Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.     

A Suppressor of snf1 Mutations Causes Constitutive High-Level Invertase Synthesis in Yeast

The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1 ). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type ( SNF1 ) genetic background and are, therefore, not merely suppressors of snf1 . Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6 .     

Genetic Interactions among Chlamydomonas Reinhardtii Mutations That Confer Resistance to anti-Microtubule Herbicides

We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1-apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1- alleles and apm2-1 suppressor mutations. Those apm1- apm2-1rev strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs+ phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D2O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1- alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1- and apm2- mutants.     

Functional Interchangeability of DNA Replication Genes in SALMONELLA TYPHIMURIUM and ESCHERICHIA COLI Demonstrated by a General Complementation Procedure

Twenty-four genes from Salmonella typhimurium that affect DNA replication were isolated from a lambda-Salmonella genomic library by lysogenic complementation of temperature-sensitive mutants of Salmonella or E. coli, using a new plaque complementation assay. The complementing lambda clones, which make red plaques in this assay, and noncomplementing mutant derivatives, which make uncolored plaques, were used to further characterize the temperature-sensitive Salmonella mutants and to establish the functional similarity of E. coli and Salmonella DNA replication genes. For 17 of 18 E. coli mutants representing distinct loci, a Salmonella gene that complemented the mutant was found. This result indicates that single Salmonella replication proteins are able to function in otherwise all E. coli replication complexes and suggests that the detailed properties of Salmonella and E. coli replication proteins are very similar. The other seven Salmonella genes that were cloned were unrelated functionally to any E. coli genes examined. --As an aid to the derivation of chromosomal mutations affecting some of the cloned genes, a general method was developed for placing a transposon in the Salmonella chromosome in a segment corresponding to cloned DNA. Chromosomal mutations were derived in Salmonella affecting a gene (dnaA) that was cloned by complementation of an E. coli mutant by using the transposon-encoded drug resistance as a selectable marker in local mutagenesis.     

A Cytogenetic and Genetic Characterization of a Group of Closely Linked Second Chromosome Mutations That Suppress Position-Effect Variegation in Drosophila Melanogaster

Characterization of a group of dominant second chromosome suppressor of position-effect variegation (PEV) (Su(var)) mutants has revealed a variety of interesting properties, including: maternal-effect suppression of PEV, homozygous lethality or semilethality and male-specific hemizygous lethality, female infecundity, acute sensitivity to the amount of heterochromatin in the cell and sensitivity to sodium butyrate. Deficiency/duplication mapping and complementation tests have revealed that eight of the mutants define at least two genes in section 31 of the left arm of chromosome 2 and they suggest that a ninth corresponds to an additional nonessential Su(var) gene within or near this region. The effects of specific deficiencies and a duplication on PEV indicate that the expression of one or more of the Su(var) genes in this region of the chromosome is dose-dependent, i.e., capable of haplo-abnormal suppression and triplo-abnormal enhancement. Interestingly, the appearance of certain visible phenotypes among a subset of the mutants suggests that they may possess antimorphic properties. Our results are consistent with the hypothesis that two of these Su(var) genes encode structural components of heterochromatin. We also report that two previously isolated mutants located in 31E and 31F-32A act as recessive suppressors of PEV.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

Mutations in the Sup-38 Gene of Caenorhabditis Elegans Suppress Muscle-Attachment Defects in Unc-52 Mutants

Mutations in the unc-52 locus of Caenorhabditis elegans have been classified into three different groups based on their complex pattern of complementation. These mutations result in progressive paralysis (class 1 mutations) or in lethality (class 2 and 3 mutations). The paralysis exhibited by animals carrying class 1 mutations is caused by disruption of the myofilaments at their points of attachment to the cell membrane in the body wall muscle cells. We have determined that mutations of this class also have an effect on the somatic gonad, and this may be due to a similar disruption in the myoepithelial sheath cells of the uterus, or in the uterine muscle cells. Mutations that suppress the body wall muscle defects of the class 1 unc-52 mutations have been isolated, and they define a new locus, sup-38. Only the muscle disorganization of the Unc-52 mutants is suppressed; the gonad abnormalities are not, and the suppressors do not rescue the lethal phenotype of the class 2 and class 3 mutations. The suppressor mutations on their own exhibit a variable degree of gonad and muscle disorganization. Putative null sup-38 mutations cause maternal-effect lethality which is rescued by a wild-type copy of the locus in the zygote. These loss-of-function mutations have no effect on the body wall muscle structure.     

P Transposon-Induced Dominant Enhancer Mutations of Position-Effect Variegation in Drosophila Melanogaster

P transposon induced modifier mutations of position-effect variegation (PEV) were isolated with the help of hybrid dysgenic crosses (π2 strain) and after transposition of the mutator elements pUChsneory(+) and P[lArB]. Enhancer mutations were found with a ten times higher frequency than suppressors. The 19 pUChsneory(+)- and 15 P[lArB]-induced enhancer mutations can be used for cloning of genomic sequences at the insertion sites of the mutator elements via plasmid rescue. Together with a large sample of X-ray-induced (48) and spontaneous (93) enhancer mutations a basic genetic analysis of this group of modifier genes was performed. On the basis of complementation and mapping data we estimate the number of enhancer genes at about 30 in the third chromosome and between 50 and 60 for the whole autosome complement. Therefore, enhancer of PEV loci are found in the Drosophila genome as frequently as suppressor genes. Many of the enhancer mutations display paternal effects consistent with the hypothesis that some of these mutations can induce genomic imprinting. First studies on the developmentally regulated gene expression of PEV enhancer genes were performed by β-galactosidase staining in P[lArB] induced mutations.     

Autosomal Mutations Affecting Adhesion between Wing Surfaces in Drosophila Melanogaster

Integrins are evolutionarily conserved transmembrane α,β heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila the position-specific (PS) integrins mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, we conducted a screen for autosomal mutations that produce blisters in somatic wing clones. We isolated 76 independent mutations in 25 complementation groups, 15 of which contain more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the β(PS) integrin gene myospheroid were investigated. Mutations in four known genes (blistered, Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion.     

Two Recessive Suppressors of SACCHAROMYCES CEREVISIAE CHO1 That Are Unlinked but Fall in the Same Complementation Group

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.     

Extragenic Suppressors of a Dynein Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed ``motor.' Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.     

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 Genetic Analysis of the 63e-64a Genomic Region of Drosophila Melanogaster: Identification of Mutations in a Replication Factor C Subunit

We have performed and F(2) genetic screen to identify lethal mutations within the 63E-64A genomic region. We have isolated 122 mutations in 20 different complementation groups. Of these groups, 16 are represented by multiple alleles. We have also established that the Rop and Ras2 genes are located within the 63E-64A genomic domain at 64A10,11. We have sequenced 10.2 kb of DNA surrounding this gene pair and find that in addition to Rop and Ras2 there is another gene located within this DNA sequence. The gene product, which we have named Rfc40, shows 68% identity to the 40-kDa subunit of replication factor C. We find that the members of one complementation group (13 alleles) derived from our screen correspond to mutations in the Rop gene, whereas the members of another (five alleles) correspond to mutations in the Rfc40 gene. In addition we have isolated 11 new mutant alleles of the disembodied gene.     

Cycloheximide-Resistant Temperature-Sensitive Lethal Mutations of Saccharomyces Cerevisiae

We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37°), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5°). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37° but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.     

Genetic Analysis of Saccharomyces Cerevisiae Chromosome I: On the Role of Mutagen Specificity in Delimiting the Set of Genes Identifiable Using Temperature-Sensitive-Lethal Mutations

In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts(-)) lethal mutations that had been induced by ethyl methanesulfonate or nitrosoguanidine were analyzed. Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously. In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes. In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts(-) lethal mutations, we isolated a new set of such mutations after mutagenesis with UV or nitrogen mustard. Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis. Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped. These results suggest that only a minority of the essential genes in yeast can be identified using Ts(-) lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.     

Screens for Extragenic Mutations That Fail to Complement Act1 Alleles Identify Genes That Are Important for Actin Function in Saccharomyces Cerevisiae

Null mutations in SAC6 and ABP1, genes that encode actin-binding proteins, failed to complement the temperature-sensitive phenotype caused by a mutation in the ACT1 gene. To identify novel genes whose protein products interact with actin, mutations that fail to complement act1-1 or act1-4, two temperature-sensitive alleles of ACT1, were isolated. A total of 14 extragenic noncomplementing mutations and 12 new alleles of ACT1 were identified in two independent screens. The 14 extragenic noncomplementing mutations represent alleles of at least four different genes, ANC1, ANC2, ANC3 and ANC4 (Actin NonComplementing). Mutations in the ANC1 gene were shown to cause osmosensitivity and defects in actin organization; phenotypes that are similar to those caused by act1 mutations. We conclude that the ANC1 gene product plays an important role in actin cytoskeletal function. The 12 new alleles of ACT1 will be useful for further elucidation of the functions of actin in yeast.     

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.     

Nuclear Mutations in Saccharomyces Cerevisiae That Affect the Escape of DNA from Mitochondria to the Nucleus

We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion. TRP1 was not phenotypically expressed within the organelle. However, this Trp(-) strain readily gave rise to respiratory competent Trp(+) clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei. Thus the Trp(+) clones arose as a result of DNA escaping from mitochondria and migrating to the nucleus. We have isolated 21 nuclear mutants in which the rate of mtDNA escape is increased by screening for increased rates of papillation to Trp(+). All 21 mutations were recessive and fell into six complementation groups, termed YME1-YME6. In addition to increasing the rate of mtDNA escape, yme1 mutations also caused a heat-sensitive respiratory deficient phenotype at 37° and a cold-sensitive growth defect on complete glucose medium at 14°. While the other yme mutations had no detectable growth phenotypes, synergistic interactions were observed in two double mutant combinations: a yme1, yme2 double mutant failed to respire at 30° and a yme4, yme6 double mutant failed to respire at all temperatures tested. None of the respiratory defects were caused by loss of functional mtDNA. These findings suggest that yme1, yme2, yme4 and yme6 mutations alter mitochondrial functions and thereby lead to an increased rate of DNA escape from the organelle.     

Dominant Suppressors of Yeast Actin Mutations That Are Reciprocally Suppressed

A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.