scholarly journals The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1493-1502
Author(s):  
Richard D Gardner ◽  
Atasi Poddar ◽  
Chris Yellman ◽  
Penny A Tavormina ◽  
M Cristina Monteagudo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Spindle Checkpoint ◽  
Essential Genes ◽  
Gene Fusions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Signaling ◽  
One Step ◽  
Diploid Cells ◽  
Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3992-3998
Author(s):  
A M Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Yeast Cells ◽  
Specific Gene ◽  
Activity Levels ◽  
Sporulation Medium ◽  
Mrna Levels ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae

2020 ◽  
Vol 104 (7) ◽  
pp. 3037-3047
Author(s):  
Shan Yang ◽  
Xuan Cao ◽  
Wei Yu ◽  
Shengying Li ◽  
Yongjin J. Zhou
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Genes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Targeted Mutation

scholarly journals Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis

2011 ◽  
Vol 22 (14) ◽  
pp. 2448-2457 ◽  
Author(s):  
Erin L. Barnhart ◽  
Russell K. Dorer ◽  
Andrew W. Murray ◽  
Scott C. Schuyler
Keyword(s):  
Chromosome Segregation ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Chromosome Loss ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Diploid Cells ◽  
Antimicrotubule Drugs

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1–Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.

scholarly journals Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (12) ◽  
pp. 6838-6844 ◽  
Author(s):  
Y Wang ◽  
D J Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Cycle Delay ◽  
Delay Cell ◽  
Antimitotic Drugs ◽  
Checkpoint Genes ◽  
Spindle Structure ◽  
Kinetochore Function

Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 3069-3073
Author(s):  
I Yamashita ◽  
S Fukui
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Transcriptional Control ◽  
Northern Blot Analysis ◽  
Insertion Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Mating Type Genes ◽  
Diploid Cells ◽  
Glucoamylase Gene

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae

1976 ◽  
Vol 22 (2) ◽  
pp. 219-242 ◽  
Author(s):  
J.B. Peterson ◽  
H. Ris
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole ◽  
Linkage Groups ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genetic Studies ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Spindle Microtubules ◽  
Diploid Cells

Mitosis in yeast Saccharomyces cerevisiae was investigated in thick (0-25-I mum) serial sections with a high voltage electron microscope and in preparations of spheroplasts spread on a water surface. Spindle microtubules originate from a plaque-like structure called the spindle pole bosis the SPB duplicates and a set of long and short microtubules develops on each SPB. The spindle arises as the SPBs separate on the nuclear membrane adense and are not individually visible. Genetic studies, however, have indicated that there are 17 linkage groups. The number of microtubules was determined in diploid and haploid spindles on serial stereo micrographs. In diploid mitosis about 40 microtubules issue from a SPB. Most are non-continuous and often they are visibly associated with a chromatin fibre. The spindle in haploid cells is similar except that the number of microtubules is about half that in diploid cells and the SPB is smaller. The pole-to-pole microtubules vary in number from spindle to spindle, but in each case enough microtubules are present to account for each linkage group being associated with a single non-continuous microtubule. We conclude that mitosis in yeast is comparable in its general aspect to that observed in typical eukaryotes.

scholarly journals Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Atasi Poddar ◽  
P. Todd Stukenberg ◽  
Daniel J. Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
In Cells

ABSTRACT Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

scholarly journals Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

1994 ◽  
Vol 14 (9) ◽  
pp. 6306-6316 ◽  
Author(s):  
A R Butler ◽  
J H White ◽  
Y Folawiyo ◽  
A Edlin ◽  
D Gardiner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Kluyveromyces Lactis ◽  
Yeast Cells ◽  
Yeast Genome ◽  
G1 Arrest ◽  
Yeast Saccharomyces Cerevisiae ◽  
Toxin Resistance ◽  
Complementation Groups ◽  
Diploid Cells

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

scholarly journals CEP3 encodes a centromere protein of Saccharomyces cerevisiae.

1995 ◽  
Vol 128 (5) ◽  
pp. 749-760 ◽  
Author(s):  
A V Strunnikov ◽  
J Kingsbury ◽  
D Koshland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Essential Gene ◽  
Selection Procedure ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Centromere Protein ◽  
Kinetochore Function ◽  
Binding Component

We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).

scholarly journals Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (9) ◽  
pp. 3992-3998 ◽  
Author(s):  
A M Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Yeast Cells ◽  
Specific Gene ◽  
Activity Levels ◽  
Sporulation Medium ◽  
Mrna Levels ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

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