scholarly journals The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle.

1994 ◽  
Vol 125 (1) ◽  
pp. 143-158 ◽  
Author(s):  
J N McMillan ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Low Temperature ◽  
Mother Cell ◽  
Significant Proportion ◽  
Nuclear Migration ◽  
Cold Sensitivity ◽  
Chain Gene ◽  
Chromosome Loss ◽  
Lacz Gene ◽  
Yeast Saccharomyces Cerevisiae

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein.

scholarly journals The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

scholarly journals GENETIC ANALYSIS OF MUTATIONS AFFECTING GROWTH OF SACCHAROMYCES CEREVISIAE AT LOW TEMPERATURE

Genetics ◽  
1974 ◽  
Vol 77 (4) ◽  
pp. 651-659
Author(s):  
Arjun Singh ◽  
T R Manney
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Low Temperature ◽  
Low Temperatures ◽  
Cold Sensitivity ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycloheximide Resistance ◽  
Control Growth ◽  
Cold Sensitive ◽  
Increased Sensitivity

ABSTRACT A large number of genes control growth of the yeast Saccharomyces cerevisiae at low temperatures (< 10°). Approximately 47 percent of the mutants selected for inability to grow at 4-5°C show increased sensitivity to cycloheximide. In 3 of 4 cases tested, supersensitivity to cycloheximide and inability to grow at the low temperature segregate together and thus appear to be effects of the same mutation. Since many cold-sensitive mutants of bacteria have been found to have altered ribosomes and since cycloheximide resistance in yeast can be caused by ribosomal changes, this suggests that the mutants having low-temperature-sensitive growth may be defective in ribosome-assembly processes at the low temperatures. Two of the lts loci, lts1 and lts3 have been located on chromosome VII and another two, lts4 and lts10 on chromosome IV. A mutation, cyh10, conferring cycloheximide resistance, but not cold sensitivity, has been located close to the centromere on chromosome II.

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.

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes

1990 ◽  
Vol 10 (1) ◽  
pp. 223-234
Author(s):  
M A Hoyt ◽  
T Stearns ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Instability ◽  
Null Alleles ◽  
Cold Sensitivity ◽  
Phenotypic Characterization ◽  
Chromosome Loss ◽  
Loss Of Function ◽  
Microtubule Stability ◽  
Chromosome Iii ◽  
Yeast Genes

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

scholarly journals Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.

1990 ◽  
Vol 10 (6) ◽  
pp. 2458-2467 ◽  
Author(s):  
R E Baker ◽  
D C Masison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Disruption ◽  
Mitotic Chromosome ◽  
Binding Protein ◽  
Chromosome Loss ◽  
Chromosome X ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unexpected Consequence ◽  
Cell Doubling Time

CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CP1. The gene codes for a protein of molecular weight 39,400. When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1. We have given the CP1 gene the genetic designation CEP1 (centromere protein 1). CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed in which most of CEP1 was deleted. Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss. An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D. Thomas, H. Cherest, and Y. Surdin-Kerjan, J. Mol. Biol. 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.

scholarly journals SUM1-1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability.

1996 ◽  
Vol 16 (8) ◽  
pp. 4281-4294 ◽  
Author(s):  
M H Chi ◽  
D Shore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Transcriptional Silencing ◽  
Chromosome Loss ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sir Proteins ◽  
Telomere Binding Protein ◽  
Single Allele ◽  
Dominant Suppressor

Transcriptional silencing in the yeast Saccharomyces cerevisiae occurs at HML and HMR mating-type loci and telomeres and requires the products of the silent information regulator (SIR) genes. Recent evidence suggests that the silencer- and telomere-binding protein Rap1p initiates silencing by recruiting a complex of Sir proteins to the chromosome, where they act in some way to modify chromatin structure or accessibility. A single allele of the SUM1gene (SUM1-1) which restores silencing at HM loci in strains mutant for any of the four SIR genes was identified a number of years ago. However, conflicting genetic results and the lack of other alleles of SUM1 made it difficult to surmise the wild-type function of SUM1 or the manner in which the SUM1-1 mutation restores silencing in sir mutant strains. Here we report the cloning and characterization of the SUM1 gene and the SUM1-1 mutant allele. Our results indicate that SUM1-1 is an unusual altered-function mutation that can bypass the need for SIR function in HM silencing and increase repression at telomeres. A sum1 deletion mutation has only minor effects on silencing in SIR strains and does not restore silencing in sir mutants. In addition to its effect on transcriptional silencing, the SUM1-1 mutation (but not a sum1 deletion) increases the rate of chromosome loss and cell death. We suggest several speculative models for the action of SUM1-1 in silencing based on these and other data.

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 Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

1997 ◽  
Vol 138 (5) ◽  
pp. 1023-1040 ◽  
Author(s):  
Todd M. DeZwaan ◽  
Eric Ellingson ◽  
David Pellman ◽  
David M. Roof
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Live Cells ◽  
Spindle Orientation ◽  
Chain Gene ◽  
Null Mutant ◽  
Bud Neck ◽  
Bud Site

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.

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