Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site.

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Download Full-text

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

Eukaryotic Cell ◽

10.1128/ec.4.4.832-835.2005 ◽

2005 ◽

Vol 4 (4) ◽

pp. 832-835 ◽

Cited By ~ 13

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5212-5221.1991 ◽

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.

Download Full-text

Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.189-199.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 189-199

Author(s):

D S Pederson ◽

T Fidrych

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Transcription Initiation ◽

Heat Shock Factor ◽

Micrococcal Nuclease ◽

Nucleosome Assembly ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

Download Full-text

Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1805 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1805-1812 ◽

Cited By ~ 58

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.

Download Full-text

3'-end-forming signals of yeast mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.5983 ◽

1995 ◽

Vol 15 (11) ◽

pp. 5983-5990 ◽

Cited By ~ 58

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.

Download Full-text

Genome-Scale Analysis Reveals Sst2 as the Principal Regulator of Mating Pheromone Signaling in the Yeast Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.5.2.330-346.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 330-346 ◽

Cited By ~ 46

Author(s):

Scott A. Chasse ◽

Paul Flanary ◽

Stephen C. Parnell ◽

Nan Hao ◽

Jiyoung Y. Cha ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transition State ◽

G Protein ◽

Common Property ◽

Rgs Proteins ◽

Homologous Proteins ◽

Yeast Saccharomyces Cerevisiae ◽

Α Subunit ◽

Pheromone Signaling

ABSTRACT A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein α subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two Gα proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.

Download Full-text

Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.4.1371 ◽

1987 ◽

Vol 7 (4) ◽

pp. 1371-1377 ◽

Cited By ~ 313

Author(s):

T Toda ◽

S Cameron ◽

P Sass ◽

M Zoller ◽

J D Scott ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Cyclic Amp ◽

Carbon Sources ◽

Regulatory Subunit ◽

Dependent Protein Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Regulatory Subunits ◽

Dependent Protein

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5679-5687.1990 ◽

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.

Download Full-text

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4215-4229.1992 ◽

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.

Download Full-text