Characterization of Null Mutants of the RAD55 Gene of Saccharomyces cerevisiae: Effects of Temperature, Osmotic Strength and Mating Type

RAD55 belongs to a group of genes required for resistance to ionizing radiation, RAD50-RAD57, which are thought to define a pathway of recombinational repair. Since all four alleles of RAD55 are temperature conditional (cold sensitive) for their radiation phenotype, we investigated the phenotype produced by null mutations in the RAD55 gene, constructed in vitro and transplaced to the yeast chromosome. The X-ray sensitivity of these null mutant strains was surprisingly suppressed by increased temperature, osmotic strength of the growth medium and heterozygosity at the mating-type locus. These first two properties, temperature conditionality and osmotic remediability, are commonly associated with missense mutations; these rad55 null mutants are unique in that they exhibit these properties although the mutant gene cannot be expressed. X-ray-induced mitotic recombination was also cold sensitive in rad55 mutant diploids. Although mitotic growth was unaffected in these strains, meiosis was a lethal event at both high and low temperatures. Whereas the phenotype of rad55 null mutants is consistent with a role of RAD55 in recombination and recombinational repair, there is evidence for considerable RAD55-independent recombination, at least in mitotic cells, which is influenced by temperature and MAT. We discuss models for the role of RAD55 in recombination to explain the unusual properties of rad55 mutants.     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Ethanol-hypersensitive and ethanol-dependent cdc? mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^– mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^– mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^– phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

The Identification of Transposon-Tagged Mutations in Essential Genes That Affect Cell Morphology in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphology--elongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis.     

The Cs Sec Mutants of Escherichia Coli Reflect the Cold Sensitivity of Protein Export Itself

We have found that temperature can have a striking effect upon protein export in Escherichia coli, suggesting that there is a cold-sensitive step in the protein export pathway. Cs mutations comprise the largest class of mutations affecting the membrane-localized Sec proteins SecD, SecE, SecF and SecY. Although some of these mutations could encode cold-labile proteins, this is unlikely to account for the Cs phenotype of most export mutants, as mutations which simply produce lower amounts of SecE protein have the same phenotype. Certain signal sequence mutations affecting maltose binding protein are also cold sensitive for export. These effects appear to arise by a specific interaction of cold with certain export defects. We believe that the Cs sec mutations are representative of a large class of conditional lethal mutations, whose conditional phenotype reflects an underlying thermal sensitivity of the process in which they are involved.     

Extragenic Suppression and Synthetic Lethality among Chlamydomonas Reinhardtii Mutants Resistant to anti-Microtubule Drugs

The antimicrotubule agents oryzalin (ORY), colchicine (COL) and taxol (TAX) were used to select three recessive, conditional lethal (ts-) mutants which defined two new essential loci, ory1 and cor1. The two ory1 mutants conferred resistance to ORY, TAX, and COL; the cor1 mutant conferred resistance only to COL. Each of the mutants displayed wild-type sensitivity to a number of unrelated inhibitors. Assembly and disassembly of flagellar microtubules in the ory1 mutants displayed wild-type sensitivity to ORY and COL, suggesting that the ory1 gene product either does not participate in these processes or the ory1 gene product alone is not sufficient to confer resistance. The ory1 locus mapped to linkage group X; cor1 was mapped to the left arm of linkage group XII. A synthetic lethal interaction was observed between ory1 and cor1 mutations, i.e., inferred ory1 cor1 double mutants were inviable at the permissive temperature. The conditional lethal phenotype of ory1-1 was used to select many spontaneous TS+ revertants, which arose at high frequencies. Genetic and phenotypic characterization of the revertants demonstrated that (1) the revertants fell into four phenotypic classes, including some which conferred supersensitivity to ORY and others which conferred cold-sensitive lethality, (2) reversion was caused in most or all cases by extragenic suppressors, (3) suppressor mutations displayed complex behavior in heterozygous (sup/+) diploids, (4) many different loci may be capable of suppressing ory1 mutants, and (5) suppressors of ory1-1 efficiently suppressed an independently isolated allele, ory1-2. Taken together the ory1, cor1, and suppressor mutations identify a number of interacting loci involved in essential cellular processes which are specifically susceptible to antimicrotubule agents.     

Cold-sensitive Mutations in Salmonella typhimurium Which Affect Ribosome Synthesis

A number of mutations (45) expressed as cold-sensitive conditional lethal pheno-types were screened by transduction for their linkage to the streptomycin-resistance locus; 7 showed such linkage. Of these, two were studied in greater detail. The sedimentation profiles of ribosomes from cultures grown at low temperature differed from wild type and from one another. Both mutants lost ribonucleic acid control at low temperature. It is suggested that a high proportion of mutants expressing a cold-sensitive phenotype harbor mutations in genes affecting ribosome synthesis or regulation.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

Overexpression of Yeast Homologs of the Mammalian Checkpoint Gene Rcc1 Suppresses the Class of α-Tubulin Mutations That Arrest with Excess Microtubules

Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in α-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.     

A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea.

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc(29DE) gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc(95E) mutants, which are extremely sensitive to caffeine. Although huc(95E) is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc(95E) gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc(29D) and huc(95E) as a first step toward molecularly characterizing the relevant genes.     

Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram‐positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA‐binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short‐chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA ‘PM1’ mutant, which carries two amino acid changes to a highly conserved region, yields a dominant‐negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg²⁺ concentrations, beginning as low as 2 mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg²⁺ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg²⁺ can be independently suppressed by specific genomic mutations.     

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes.

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.     

Yeast resistance to polyene antibiotics. I. Production of Saccharomyces cerevisiae mutants resistant to nystatin and their genetic analysis]

239 nistatin-resistant mutants were selected after UV-irradiation of yeasts. Phenotypical analysis has revealed two main groups of the mutants: 1) resistant to nistatin and resistant or sensitive (in different combinations) to haptaens; 2) resistant to nistatin and having an increased resistance to haptens. It is found that the sensitivity dominates over the resistance and hyper-resistance. Genetic analysis of the mutant collection has shown that the resistance to nistatin is determined by 5 nuclear genes (hysr). Hyper-resistance is controlled by mutations in other genes, which are not connected with stable phenotype. Genes of hyper-resistance can be considered as minus-modificators of pleiothrophic cross-resistance, characteristic of hysr genes. Plus-modificator genes of polyenic resistance are described. The gene hysr1 is linked with its chromosome.     

Hypersensitivity to heavy water: a new conditional phenotype

Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D2O. Using chemical mutagenesis we obtained a number of strains that grow on H2O-containing media but not on otherwise identical media containing 90% D2O. The frequency of these D2O-sensitive (d) mutants is comparable to the frequency of conventional temperature-sensitive (ts) mutants in the same mutagenized sample, and the ds mutations are distributed over a large number of complementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle ts mutants tested, only 2 are also ds. Thus, the ds technique should be useful for producing conditional mutations in genes that are not amenable to the ts and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the advantages of conditional phenotypes to mammalian and avian genetics.     

Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants.     

Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae.

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.     

Yeast Mutants Sensitive to Antimicrotubule Drugs Define Three Genes That Affect Microtubule Function

Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.     

A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins.

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.     

Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase

Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase.