scholarly journals Sli15 Associates with the Ipl1 Protein Kinase to Promote Proper Chromosome Segregation in Saccharomyces cerevisiae

1999 ◽  
Vol 145 (7) ◽  
pp. 1381-1394 ◽  
Author(s):  
Jae-hyun Kim ◽  
Jung-seog Kang ◽  
Clarence S.M. Chan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Chromosome Segregation ◽  
Genetic Interaction ◽  
Spindle Pole ◽  
Mutant Phenotype ◽  
Yeast Saccharomyces Cerevisiae ◽  
Synthetic Lethal ◽  
Mutant Cells

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole–associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.

scholarly journals Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation.

1994 ◽  
Vol 14 (7) ◽  
pp. 4731-4740 ◽  
Author(s):  
L Francisco ◽  
W Wang ◽  
C S Chan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
High Fidelity ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
Mutant Cells ◽  
Putative Protein Kinase

The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomyces cerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the later part of each cell cycle. At 26 degrees C, the permissive growth temperature, ipl1 mutant cells are defective in the recovery from a transient G2/M-phase arrest caused by the antimicrotubule drug nocodazole. In an effort to identify additional gene products that participate with the Ipl1 protein kinase in regulating chromosome segregation in yeast, a truncated version of the previously identified DIS2S1/GLC7 gene was isolated as a dosage-dependent suppressor of ipl1ts mutations. DIS2S1/GLC7 is predicted to encode a catalytic subunit (PP1C) of type 1 protein phosphatase. Overexpression of the full-length DIS2S1/GLC7 gene results in chromosome missegregation in wild-type cells and exacerbates the mutant phenotype in ipl1 cells. In addition, the glc7-1 mutation can partially suppress the ipl1-1 mutation. These results suggest that type 1 protein phosphatase acts in opposition to the Ipl1 protein kinase in vivo to ensure the high fidelity of chromosome segregation.

Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation

1994 ◽  
Vol 14 (7) ◽  
pp. 4731-4740
Author(s):  
L Francisco ◽  
W Wang ◽  
C S Chan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
High Fidelity ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
Mutant Cells ◽  
Putative Protein Kinase

The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomyces cerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the later part of each cell cycle. At 26 degrees C, the permissive growth temperature, ipl1 mutant cells are defective in the recovery from a transient G2/M-phase arrest caused by the antimicrotubule drug nocodazole. In an effort to identify additional gene products that participate with the Ipl1 protein kinase in regulating chromosome segregation in yeast, a truncated version of the previously identified DIS2S1/GLC7 gene was isolated as a dosage-dependent suppressor of ipl1ts mutations. DIS2S1/GLC7 is predicted to encode a catalytic subunit (PP1C) of type 1 protein phosphatase. Overexpression of the full-length DIS2S1/GLC7 gene results in chromosome missegregation in wild-type cells and exacerbates the mutant phenotype in ipl1 cells. In addition, the glc7-1 mutation can partially suppress the ipl1-1 mutation. These results suggest that type 1 protein phosphatase acts in opposition to the Ipl1 protein kinase in vivo to ensure the high fidelity of chromosome segregation.

In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation

1991 ◽  
Vol 11 (10) ◽  
pp. 5212-5221
Author(s):  
B Jehn ◽  
R Niedenthal ◽  
J H Hegemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Artificial Chromosome ◽  
Complete Information ◽  
Saturation Mutagenesis ◽  
Chromosome Loss ◽  
Single Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Base

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

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.

The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae

1978 ◽  
Vol 30 (1) ◽  
pp. 331-352 ◽  
Author(s):  
B. Byers ◽  
K. Shriver ◽  
L. Goetsch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Structural Differentiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Microtubule Polymerization ◽  
Spindle Pole Bodies ◽  
Osmotic Lysis

The spindle poles of the budding yeast, Saccharomyces cerevisiae, have been removed from mitotic and meiotic cells by osmotic lysis of spheroplasts. The spindle pole bodies (SPBs)—diskoidal structures also termed ‘spindle plaques’—have been analysed for their ability to potentiate the polymerization of microtubules in vitro. Free SPBs were completely deprived of any detectable native microtubules by incubation in the absence of added tubulin and were then challenged with chick neurotubulin, which had been rendered partially defective in self-initiation of repolymerization. Electron microscopy revealed that these SPBs served as foci for the initiation of microtubule polymerization in vitro. Because the attached microtubules elongated linearly with time but did not increase in numbers after the first stage of the reaction, it is apparent that there are a limited number of sites for initiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysis of protein but not of DNA. The microtubule end proximal to the site of initiation on the SPB is distinguished by a ‘closed’ appearance because of a terminal component which is continuous with the microtubule wall, whereas the distal end has the ‘open’ appearance characteristic of freely repolymerized neurotubules. SPBs which were partially purified on sucrose gradients retained their ability to initiate the assembly of microtubules with the same structural differentiation of their ends. The occurrence of closed proximal ends on native yeast microtubules suggests that closed ends may play a role in the initiation of microtubule polymerization in vivo, as well as in vitro.

scholarly journals In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation.

1991 ◽  
Vol 11 (10) ◽  
pp. 5212-5221 ◽  
Author(s):  
B Jehn ◽  
R Niedenthal ◽  
J H Hegemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Artificial Chromosome ◽  
Complete Information ◽  
Saturation Mutagenesis ◽  
Chromosome Loss ◽  
Single Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Base

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

scholarly journals Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1.

1995 ◽  
Vol 15 (11) ◽  
pp. 6064-6074 ◽  
Author(s):  
H Y Tung ◽  
W Wang ◽  
C S Chan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Site Directed Mutagenesis ◽  
Protein Phosphatase 1 ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Phenotype

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167 ◽  
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

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