Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (5) ◽  
pp. 2308-2314
Author(s):  
T M Dunn ◽  
D Shortle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Normal Growth ◽  
Null Alleles ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Actin Assembly ◽  
New Gene ◽  
Cold Sensitive ◽  
Sensitive Allele ◽  
Extragenic Suppressors

Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin. One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted. Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures. Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2. These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.

scholarly journals Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (5) ◽  
pp. 2308-2314 ◽  
Author(s):  
T M Dunn ◽  
D Shortle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Normal Growth ◽  
Null Alleles ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Actin Assembly ◽  
New Gene ◽  
Cold Sensitive ◽  
Sensitive Allele ◽  
Extragenic Suppressors

Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin. One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted. Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures. Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2. These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.

scholarly journals SEX DETERMINATION IN THE NEMATODE C. ELEGANS: ANALYSIS OF tra-3 SUPPRESSORS AND CHARACTERIZATION OF fem GENES

Genetics ◽  
1986 ◽  
Vol 114 (1) ◽  
pp. 15-52
Author(s):  
Jonathan Hodgkin
Keyword(s):  
Sex Determination ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Female Development ◽  
C Elegans ◽  
Male Development ◽  
Maternal Contribution ◽  
New Gene ◽  
Extragenic Suppressors

ABSTRACT Mutations of the gene tra-3 result in partial masculinization of XX animals of C. elegans, which are normally hermaphrodites (males are XO). A total of 43 tra-3 revertants (one intragenic, 42 extragenic) have been isolated and analyzed, in the hope of identifying new sex-determination loci. Most (38) of the extragenic suppressors cause partial or complete feminization of XX and XO animals; the remaining four are weak suppressors. The feminizing suppressors are mostly alleles of known sex-determining genes: tra-1 (11 dominant alleles), tra-2 (one dominant allele), fem-1 (four alleles) and fem-2 (four alleles), but 18 are alleles of a new gene, fem-3. Additional alleles have been isolated for the fem-2 and fem-3 genes, as well as fem-3 deficiencies. Mutations in fem-3 resemble alleles of fem-1 (previously characterized): putative null alleles result in complete feminization of XX and XO animals, transforming them into fertile females. Severe alleles of fem-2 also cause complete feminization of XX animals at all temperatures, but feminization of fem-2 XO animals is temperature-sensitive: complete at 25°, incomplete at 20°. As with fem-1, severe mutations of fem-2 and fem-3 are wholly epistatic to masculinizing alleles of tra-2 and tra-3, and epistatic to tra-1 masculinizing alleles in the germline, but not in the soma. All three fem genes are essential for male development and appear to have a dual role in promoting spermatogenesis and repressing tra-1 activity. All three fem genes exhibit strong maternal effects; the maternal contribution of fem gene products may be inactivated in XX animals by a posttranscriptional mechanism. Maternal contributions of wild-type fem-3 product are necessary for normal XO male development and XX hermaphrodite (as opposed to female) development.

scholarly journals Isolation and characterization of extragenic suppressors of mutations in the SSA hsp70 genes of Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 131 (2) ◽  
pp. 277-285 ◽  
Author(s):  
R J Nelson ◽  
M F Heschl ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Regulatory Protein ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Transcriptional Regulatory ◽  
Extragenic Suppressors

Abstract Saccharomyces cerevisiae strains that contain null alleles of two hsp70 genes, SSA1 and SSA2, are temperature sensitive for growth. In this study, extragenic suppressors of ssa1 ssa2 have been isolated. Suppression is due to mutations at nuclear loci designated EXA1, EXA2 and EXA3 for EXtragenic suppressor hsp70 subfamily A. Two of the four EXA1 alleles are dominant as is EXA3-1. The other two EXA1 alleles as well as the sole EXA2 allele are recessive. EXA1 mutations lead to accumulation of a previously uncharacterized form of hsp70. EXA2 and EXA3 mutations affect the regulation of the stress response. In exa2-1 ssa1 ssa2 strains the gene products of the remaining SSA hsp70 genes, SSA3 and SSA4 (Ssa3/4p), accumulate to higher levels. The EXA3-1 mutation results in increased accumulation of both Ssa3/4p and the hsp70s encoded by the SSB1 and SSB2 genes (Ssb1/2p), suggesting that the EXA3 gene product plays a central role in the yeast stress response. Consistent with this hypothesis, EXA3-1 is tightly linked to HSF1, the gene encoding the transcriptional regulatory protein known as "heat shock factor." All of the genes identified in this study seem to be involved in regulating the expression of SSA3 and SSA4 or the activity of their protein products.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 833-841
Author(s):  
Yu Jiang ◽  
Al Scarpa ◽  
Li Zhang ◽  
Shelly Stone ◽  
Ed Feliciano ◽  
...  
Keyword(s):  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Suppression ◽  
Golgi Transport ◽  
New Gene ◽  
Insight Into ◽  
Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1389-1400
Author(s):  
Xiao Ming Zuo ◽  
G Desmond Clark-Walker ◽  
Xin Jie Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Nucleoids ◽  
Mtdna Replication ◽  
Sensitive Allele ◽  
Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase

1991 ◽  
Vol 11 (11) ◽  
pp. 5767-5780
Author(s):  
A M Healy ◽  
S Zolnierowicz ◽  
A E Stapleton ◽  
M Goebl ◽  
A A DePaoli-Roach ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein A ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
B Subunit ◽  
Bud Emergence ◽  
New Gene ◽  
Cold Sensitive ◽  
Polymerase Chain

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

scholarly journals Characterization of mutations that suppress the temperature-sensitive growth of the hpr1 delta mutant of Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 945-956 ◽  
Author(s):  
H Y Fan ◽  
H L Klein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Lethality ◽  
Direct Repeat ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Rad Genes ◽  
Complementation Groups ◽  
Rad Mutants ◽  
Extragenic Suppressors

Abstract The hpr1 delta 3 mutant of Saccharomyces cerevisiae is temperature-sensitive for growth at 37 degrees and has a 1000-fold increase in deletion of tandem direct repeats. The hyperrecombination phenotype, measured by deletion of a leu2 direct repeat, is partially dependent on the RAD1 and RAD52 gene products, but mutations in these RAD genes do not suppress the temperature-sensitive growth phenotype. Extragenic suppressors of the temperature-sensitive growth have been isolated and characterized. The 14 soh (suppressor of hpr1) mutants recovered represent eight complementation groups, with both dominant and recessive soh alleles. Some of the soh mutants suppress hpr1 hyperrecombination and are distinct from the rad mutants that suppress hpr1 hyperrecombination. Comparisons between the SOH genes and the RAD genes are presented as well as the requirement of RAD genes for the Soh phenotypes. Double soh mutants have been analyzed and reveal three classes of interactions: epistatic suppression of hpr1 hyperrecombination, synergistic suppression of hpr1 hyperrecombination and synthetic lethality. The SOH1 gene has been cloned and sequenced. The null allele is 10-fold increased for recombination as measured by deletion of a leu2 direct repeat.

A DrosophilaSNAP-25Null Mutant Reveals Context-Dependent Redundancy WithSNAP-24in Neurotransmission

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 259-271 ◽  
Author(s):  
Ilya Vilinsky ◽  
Bryan A Stewart ◽  
James Drummond ◽  
Iain Robinson ◽  
David L Deitcher
Keyword(s):  
Synaptic Transmission ◽  
Larval Stage ◽  
Neurotransmitter Release ◽  
Synaptic Proteins ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
F1 Progeny ◽  
Sensitive Allele ◽  
Pharate Adult

AbstractThe synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role in synaptic transmission. In this study, we performed a mutagenesis screen to identify new SNAP-25 alleles that fail to complement our previously isolated recessive temperature-sensitive allele of SNAP-25, SNAP-25ts. In a screen of 100,000 flies, 26 F1 progeny failed to complement SNAP-25ts and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25ts larvae exhibit a much stronger synaptic phenotype. Our evidence indicates that a related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution.

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae

1996 ◽  
Vol 109 (9) ◽  
pp. 2311-2318 ◽  
Author(s):  
N. Nakashima ◽  
N. Hayashi ◽  
E. Noguchi ◽  
T. Nishimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Importin Alpha ◽  
Nucleotide Exchange Factor ◽  
Cold Sensitive ◽  
Gtpase Cycle

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

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