scholarly journals Two Classes of sir3 Mutants Enhance the sir1 Mutant Mating Defect and Abolish Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 509-522 ◽  
Author(s):  
Elisa M Stone ◽  
Cheryl Reifsnyder ◽  
Mitch McVey ◽  
Brandy Gazo ◽  
Lorraine Pillus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Control ◽  
Replication Initiation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Protein Sequence Analysis ◽  
Dna Replication Initiation ◽  
Sir Proteins ◽  
Telomeric Silencing

Abstract Silent information regulators, or Sir proteins, play distinct roles in chromatin-mediated transcriptional control at the silent mating-type loci, telomeres, and within the rDNA repeats of Saccharomyces cerevisiae. An unusual collection of sir3 mutant alleles was identified in a genetic screen for enhancers of the sir1 mutant mating-defective phenotype. These sir3-eso mutants, like the sir1 mutant, exhibit little or no mating defects alone, but the sir1 sir3-eso double mutants are essentially nonmating. All of the sir3-eso mutants are defective in telomeric silencing. In some mutants, this phenotype is suppressed by tethering Sir1p to telomeres; other mutants are dominant for mating and telomeric silencing defects. Additionally, several sir3-eso mutants are nonmating in combination with the nat1 N-terminal acetyltransferase mutant. The temperature-sensitive allele sir3-8 has an eso phenotype at permissive temperature, yet acts as a null allele at restrictive temperature due to loss of sir3-8 protein. Sequence analysis showed that eight of the nine sir3-eso alleles have mutations within the N-terminal region that is highly similar to the DNA replication initiation protein Orc1p. Together, these data reveal modular domains for Sir3p and further define its function in silencing chromatin.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

scholarly journals end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (1) ◽  
pp. 55-65 ◽  
Author(s):  
S Raths ◽  
J Rohrer ◽  
F Crausaz ◽  
H Riezman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Cell Surface Receptor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Fluid Phase Endocytosis ◽  
Alpha Factor

alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). After internalization, alpha-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H. Riezman. 1990. J. Cell Biol. 110:1911-1922; Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). We have isolated two mutants that are defective in the internalization process. Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect. Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-alpha-factor. The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed. Furthermore, the end4 mutation only affects alpha-factor internalization and not the later delivery of alpha-factor to the vacuole. Other vesicle-mediated processes seem to be normal in end3 and end4 mutants. END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast.

scholarly journals ARE MITOTIC FUNCTIONS REQUIRED IN MEIOSIS?

Genetics ◽  
1974 ◽  
Vol 76 (4) ◽  
pp. 745-753
Author(s):  
G Simchen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Division ◽  
Mitotic Cell ◽  
Intragenic Recombination ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitotic Cell Cycle ◽  
Permissive Temperature ◽  
Meiotic Behavior ◽  
Group I

ABSTRACT Sporulation of diploid yeasts (Saccharomyces cerevisiae), homozygous or heterozygous for temperature-sensitive mitotic cell-cycle mutations, was examined at the restrictive and permissive temperatures. Twenty genes, represented by 32 heterozygotes and 60 homozygotes, were divided into three groups, showing (i) normal sporulation, (ii) no sporulation at the restrictive temperature but normal sporulation at the permissive temperature, (iii) no sporulation at both temperatures. Group (i) as well as several other strains were tested for their meiotic behavior with regard to intragenic recombination and haploidization. The conclusion reached was that all the mitotic nuclear-division and DNA-synthesis functions were required in meiosis. The only cell-division mutations not to affect meiosis were in three cytokinesis loci and in one budemergence locus.

scholarly journals Evidence That the pre-mRNA Splicing Factor Clf1p Plays a Role in DNA Replication in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1319-1333
Author(s):  
Wenge Zhu ◽  
Irene R Rainville ◽  
Min Ding ◽  
Margaret Bolus ◽  
Nicholas H Heintz ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Initiation ◽  
Mrna Splicing ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Direct Role ◽  
Dna Replication Initiation ◽  
Single Nucleus ◽  
2C Dna Content

Abstract Clf1p is an essential, highly conserved protein in S. cerevisiae that has been implicated in pre-mRNA splicing. Clf1p's ortholog in Drosophila, Crn, is required for normal cell proliferation. Cells depleted of Clf1p arrest primarily with large buds, a single nucleus, a 2C DNA content, and a short, intact mitotic spindle. We isolated temperature-sensitive clf1 mutants that exhibit similar mitotic defects when released to the restrictive temperature from an early S-phase block. While these mutants also accumulate unspliced pre-mRNA at the restrictive temperature, the mitotic arrest does not appear to result from a failure to splice tubulin pre-mRNA. Moreover, the same mutants exhibit a delayed entry into S phase when released to the restrictive temperature from a G1 phase block. This delay could not be suppressed by disruption of the S-phase CDK inhibitor SIC1, suggesting that Clf1p is involved in DNA replication. Consistent with this possibility, we find that Clf1p (but not the mutant clf1p) interacts with the DNA replication initiation protein Orc2p in two-hybrid and co-immunoprecipitation assays, that Clf1p preferentially associates with origins of DNA replication, and that this association is Orc2p dependent. These observations suggest that Clf1p plays a direct role in the initiation of DNA replication.

scholarly journals DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360 ◽  
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

scholarly journals Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 103-118 ◽  
Author(s):  
Janet R Mullen ◽  
Vivek Kaliraman ◽  
Samer S Ibrahim ◽  
Steven J Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Protein Complexes ◽  
Ring Finger ◽  
Dna Helicase ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Helicases ◽  
Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product

1987 ◽  
Vol 7 (10) ◽  
pp. 3857-3861
Author(s):  
J Daniel ◽  
J M Becker ◽  
E Enari ◽  
A Levitzki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Gene Product ◽  
Ternary Complex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Terminal Portion ◽  
Altered Regulation ◽  
Cdc25 Protein

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

Sign in / Sign up

Export Citation Format

Share Document