations begin to revert to the wild type. Of the revertants 50% have double reversions. Our experiments revealed a high specificity of insertion sites depending on the nature of transposon and the strain genotype. A possible role played by the burst of transposition in the evolution and possible mechanisms of transposition specificity are discussed.     

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.     

The influence of temperature on the ultraviolet induced revertant frequencies of two auxotrophs ofNeurospora crassa

Summary The frequency of ultraviolet-induced adenine reversions relative to inositol reversions in the strainK3/17 ad-3A 38701: inos 37401 is influenced by temperature. Fewer adenine revertants are obtained from a given ultraviolet dose adminstered at 30°C than from the same dose administered at 2°C. The inositol reversion frequency and the percentage survival are not greatly affected by these differences in temperature. Temperature differences are only effective if a) applied during irradiation b) higher temperatures above 15°C are used. Possible explanations for these results are considered.With 2 Figures in the Text     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3() strain. Although the constitutive reversions could be transduced by , the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by .In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3() strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3() strain, and not in gal ⁺, gal ⁺(), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.A gal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (31). Electron micrographs of gal ⁺ and gal ^c 200 31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage ; and (iii) the gal3 insertion appears to inhibit the production of gal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.     

Induced mutagenesis in Ustilago maydis

Summary A UV-revertible mutant of the nar1 structural gene for nitrate reductase was isolated in wildtype (nar ⁺ nir ⁺) Ustilago maydis. It proved to be vigorously revertible by gamma rays as well. Genetic analysis revealed that the strain carried a single, nonleaky, recessive allele (nar1-m) with an unusually high spontaneous reversion rate (3×10^-5/div.). Reliable reversion frequencies were determined with a special agar medium that reduced the normally high level of residual growth observed on nitrate minimal agar. Radiation-induced reversion frequencies in the homozygous diploid were approximately twice those in the hapliod. Following crosses to wild type, two revertants (one spontaneous and one UV-induced) were found to map at nar1. Although the molecular basis of nar1-m reversion is not known, available data suggest that some form of point mutation is involved.     

The effect of a histidine operator-constitutive mutation on UV-induced mutability within the histidine operon ofSalmonella typhimurium

Summary The presence of the histidine operator-constitutive mutationhis01242 increases UV-induced mutability within the histidine operon ofSalmonella typhimurium. The rate of reversion ofhisC andhisF ochre and frameshift mutants is increased 5- to 8-fold when these mutations are coupled withhis01242 which causes 15-fold derepression of the operon. The effect does not extend to the whole chromosome since the rate of UV-induced mutability at the unlinked streptomycin locus is the same in the strains carryinghis0 ⁺ orhis01242 alleles. The same phenomenon was observed in Hcr⁻ strains.     

Cell volume change of Escherichia coli under stringent control apparent increase of K in rel cells

With several pairs of rel⁺ and rel⁻ strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K⁺ and the rate of uptake of ⁴²K⁺ were investigated. In the early phase of the experiments, the intracellular concentration of K⁺ was estimated by the conventional method in which the cell volume per A₆₆₀ value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K⁺ concentration of rel⁺ cells was kept almost constant, while that of rel⁻ cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A₆₆₀ changed only slightly in CP78 (rel⁺) cells, while it increased markedly in CP79 (rel⁻) cells after the exposure to amino acid starvation. Reestimation of the K⁺ concentrations based on the estimated respective values of cell volumes per A₆₆₀ revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel⁺ cells was arrested upon amino acid starvation whereas that in the rel⁻ cells was not. The ⁴²K⁺ uptake by the rel⁺ cells was depressed upon amino acid starvation, whereas that by the rel⁻ cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K⁺ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A₆₆₀ of the culture, was pointed out.     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin, hemolysin, and extracellular serine protease (i.e., aerA, hlyA, and ahpA, respectively). These genes were investigated using polymerase chain reaction (PCR) with specific primers for each gene. And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed. PCR results demonstrated that the distribution patterns of aerA, hlyA, and ahpA were different in these strains. 6/9 of A. hydrophila strains were aerA positive, 8/9 of strains hlyA positive, 7/9 of strains ahpA positive, respectively. However, the assay for pathogenesis showed that two strains (A. hydrophila XS91-4-1 and C2) were strong virulent, two strains (A. hydrophila ST78-3-3 and 58-20-9) avirulent and the rest middle virulent was to the fish. In conclusion, there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio. All strong virulent A. hydrophila strains were aerA ⁺ hlyA ⁺ ahpA ⁺ genotype, and all aerA ⁺ hlyA ⁺ ahpA ⁺ strains were virulent. Strains with the genotype of aerA ⁻ hlyA ⁻ ahpA ⁺ have middle pathogenicity. In the present study, we found for the first time that all A. hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio. Additionally, there was a positive correlation between the virulence of A. hydrophila and the presence of aerA and ahpA. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(1): 82–85 [译自: 中山大学学报 (自然科学版)]     

Base pair substitutions caused by the uvr502 mutation affecting mutation rates and UV-sensitivity of Escherichia coli

Summary The types of base pair substitutions induced by the uvr502 mutator activity were studied using the isogenic uvr ⁺ and uvr502 strains bearing an ochre or missense mutations in the trp operon. It was found that the uvr502 mutation increased the frequency of both structural gene (true) reversions and suppressor mutations in the trp _oc ^– mutant. The trpA58 missense mutation was also reverted by the uvr502 allele and 5-methyl tryptophane resistant as well as 5-methyl tryptophane sensitive Trp⁺ revertants were formed. However the uvr502 mutation was unable to increase significantly the frequency of Trp⁺ revertants in the rpA78 mutant. With the help of key of Yanofsky et al. (1966b) and codon catalogue it could be concluded that the uvr502 mutation induces transitions in both directions but not A:TC:G and probably not G:CT:A transversions. Incubation of the uvr502 mutant with either of four deoxyribonucleosides has no effect on its spontaneous mutability while deoxyguanosine and deoxyadenosine reduce the mutagenic effect of 2-aminopurine in the uvr ⁺ strain, suggesting that the mutator effect of the uvr502 mutation has nothing to do with the formation of mutagenic base analogue or insufficient synthesis of bases.