Molecular and genetic analysis of the SNF7 gene in Saccharomyces cerevisiae.

Abstract Mutations in the SNF7 gene of Saccharomyces cerevisiae prevent full derepression of the SUC2 (invertase) gene in response to glucose limitation. We report the molecular cloning of the SNF7 gene by complementation. Sequence analysis predicts that the gene product is a 27-kDa acidic protein. Disruption of the chromosomal locus causes a fewfold decrease in invertase derepression, a growth defect on raffinose, temperature-sensitive growth on glucose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf7 null mutation with ssn6 and spt6/ssn20 suppressor mutations distinguished SNF7 from the SNF2, SNF5 and SNF6 genes. The snf7 mutation also behaved differently from mutations in SNF1 and SNF4 in that snf7 ssn6 double mutants displayed a synthetic phenotype of severe temperature sensitivity for growth. We also mapped SNF7 to the right arm of chromosome XII near the centromere.

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Genetic exploration of interactive domains in RNA polymerase II subunits

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.1908-1914.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 1908-1914

Author(s):

C Martin ◽

S Okamura ◽

R Young

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Temperature Sensitive ◽

Suppressor Mutations ◽

Region I ◽

Temperature Sensitive Mutation ◽

Allele Specific ◽

Separate Protein

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

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SUPPRESSORS OF snf 2 MUTATIONS RESTORE INVERTASE DEREPRESSION AND CAUSE TEMPERATURE-SENSITIVE LETHALITY IN YEAST

Genetics ◽

10.1093/genetics/112.4.741 ◽

1986 ◽

Vol 112 (4) ◽

pp. 741-753

Author(s):

Lenore Neigeborn ◽

Kenneth Rubin ◽

Marian Carlson

Keyword(s):

Genetic Background ◽

Glucose Deprivation ◽

Temperature Sensitive ◽

Wild Type ◽

Suppressor Mutations ◽

Invertase Gene ◽

Selection For ◽

Galactose Utilization ◽

Yeast Genes ◽

Recessive Defect

ABSTRACT Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.

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Suppressor Mutations indegSOvercome the Acute Temperature-Sensitive Phenotype of ΔdegPand ΔdegPΔtol-palMutants ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00742-18 ◽

2019 ◽

Vol 201 (11) ◽

Cited By ~ 4

Author(s):

Brea Kern ◽

Owen P. Leiser ◽

Rajeev Misra

Keyword(s):

Protease Activity ◽

Outer Membrane Proteins ◽

Critical Role ◽

Pdz Domain ◽

Growth Defect ◽

Temperature Sensitive ◽

Salt Bridges ◽

Suppressor Mutations ◽

Growth Phenotype ◽

Complex Display

ABSTRACTInEscherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in thedegSgene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS’s protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS’s basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS’s protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolBΔdegPmutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCEThe Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP’s periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.

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PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3843 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3843-3856 ◽

Cited By ~ 20

Author(s):

J P O'Connor ◽

C L Peebles

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Genomic Library ◽

Essential Gene ◽

Growth Defect ◽

Temperature Sensitive ◽

Putative Open Reading Frame ◽

Reading Frame ◽

Trna Processing ◽

Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

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Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.9.5001 ◽

1997 ◽

Vol 17 (9) ◽

pp. 5001-5015 ◽

Cited By ~ 67

Author(s):

N I Zanchin ◽

P Roberts ◽

A DeSilva ◽

F Sherman ◽

D S Goldfarb

Keyword(s):

Saccharomyces Cerevisiae ◽

Fluorescent Protein ◽

Open Reading Frames ◽

Growth Defect ◽

Temperature Sensitive ◽

Protein Fusion ◽

Acid Protein ◽

Conserved Gene ◽

Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

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Alteration of the Protein Kinase Binding Domain Enhances Function of the Saccharomyces cerevisiae Molecular Chaperone Cdc37

Eukaryotic Cell ◽

10.1128/ec.00165-07 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1363-1372 ◽

Cited By ~ 5

Author(s):

Min Ren ◽

Arti Santhanam ◽

Paul Lee ◽

Avrom Caplan ◽

Stephen Garrett

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Protein Kinases ◽

Molecular Chaperone ◽

Binding Domain ◽

Growth Defect ◽

Temperature Sensitive ◽

Physical Interaction ◽

General Function ◽

Amino Terminal

ABSTRACT Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative “protein kinase binding domain” of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

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Bni1p Regulates Microtubule-Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8016 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8016-8027 ◽

Cited By ~ 30

Author(s):

Takeshi Fujiwara ◽

Kazuma Tanaka ◽

Eiji Inoue ◽

Mitsuhiro Kikyo ◽

Yoshimi Takai

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Nuclear Migration ◽

Growth Defect ◽

Temperature Sensitive ◽

Cortical Actin ◽

Synthetic Lethal ◽

Downstream Target ◽

Synthetically Lethal ◽

Mother Cells

ABSTRACT The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identifiedPAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150Glued, a component of the dynein-activated dynactin complex. Disruption ofBNI1 in either the pac1 or nip100mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

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AN ENDOMITOTIC EFFECT OF A CELL CYCLE MUTATION OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/97.3-4.551 ◽

1981 ◽

Vol 97 (3-4) ◽

pp. 551-562 ◽

Cited By ~ 1

Author(s):

David Schild ◽

Honnavara N Ananthaswamy ◽

Robert K Mortimer

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Analysis ◽

Nuclear Division ◽

Pedigree Analysis ◽

Visual Observation ◽

Temperature Sensitive ◽

Cell Stage ◽

Haploid Strain ◽

A Cell ◽

Dna Measurements

ABSTRACT A recessive temperature-sensitive mutation of Saccharomyces cerevisiae has been isolated and shown to cause an increase in ploidy in both haploids and diploids. Genetic analysis revealed that the strain carrying the mutation was an aa diploid, although MNNG mutagenesis had been done on an a haploid strain. When the mutant strain was crossed with an aa diploid and the resultant tetraploid sporulated, some of the meiotic progeny of this tetraploid were themselves tetraploid, as shown by both genetic analysis and DNA measurements, instead of diploid as expected of tetraploid meiosis. The ability of these tetraploids to continue to produce tetraploid meiotic progeny was followed for four generations. Homothallism was excluded as a cause of the increase in ploidy; visual pedigree analysis of spore clones to about the 32-cell stage failed to reveal any zygotes, and haploids that diploidized retained their mating type. An extra round of meiotic DNA synthesis was also considered and excluded. It was found that tetraploidization was independent of sporulation temperature, but was dependent on the temperature of germination and the growth of the spores. Increase in ploidy occurred when the spores were germinated and grown at 30°, but did not occur at 23°. Two cycles of sporulation and growth at 23° resulted in haploids, which were shown to diploidize within 24 hr when grown at 30°. Visual observation of the haploid cells incubated at 36° revealed a celldivisioncycle phenotype characteristic of mutations that affect nuclear division; complementation analysis demonstrated that the mutation, cdc31-2, is allelic to cdc31-1, a mutation isolated by HARTWEeLL et al.(1973) and characterized as causing a temperature-sensitive arrest during late nuclear division. The segregation of cdc31-2 in heterozygous diploids was 2:2 and characteristic of a noncentromere-linked gene.

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Dominant suppressors of yeast actin mutations that are reciprocally suppressed.

Genetics ◽

10.1093/genetics/121.4.675 ◽

1989 ◽

Vol 121 (4) ◽

pp. 675-683

Author(s):

A E Adams ◽

D Botstein

Keyword(s):

High Temperature ◽

Linkage Group ◽

Growth Defect ◽

Temperature Sensitive ◽

Mutant Library ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

New Gene ◽

Allele Specificity

Abstract 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.

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An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.1.33 ◽

1998 ◽

Vol 148 (1) ◽

pp. 33-47 ◽

Cited By ~ 2

Author(s):

Jeffrey S Flick ◽

Jeremy Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Phospholipase C ◽

Growth Defect ◽

Temperature Sensitive ◽

Complete Medium ◽

Restrictive Temperature ◽

Dependent Protein Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Dependent Protein

Abstract The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1Δ cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80Δ) mutation and growth on low-phosphate medium, also permitted growth of plc1Δ cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1Δ cells by pho80Δ does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81Δ and spl2Δ show a synthetic phenotype in combination with plc1Δ. Unlike single mutants, plc1Δ pho81Δ and plc1Δ spl2Δ double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

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