Genetic Analysis of Mutations Affecting Growth of SACCHAROMYCES CEREVISIAE at Low Temperature

A large number of genes control growth of the yeast Saccharomyces cerevisiae at low temperatures (< 10 degrees ). Approximately 47 percent of the mutants selected for inability to grow at 4-5 degrees C show increased sensitivity to cycloheximide. In 3 of 4 cases tested, supersensitivity to cycloheximide and inability to grow at the low temperature segregate together and thus appear to be effects of the same mutation. Since many cold-sensitive mutants of bacteria have been found to have altered ribosomes and since cycloheximide resistance in yeast can be caused by ribosomal changes, this suggests that the mutants having low-temperature-sensitive growth may be defective in ribosome-assembly processes at the low temperatures. Two of the lts loci, lts1 and lts3 have been located on chromosome VII and another two, lts4 and lts10 on chromosome IV. A mutation, cyh10, conferring cycloheximide resistance, but not cold sensitivity, has been located close to the centromere on chromosome II.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Direct selection for gene replacement events in yeast

A method that facilitates gene replacement at the HIS3 locus of Saccharomyces cerevisiae (yeast) has been developed. First, an internal region of the cloned HIS3 gene was replaced by a DNA segment containing the wild-type ribosomal protein gene, CYH2. Second, by using standard yeast transformation methods, the wild-type HIS3 locus of a cycloheximide resistant strain (cyh2r) was replaced by this his3-CYH2 substitution. The resulting strain is sensitive to cycloheximide because CYH2 is dominant to cyh2r. Third, his3 mutations cloned into integrating or replicating vectors were introduced into this strain by selecting transformants via the vector-encoded marker. Selection for cycloheximide-resistant colonies resulted in the replacement of the his3-CYH2 allele by newly introduced his3 alleles. Thus, this scheme provides for the direct selection of gene replacement events at the HIS3 locus independently of the phenotype of the cloned his3 derivatives. In principle, it can be extended to any region of the yeast genome.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

Genetic analysis of antisuppressor mutants in the fission yeast Schizosaccharomyces pombe.

Fourteen unlinked sin genes could be mutated to recessive antisuppressor alleles preventing the expression of suppressors in the fission yeast Schizosaccharomyces pombe. cyh1 alleles, resistant to the ribosomal inhibitor cycloheximide, also have some antisuppressor effect. The genetical and physiological characterization of these mutants is consistent with the hypothesis that they affect components of the messenger RNA translation machinery such as tRNA modifying enzymes or ribosomal proteins.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

Genetic instability of heterozygous, hybrid, natural wine yeasts

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2(R)/cyh2(R) mutants resistant to high levels of cycloheximide can be directly isolated from cyh2(S)/cyh2(S) wine yeasts. Heterozygous cyh2(R)/cyh2(S) hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2(R)/cyh2(R) homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells, ts 13A and ts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and the ts lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae.

Summary Mutants of Schizosaccharomyces pombe were isolated as resistant either to trichodermin or to anisomycin. Growth tests showed that the majority of mutants isolated were cross resistant to both drugs and also to cycloheximide. A limited genetic analysis showed that mutants at least four loci, tri3, tri4, ani1 and ani2, had this phenotype as was also the case for mutants at three cycloheximide resistant loci, cyh2, cyh3 and cyh4 reported previously (Ibrahim and Coddington, 1976). Allelism tests showed that the tri3, ani2 and cyh4 strains were allelic. A mutant at another trichodermin resistant locus, tri5, was cross resistant to anisomycin but sensitive to cycloheximide.Ribosomes from wild type and selected strains were analysed in a poly U directed cell free protein synthesising system. Three strains, cyh1-C7, ani1-F1 and tri-N15 (probably a tri5 allele) possessed ribosomes which were more resistant than the wild type to the drugs used in their isolation. In each case the site of the resistance was in the 60S subunit. Ribosomes from the cyh2, cyh3 and cyh4 strains were as sensitive to cycloheximide as those from wild type.     

Induction of Cycloheximide-Resistant Mutants in Saccharomyces cerevisiae with N-Methyl-N′-Nitro-N-Nitrosoguanidine and ICR-170

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.     

Resistance of Pseudomonas to Quaternary Ammonium Compounds: II. Cross-Resistance Characteristics of a Mutant of Pseudomonas aeruginosa

Tube dilution experiments showed that benzalkonium chloride (BC)-resistant mutants of Pseudomonas aeruginosa grown in the presence of 1,000 mug of BC per ml were at least 20 times more sensitive to polymyxin B and colistin sulfate than the BC-sensitive (BCS) parent strain. BCS cells selected for resistance to 500 mug of polymyxin B per ml remained sensitive to BC. There was little difference in the amount of carbenicillin, gentamicin sulfate, or rifampin needed to prevent growth of either the BCS or BC-resistant (BCR) strains. Growth of BCR cells was inhibited by ethylenediaminetetraacetate at a concentration of 400 mug/ml or less, whereas the BCS strain grew at ethylenediaminetetraacetate levels of 10,000 mug/ml. Phenylmercuric acetate and thimerosal inhibited growth of BCR and BCS cells at concentrations of 10 mug/ml or less. BCR cells were cross-resistant to >1,000 mug/ml concentrations of five other quaternary ammonium compounds, including three with C(16) alkyls and two with alkyl groups of shorter length. The BCS strain was also resistant to >1,000 mug/ml concentrations of the three quaternary ammonium compounds with C(16) alkyl groups but, in addition to BC, was inhibited by 200 mug/ml levels or less of the two quaternary ammonium compounds containing alkyl groups of less than 16 carbon atoms.     

Herpes simplex virus-1-specific proteins are involved in alteration of polyphosphoinositide metabolism in baby-hamster kidney cells.

Herpes simplex virus type 1 (HSV-1) induces altered phosphoinositide metabolism in baby hamster kidney (BHK) cells, measured as incorporation of [3H]inositol or [32P]Pi [Langeland, Haarr & Holmsen (1986) Biochem. J. 237, 707-712]. We now report that this response in the inositol phospholipids is dependent on virus-specific proteins synthesized in the beta (early) stage of virus protein synthesis. This was demonstrated both by resistance to the inhibitory effect of cycloheximide after this stage of infection, and by the use of temperature-sensitive (ts) mutants of HSV-1; ts mutants in which protein synthesis was blocked so that only the alpha proteins were expressed showed a PIP2/PIP (phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 4-monophosphate) ratio similar to uninfected cells, while ts mutants which were defective in protein synthesis at a late beta stage or later showed increased PIP2/PIP ratios similar to cells infected by wild type HSV-1.     

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.     

Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.     

Recombination-deficient mutants of colicinogenic Salmonella typhimurium detected by their failure to produce colicin

Survivors of nitrosoguanidine-treated cultures of a colicinogenic strain of Salmonella typhimurium were tested for spontaneous production of colicin E1. Of about 1,000 colonies tested, 13 produced no (or very narrow) colicin zones. Four of these isolates proved to be more sensitive to ultraviolet (UV) light, X rays, and methyl methane sulfonate than the parent strain and did not show enhanced production of colicin when treated with mitomycin C (which acts as an inducer on wild-type cells). Further studies showed that these isolates were of two classes. Three mutants were extremely sensitive to UV, failed to show spontaneous release of two temperate phages, and were infertile as recipients in transduction or in an Hfr cross although they accepted an F' factor normally. These independently isolated mutants were inferred to be recombination-deficient; one of them had the additional property of increased spontaneous mutability at two loci. The other colicin-nonreleasing isolate was only moderately sensitive to UV, showed enhanced spontaneous release of two temperate phages, and was of approximately normal fertility as a recipient in transduction or conjugation.     

Increased Spontaneous Mitotic Segregation in Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (canr/+) diploids by 13-to 170-fold. The mms8-1 mutant is MMS and X-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to X rays and UV, respectively, in addition of MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, X rays and UV and increases the rate of spontaneous mitotic segregation 23-fold.