Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.     

Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability]

To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained. Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation. Such mutations were called hsm (high spontaneous mutagenesis). Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations. It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7. Genetical analysis revealed that mutation hsm3-1 is recessive. The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors. Possible nature of hsm mutations is discussed.     

RBE of densely ionizing radiation for wild-type and radiosensitive mutants of yeast

Survival curves were obtained for haploid and diploid yeasts, Saccharmyces cerevisiae, of wild-type strains and radiosensitive mutants exposed to γ-rays and α-particles. A correlation between the values of the relative biological effectiveness (RBE) of high-LET radiation and cell-repair capacity was found. The difference in radiosensitivities of the wild-type diploid strain and homozygous rad mutants incapable of recovery was significantly higher after low-LET radiation than after high-LET radiation. Possible reasons for the observed radiation responses to low- and high-LET exposure of yeast cells with various genotype are discussed.     

alpha-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants.

In contrast to wild-type strains of the yeast Saccharomyces cerevisiae, lys2 and lys5 mutants are able to utilize alpha-aminoadipate as a primary source of nitrogen. Chattoo et al. (B. B. Chattoo, F. Sherman, D. A. Azubalis, T. A. Fjellstedt, D. Mehnert, and M. Ogur, Genetics 93:51-65, 1979) relied on this difference in the effective utilization of alpha-aminoadipate to develop a procedure for directly selecting lys2 and lys5 mutants. In this study we used a range of mutant strains and various media to determine why normal strains are unable to utilize alpha-aminoadipate as a nitrogen source. Our results demonstrate that the anabolism of high levels of alpha-aminoadipate through the biosynthetic pathway of lysine results in the accumulation of a toxic intermediate and, furthermore, that lys2 and lys5 mutants contain blocks leading to the formation of this intermediate.     

Biochemical basis of varying nystatin resistance of Saccharomyces cerevisiae and Candida maltosa mutants]

Six groups of nystatin resistant mutants of C. maltosa and of haploid and diploid Saccharomyces cerevisiae strains were obtained with the help of genetic and biochemical analysis. It has been shown that every group of the mutants was characterized by a specific level of resistance to nystatin. The dependence of the resistance level upon sterol content has been established. It has been shown that the more the structure of the sterol present differed from ergosterol the higher was the resistance level. The results obtained in vivo permit to make conclusions about the role of different functional groups of sterol molecule in the interaction with nystatin.     

Isolation of mutants with altered pigments after irradiating haploid protoplasts from Datura innoxia Mill. with X-rays

Summary Ten different mutants with altered pigment patterns were isolated following X-irradiation of approximately 10⁵ haploid protoplasts of Datura innoxia Mill. Seven of the selected strains gave rise to shoots and 3 to leaves only. The mutants were selected from light green or white calli, which had developed 4 weeks after transfer of developing cell clusters onto B₅ agar medium containing 0.5 mg/l BAP (Gamborg et al., 1968). Of the 10 mutant strains 5 were light green, two were yellow, one was pale yellow and one was white. One additional strain does not possess anthocyanin in its stems; a feature chracteristic of the wildtype is the possession of anthocyanin. This strain is able to grow in soil and has now flowered. None of the mutants obtained is haploid. Nine are diploid and the other is tetraploid. The chlorophyll deficient strains can be propagated on B₅ agar medium supplemented with higher concentration of sucrose than normally required for the growth of the wild-type.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

Genetic analysis of methylotrophic yeastCandida boidinii PLD1

This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeastCandida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA²⁺ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutationslys2-8-met-3 from one hand andade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.     

Frequency of induced mutations at the haploid and diploid levels inNicotiana tabacum L.

The frequencies of chlorophyll mutants were investigated in anther cultures derived from mutagen-treated plants ofN. tabacum cv. Samsun (haploid level) and in the seed offspring from the same treated plants (diploid level). Comparison of the induced mutation frequencies at the haploid and diploid levels demonstrated that selection existed against the haploid embryoids with induced chlorophyll deficient mutations. The diploid vegetative stage with phenotypic expression of the chlorophyll mutation was more vital than the haploid one. The suitability of anther cultures for studying induced mutagenesis is discussed.     

Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development.

The ascomycete Aspergillus nidulans produces green conidia (asexual spores). Recessive mutants which produce yellow conidia have been previously isolated from haploid strains and have been shown to be deficient in laccase (diphenol oxidase), an enzyme that requires copper for activity. Using a diploid parent strain, we isolated dominant yellow conidial mutants which, in the haploid state, produced even less laccase activity than a recessive mutant. Three isolates of such mutants behaved similarly and define a single complementation group (yB) on chromosome VIII distinct from the yA locus on chromosome I defined by recessive mutants. Unlike yA mutants, whose only discernable phenotype is their conidial color, yB mutants are pleiotropic: conidial germination was delayed relative to the wild type, and sexual development was blocked at an early stage. The three phenotypes of yB mutants were expressed on yeast extract-glucose medium containing 1.6 microM of added copper. When copper was added to above 5 microM, all three phenotypes were remediated, and near wild-type levels of laccase were produced. We conclude that yB mutants have a reduced availability of copper. The dominance of yB mutants could result, for example, from an alteration in transport or storage of copper. Using an immunological assay, we detected no laccase antigenic cross-reacting material in yB mutants grown on medium of low copper content. We conclude that either the synthesis or the stability of laccase is copper dependent.     

Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE

Normal strains of Saccharomyces cerevisiae do not use alpha-aminoadipate as a principal nitrogen source. However, alpha-aminoadipate is utilized as a nitrogen source by lys2 and lys5 strains having complete or partial deficiencies of alpha-aminoadipate reductase and, to a limited extent, by heterozygous lys2/+ strains. Lys2 mutants were conveniently selected on media containing alpha-aminoadipate as a nitrogen source, lysine, and other supplements to furnish other possible auxotrophic requirements. The lys2 mutations were obtained in a variety of laboratory strains containing other markers, including other lysine mutations. In addition to the predominant class of lys2 mutants, low frequencies of lys5 mutants and mutants not having any obvious lysine requirement were recovered on alpha-aminoadipate medium. The mutants not requiring lysine appeared to have mutations at the lys2 locus that caused partial deficiencies of alpha-aminoadipate reductase. Such partial deficiencies are believed to be sufficiently permissive to allow lysine biosynthesis, but sufficiently restrictive to allow for the utilization of alpha-aminoadipate. Although it is unknown why partial or complete deficiencies of alpha-aminoadipate reductase cause utilization of alpha-aminoadipate as a principal nitrogen source, the use of alpha-aminoadipate medium has considerable utility as a selective medium for lys2 and lys5 mutants.     

Isolation and characteristics of rifampicin-resistant mutants of Streptomyces erythraeus strain

Erythromycin-producing strains of S. erythraeus were characterized with respect to formation of spontaneous and induced rifampicin-resistant mutants. It was shown that the frequency of spontaneous rifampicin-resistant mutants formed by various strains amounted to 0.9.10(-8) = 9.1.10(-7). In some events the exposure to nitrosoguanidine increased the frequency of such mutants by 2 orders of magnitude. The rifampicin-resistant mutants differed in antibiotic resistance. It was found that a significant part of the rifampicin-resistant mutants became sensitive to heating (19.1 per cent) and lost the ability to form aeromycelium (21.8 per cent).     

酿酒酵母单倍体与双倍体原生质体的融合*

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation.

This paper reports a phenotypic characterization of ggp1 mutants. The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement. The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40%. Cells are round, with more than one bud, and extensively vacuolized. In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability. The observed phenotype suggests defects in cell separation. Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth. Conjugation and sporulation are not affected. The exocellular location of gp115 led us to examine cell wall properties. Cell wall and septum ultrastructure of abnormally budded cells was analyzed by electron microscopy analysis, and no appreciable differences from wild-type cells were found. Microscopic analysis revealed an increase in chitin content and delocalization. In comparison with control cells, ggp1 null mutants are shown to be resistant to Zymolyase during the exponential growth phase. A fivefold overexpression of gp115 does not bring about any effects on cell growth parameters and cell wall properties.     

酿酒酵母单倍体与双倍体原生质体的融合

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.