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.

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 Separable Functions of ORC5 in Replication Initiation and Silencing in Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1053-1062 ◽  
Author(s):  
Andrew Dillin ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Separable Functions ◽  
Different Origins ◽  
Replication Initiator ◽  
Origin Recognition

Origin recognition complex (ORC) is a six subunit complex that functions as the replication initiator and is required for silencing the HML and HMR loci in the yeast Saccharomyces cerevisiae. The roles of ORC5 in replication initiation and silencing were investigated to determine whether the two roles were mechanistically coincident or separable. Some spontaneous revertants of orc5-1 were functional for replication initiation, but not silencing. Other alleles of ORC5 were obtained that were nonfunctional for replication initiation, but fully competent for silencing. The two types of alleles, when put in the same cell, complemented, establishing two separable functions for ORC5. These data implied that replication initiation at HMR-E was not required for silencing. The data were consistent with a model in which different ORC species functioned at different origins within the genome and that only one Orc5p subunit functioned at any given origin.

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