scholarly journals Stably denatured regions in chromosomal DNA from the cdc2 Saccharomyces cerevisiae cell cycle mutant.

1983 ◽  
Vol 3 (9) ◽  
pp. 1665-1669 ◽  
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Early Stage ◽  
Dna Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Cdc2 Gene ◽  
Initiation Of Dna Replication

DNA isolated from Saccharomyces cerevisiae strains carrying temperature-sensitive mutations in the CDC2 gene after incubation at the restrictive temperature contains multiple stably denatured regions 200 to 700 base pairs long. These regions are probably stabilized by a DNA-binding protein. They are found in both replicated and unreplicated portions of DNA molecules, suggesting that they are not an early stage in the initiation of DNA replication.

Stably denatured regions in chromosomal DNA from the cdc2 Saccharomyces cerevisiae cell cycle mutant

1983 ◽  
Vol 3 (9) ◽  
pp. 1665-1669
Author(s):  
M N Conrad ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Early Stage ◽  
Dna Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Cdc2 Gene ◽  
Initiation Of Dna Replication

DNA isolated from Saccharomyces cerevisiae strains carrying temperature-sensitive mutations in the CDC2 gene after incubation at the restrictive temperature contains multiple stably denatured regions 200 to 700 base pairs long. These regions are probably stabilized by a DNA-binding protein. They are found in both replicated and unreplicated portions of DNA molecules, suggesting that they are not an early stage in the initiation of DNA replication.

scholarly journals MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (2) ◽  
pp. 387-403 ◽  
Author(s):  
M T Brown ◽  
L Goetsch ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
In Vitro Mutagenesis ◽  
Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

scholarly journals Residual cell division measurements are unreliable as indicators of the timing of events in the Saccharomyces cerevisiae cell cycle

1983 ◽  
Vol 64 (1) ◽  
pp. 307-322
Author(s):  
K.M. Richmond ◽  
D.H. Williamson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Temperature Shift ◽  
Growth Conditions ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Point Data

We report here an analysis of the execution point of the temperature-sensitive Saccharomyces cerevisiae cell cycle mutant, cdc27-47. When a logarithmically growing culture was shifted from standard growth conditions (strain 27.8B growing in YEPD at 25 degrees C) to the restrictive temperature cell division ceased abruptly and reproducibly within one population doubling time, the extent of cell division indicating an execution point early in the cell cycle. Approximately 50% of stationary-phase cells were able to divide when refed with fresh medium at 37 degrees C, showing that the execution point could be passed before ‘start’. This makes the sharp cut-off in cell division difficult to explain. This difficulty was compounded by observations of the cell cycle stage at which individual cells acquired the capacity to divide at 37 degrees C. Half the cells that were budded at the time of a temperature shift-up formed three division-blocked cells, and in 11 of these 13 cases, two were descended from the original mother cell and one from the original bud. Thus, mother and daughter cells pass the execution point independently; daughters usually during G1, and mothers usually in the budded phase of the previous cycle. The sharp cut-off in cell division is therefore spurious, and a mechanism is proposed to account for it, which has implications for the interpretation of the execution points of other cdc mutants. In addition, the expression of the cdc27-47 execution point was modified by both genetic and environmental factors, being affected by carbon source, by the petite condition, and by genetic background. This illustrates the difficulties of interpreting execution point data and the dangers of extrapolation of cell cycle parameters between strains and growth conditions.

scholarly journals CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 111 (1) ◽  
pp. 131-142 ◽  
Author(s):  
A E Adams ◽  
D I Johnson ◽  
R M Longnecker ◽  
B F Sloat ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitotic Spindles ◽  
Yeast Saccharomyces Cerevisiae ◽  
Continue Growth

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.

A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5

1993 ◽  
Vol 13 (7) ◽  
pp. 4445-4457
Author(s):  
K Kitada ◽  
A L Johnson ◽  
L H Johnston ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Suppressor Gene ◽  
Amino Acid Sequence Similarity ◽  
Nucleotide Sequence Analysis ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Chromosomal Dna ◽  
Multicopy Suppressor

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

scholarly journals A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5.

1993 ◽  
Vol 13 (7) ◽  
pp. 4445-4457 ◽  
Author(s):  
K Kitada ◽  
A L Johnson ◽  
L H Johnston ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Suppressor Gene ◽  
Amino Acid Sequence Similarity ◽  
Nucleotide Sequence Analysis ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Chromosomal Dna ◽  
Multicopy Suppressor

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

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.

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.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

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