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.

scholarly journals Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 499-506
Author(s):  
A Sakai ◽  
Y Shimizu ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Temperature Sensitive ◽  
Cellular Functions ◽  
Isolation And Characterization ◽  
Gene Coding ◽  
Complementation Groups ◽  
Homozygous Diploid ◽  
Diploid Cells

Abstract We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

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 Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2583-2592 ◽  
Author(s):  
C C Dykstra ◽  
K Kitada ◽  
A B Clark ◽  
R K Hamatake ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intragenic Recombination ◽  
Temperature Sensitive ◽  
Strand Transfer ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Prolonged Incubation ◽  
Transfer Protein ◽  
Dna Strand

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

scholarly journals Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

1992 ◽  
Vol 119 (3) ◽  
pp. 583-593 ◽  
Author(s):  
R E Palmer ◽  
D S Sullivan ◽  
T Huffaker ◽  
D Koshland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mother Cell ◽  
Fold Increase ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Temperature Sensitive ◽  
Chromosome Movement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoskeletal Elements

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

scholarly journals Temperature-Sensitive Lethal Pseudorevertants of ste Mutations in Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 627-636
Author(s):  
Margaret E Katz ◽  
Jill Ferguson ◽  
Steven I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Complementation Group ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Pheromone Response ◽  
Cycle Arrest ◽  
Homogeneous Cell ◽  
Complementation Groups ◽  
Mutant Cells

ABSTRACT A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

scholarly journals Saccharomyces cerevisiae HOC1, a Suppressor of pkc1, Encodes a Putative Glycosyltransferase

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Aaron M Neiman ◽  
Vijay Mhaiskar ◽  
Vladimir Manus ◽  
Francis Galibert ◽  
Neta Dean
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Lysis ◽  
Integral Membrane Protein ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Calcofluor White ◽  
Kinase C

The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

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.

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