scholarly journals Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

1998 ◽  
Vol 9 (9) ◽  
pp. 2393-2405 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Masaoki Kawasumi ◽  
Marie Fujino ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Cdk Inhibitor ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cdk Phosphorylation ◽  
Cdc28 Kinase

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

scholarly journals Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

2006 ◽  
Vol 399 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Chantelle Sedgwick ◽  
Matthew Rawluk ◽  
James Decesare ◽  
Sheetal Raithatha ◽  
James Wohlschlegel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phase Boundary ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb–Cdc28 inhibitor Sic1. In proliferating cells Cln–Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCFCdc4 and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCFCdc4 was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCFCdc4. In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

scholarly journals Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1805-1812 ◽  
Author(s):  
J Zhu ◽  
R H Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Aberrations ◽  
Topoisomerase I ◽  
Wild Type Strain ◽  
Hot Spot ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4215-4229
Author(s):  
S Heidmann ◽  
B Obermaier ◽  
K Vogel ◽  
H Domdey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Adh1 Gene ◽  
Polyadenylation Sites ◽  
Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

scholarly journals A new class of histone H2A mutations in Saccharomyces cerevisiae causes specific transcriptional defects in vivo.

1995 ◽  
Vol 15 (4) ◽  
pp. 1999-2009 ◽  
Author(s):  
J N Hirschhorn ◽  
A L Bortvin ◽  
S L Ricupero-Hovasse ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Activation Function ◽  
Histone H2a ◽  
Yeast Saccharomyces Cerevisiae ◽  
New Class ◽  
Suc2 Promoter ◽  
Encoding Gene ◽  
Transcription Activation Function

Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N- and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.

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.

Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

scholarly journals Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743 ◽  
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

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