3'-end-forming signals of yeast mRNA.

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

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

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

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836-7849.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

Download Full-text

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals—Illustrated with Four Actin Cytoskeleton Proteins

Cells ◽

10.3390/cells9030672 ◽

2020 ◽

Vol 9 (3) ◽

pp. 672 ◽

Cited By ~ 1

Author(s):

Zain Akram ◽

Ishtiaq Ahmed ◽

Heike Mack ◽

Ramandeep Kaur ◽

Richard C. Silva ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Molecular Mechanisms ◽

Budding Yeast ◽

Animal Cell ◽

Molecular Genetic ◽

In Vivo Studies ◽

Yeast Saccharomyces Cerevisiae ◽

Higher Eukaryotes

The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: (1) yeast Hof1p/mammalian PSTPIP1, (2) yeast Rvs167p/mammalian BIN1, (3) yeast eEF1A/eEF1A1 and eEF1A2 and (4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.

Download Full-text

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2482 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2482-2489 ◽

Cited By ~ 27

Author(s):

B J Andrews ◽

M McLeod ◽

J Broach ◽

P D Sadowski

Keyword(s):

Saccharomyces Cerevisiae ◽

Core Region ◽

Target Sequence ◽

Repeat Elements ◽

Flp Recombinase ◽

Target Sequences ◽

2 Micron Plasmid ◽

A Site

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.

Download Full-text

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849 ◽

Cited By ~ 32

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.1999 ◽

1995 ◽

Vol 15 (4) ◽

pp. 1999-2009 ◽

Cited By ~ 71

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.

Download Full-text

The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae

Journal of Cell Science ◽

10.1242/jcs.30.1.331 ◽

1978 ◽

Vol 30 (1) ◽

pp. 331-352 ◽

Cited By ~ 1

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.

Download Full-text