scholarly journals Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation

1983 ◽  
Vol 3 (10) ◽  
pp. 1730-1737
Author(s):  
C L Kuo ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Recombinant Plasmid ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
Deletion Mapping ◽  
Nonpermissive Temperature ◽  
Recombinant Plasmids ◽  
Selection For ◽  
Cloned Gene

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.

scholarly journals Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

1983 ◽  
Vol 3 (10) ◽  
pp. 1730-1737 ◽  
Author(s):  
C L Kuo ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Recombinant Plasmid ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
Deletion Mapping ◽  
Nonpermissive Temperature ◽  
Recombinant Plasmids ◽  
Selection For ◽  
Cloned Gene

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

1982 ◽  
Vol 187 (1) ◽  
pp. 42-46 ◽  
Author(s):  
Lawrence B. Dumas ◽  
Joan P. Lussky ◽  
Elizabeth J. McFarland ◽  
Janis Shampay
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants

scholarly journals GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

1993 ◽  
Vol 13 (4) ◽  
pp. 2152-2161 ◽  
Author(s):  
P Belhumeur ◽  
A Lee ◽  
R Tam ◽  
T DiPaolo ◽  
N Fortin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Essential Gene ◽  
Negative Control ◽  
Temperature Sensitive ◽  
Vitro Assay ◽  
Gtp Binding ◽  
Genetic Suppressors ◽  
Multicopy Suppressors

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

scholarly journals Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.

1983 ◽  
Vol 3 (6) ◽  
pp. 1000-1012 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Electron Microscopic ◽  
Dna Strands ◽  
Total Dna ◽  
Random Fraction

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.

Apoptosis Was Promoted at a Nonpermissive Temperature in DNA Replication-Defective Temperature-Sensitive Mutants of Mouse FM3A Cells

1998 ◽  
Vol 238 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Yukika Yamauchi ◽  
Akiko Tanaka ◽  
Kazunori Yamaguchi ◽  
Masayuki Kobayashi ◽  
Seiichi Shimamura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Fm3a Cells

scholarly journals TEN1 Is Essential for CDC13-Mediated Telomere Capping

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 793-810 ◽  
Author(s):  
Ling Xu ◽  
Ruben C. Petreaca ◽  
Hovik J. Gasparyan ◽  
Stephanie Vu ◽  
Constance I. Nugent
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
High Temperatures ◽  
De Novo ◽  
Temperature Sensitive ◽  
Single Stranded Dna ◽  
Growth Defects ◽  
Telomere Capping ◽  
Telomere Replication ◽  
Essential Function

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

scholarly journals DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 365-376
Author(s):  
M E Budd ◽  
K D Wittrup ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
X Ray ◽  
Temperature Sensitive Mutants ◽  
Polymerase I ◽  
Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

scholarly journals Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

scholarly journals A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5778-5784
Author(s):  
S W Peltz ◽  
J L Donahue ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Steady State ◽  
Relative Number ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temperature Sensitive Mutants ◽  
Steady State Levels

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

scholarly journals The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression

2000 ◽  
Vol 113 (7) ◽  
pp. 1199-1211
Author(s):  
G. Buscemi ◽  
F. Saracino ◽  
D. Masnada ◽  
M.L. Carbone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Actin Cytoskeleton ◽  
Cell Cycle Progression ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Cycle Progression ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

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