Mutagenesis of Saccharomyces cerevisiae by sodium azide activated in barley.

Concentrated dialysate of the extract prepared from barley seeds treated with sodium azide increased up to 100--200 times the frequency of forward mutations to cycloheximide resistance in the excision-deficient UV-sensitive heploid strain rad2-5 of Saccharomyces cerevisiae, when applied to growing cells in complete medium at pH 4.2. Only a slight increase of mutation frequency (less than 4 times) was found in the haploid RAD+ strain treated in the same way as well as in haploid RAD+ and rad2-5 strains treated directly by sodium azide. In contrast with the barley-activated sodium azide, UV irradiation was more effective in the induction of cycloheximide resistance in the RAD+ strain than in the RAD2-5 mutant. The dialysate from azide-treated barley seeds, applied at both pH 4.2 and pH 9, also significantly increased the frequency of locus-specific suppressor mutations to isoleucine independence and -- to a lesser extent -- reversions and/or gene conversions in the trp5 locus in growing cells of the diploid strain D7. The dialysate was also mutagenic in resting cells of strains D7 and rad2-5 but with lower effectiveness.     

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH

The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S. cerevisiae. In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.     

Suppression of Two Missense Alleles of the TRP5 Locus of SACCHAROMYCES CEREVISIAE

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cerevisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUP101. SUP101 does not suppress ochre alleles ade2-1, his5-2, arg4-17, lys1-1, amber alleles trp1-1, tyr7-1, or unclassified alleles at a number of other loci. These results indicate SUP101 is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozygous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the complete medium always segregated with the suppressor.     

Spontaneous Point Mutations That Occur More Often When Advantageous than When Neutral

Recent reports have called into question the widespread belief "that mutations arise continuously and without any consideration for their utility" (in the words of J. Cairns) and have suggested that some mutations (which Cairns called "directed" mutations) may occur as specific responses to environmental challenges, i.e., they may occur more often when advantageous than when neutral. In this paper it is shown that point mutations in the trp operon reverted to trp+ more frequently under conditions of prolonged tryptophan deprivation when the reversions were advantageous, than in the presence of tryptophan when the reversions were neutral. The overall mutation rate, as determined from the rates of mutation to valine resistance and to constitutive expression of the lac operon, did not increase during tryptophan starvation. The trp reversion rate did not increase when the cells were starved for cysteine for a similar period, indicating that the increased reversion rate was specific to conditions where the reversions were advantageous. Two artifactual explanations for the observations, delayed growth of some preexisting revertants and cryptic growth by some cells at the expense of dying cells within aged colonies, were tested and rejected as unlikely. The trp+ reversions that occurred while trp- colonies aged in the absence of tryptophan were shown to be time-dependent rather than replication-dependent, and it is suggested that they occur by mechanisms different from those that have been studied in growing cells. A heuristic model for the molecular basis of such mutations is proposed and evidence consistent with that model is discussed. It is suggested that the results in this and previous studies can be explained on the basis of underlying random mechanisms that act during prolonged periods of physiological stress, and that "directed" mutations are not necessarily the basis of those observations.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

Effect of a water soluble derivative of alpha-tocopherol on radiation response of Saccharomyces cerevisiae

The radioprotection conferred by a highly water soluble glucose derivative of alpha-tocopherol, namely, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) in Saccharomyces cerevisiae was studied. Cells grown in standard YEPD-agar medium and irradiated in the presence of TMG showed a concentration dependent higher survival up to 10 mM of TMG in comparison to cells irradiated in distilled water. Treatment of TMG to cells given either before or immediately after irradiation but not during irradiation, had no effect on their radiation response. S. cerevisiae strain LP1383 (rad52) which is defective in recombination repair showed enhanced radioresistance only when subjected to irradiation in presence of TMG. Cells of rad52 strain grown in the medium containing TMG showed a radiation response similar to that of cells grown in the medium without TMG. The nature of TMG dependent enhanced radioresistance was studied by scoring the mutations in the strain D-7, which behaved like wild type strain in complete medium, at trp and ilv loci. Our study indicated that TMG confers radioresistance in S. cerevisiae possibly by two mechanisms viz. (i), by eliminating radiation induced reactive free radicals when the irradiation is carried out in the presence of TMG and (ii), by activating an error prone repair process involving RAD52 gene, when the cells are grown in the medium containing TMG.     

High mutagenic activity of 3-azido-1,2-propanediol (azidoglycerol, AG) in strain D7 of Saccharomyces cerevisiae

3-Azido-1,2-propanediol (azidoglycerol, AG) showed a high mutagenicity in strain D7 of Saccharomyces cerevisiae. At 5 mM it increased the spontaneous frequency of isoleucine revertants 3500 times and the frequency of gene convertants 3000 times during 24 h of growth, reducing the growth rate to 30%. In non-growth conditions, treatment with 150 mM of AG for 3 h reduced cell survival to 60% and enhanced the frequency of isoleucine revertants 490 times and tryptophan-independent convertants 50 times. At equal survival levels, AG was found to be 3000-fold more mutagenic and 200-fold more convertogenic than sodium azide.     

酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。     

Base-substitution and frameshift mutagenesis by sodium chloride and potassium chloride in Saccharomyces cerevisiae

Sodium chloride (NaCl) and potassium chloride (KCl) are both capable of inducing lethality and mutations when each is administered at a molarity of two for different lengths of time to logarithmic phase cells of the yeast Saccharomyces cerevisiae. Analysis of the revertants indicates that the reversions can be base substitutions, of both the transition and the transversion type, as well as frameshift mutations. At equal molarity, with the frequency of mutations as the criterion, KCl and NaCl are equally efficient in inducing all types of mutations.     

Analysis of factors responsible for the regeneration to intact cells from sphaeroplasts of Saccharomyces cerevisiae

The regeneration of Saccharomyces cerevisiae, NCIM 3288, cells from its sphaeroplasts were found to be influenced by a number of factors. The most suitable conditions of regeneration were also dependent on growth medium, that is, using malt-extractglucose-yeast extract-peptone (MGYP) medium: mannitol 0.7 M, pH 6.5, 30 °C and using yeast extract-peptone-glucose (YPG) medium: sucrose 0.7 M, pH 5.0 and 30 °C. The maximum regeneration frequency was observed in YPG medium.     

Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells

Resting cells experience mutations without apparent external mutagenic influences. Such DNA replication-independent mutations are suspected to be a consequence of processing of spontaneous DNA lesions. Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double-strand break (DSB) repair on the frequency of replication-independent mutations. The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non-homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication-independent mutation frequency in haploid cells. Sequencing indicated that typical NHEJ-dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors. Experiments with diploid and RAD52- or RAD54-deficient strains confirmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication-independent frameshift mutations in haploid and diploid NHEJ non-repressed cells. Thus our results provide evidence that G(0) cells with unrepressed NHEJ capacity pay for a large-scale chromosomal stability with an increased frequency of small-scale mutations, a finding of potential relevance for carcinogenesis.     

Phosphatidylserine synthesis required for the maximal tryptophan transport activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 micrograms/ml of L-tryptophan, a concentration that met the requirements of tryptophanauxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.     

Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

Effects of the antimutagen cinnamaldehyde on reversion and survival of selected Salmonella tester strains

The antimutagenic activity of trans-cinnamaldehyde (C6H5CH = CHCHO) on chemically induced mutagenesis has been shown in E. coli. Using the Ames Salmonella typhimurium tester strains TA1535 (hisG46 uvrB rfa) and TA100 (TA1535/pKM101), the effects of cinnamaldehyde on spontaneous reversions and reversions induced by 4-nitroquinoline-N-oxide(4NQO) and ethyl methanesulfonate (EMS) have been examined. To observe the effect of cinnamaldehyde in the absence of functional muc genes, a third strain, TA1535/pGW201 (pKM101 muc140: :Tn5) was included in the testing. Modifications of the standard Ames test procedures and direct-plating techniques were employed to study the "antimutagenic" response exerted by cinnamaldehyde. In all strains tested, concentrations of cinnamaldehyde up to 25 micrograms/ml slightly decreased the number of spontaneous reversions and induced reversions were more markedly reduced. The decreases in the numbers of 4NQO-induced revertants were greater than those decreases which occurred for EMS-induced reversions. There was no effect on viability in 1% (v/v) nutrient broth supplemented minimal medium containing 5-25 micrograms/ml of cinnamaldehyde. Cinnamaldehyde did not display any mucAB dependent or independent specificity against the mutagens used. On minimal medium supplemented with histidine and biotin, concentrations of cinnamaldehyde above 10 micrograms/ml were lethal for the strains tested. When the test medium was supplemented with 1-5% (v/v) liquid nutrient broth, viability was not affected at concentrations up to 25 micrograms/ml. For both TA100 and TA1535 the presence of 20 micrograms/ml of cinnamaldehyde in 1% (v/v) liquid nutrient broth-supplemented minimal glucose broth extended the lag phase for 2-4 h with no effect on survival. Depending on the test procedure employed, decreases in numbers of revertants may reflect lethality rather than antimutagenesis. When used to test for antimutagenesis rather than mutagenesis, modifications of the standard Ames test procedure may mimic an antimutagenic response due to a decrease in the total number of revertants seen even though enough cells survive to produce a background lawn.     

Non-homologous end joining dependency of gamma-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by gamma-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the gamma-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.     

Fungal β-glucosidase expression in Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae strains expressing β-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best β-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB?+?Lac12]. The recombinant extracellular β-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75?°C. The S.?cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more β-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8?g?l(-1) after 24?h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB?+?Lac12] exhibited limited growth.     

Instability kinetics of trp operon plasmid ColEl-trp in recombinant Escherichia coli MV 12[pVH5] and MV 12trp R [pVH5

In studying plasmid instability in recombinant microorganisms, Escherichia coli MV 12[p VH5] and MV 12 trpR[p VH5] harboring trp operon were used as experimental model systems. The host with the trp repression system was partially derepressed by 3-beta-indoleacrylic acid. The results from kinetic analysis of plasmid instability showed that the stability of p VH5 and the growth rate of MV 12[p VH5] decreased rapidly in presence of 3-beta-indoleacrylic acid at a concentration higher than 10 mug/mL, but beyond 30 g/mL no significant change was observed. This suggests that Trp(-) variants from MV 12[p VH5] could be produced from the host cells at a different frequency depending on the physiological condition. In another system, MV 12 trpR[p VH5] which was constructed by conjugation of E. coli MV 12[p VH5] with E. coli CSH61, the plasmid stability was much lower and the frequency of Trp(-) cell production was about 10 times higher as compared with the MV 12[p VH5] when treated with 3-beta-indoleacrylic acid. A kinetic model representing the plasmid instability was derived, and a fairly good agreement with the experimental results was found. The fraction of plasmid-free cell (or negative variant) shows a different time course profile depending on the segregation coefficient (a, production rate of negative variants from positive cells during one generation), growth ratio (G, the ratio of growth rates of negative variants to positive cells), and other parameters. The negative variant fraction continues to increase ("run away" type) or it approaches to a finite value ("settling" type) depending on the relative values of a and G.