scholarly journals Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

2020 ◽  
Vol 8 (10) ◽  
pp. 1495
Author(s):  
Scott C. Schuyler ◽  
Lin-Ing Wang ◽  
Yi-Shan Ding ◽  
Yi-Chieh Lee ◽  
Hsin-Yu Chen
Keyword(s):  
Budding Yeast ◽  
Retention Rate ◽  
Retention Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosome ◽  
Copy Numbers ◽  
Clone Differences ◽  
Linear Chromosomes

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air

1978 ◽  
Vol 24 (6) ◽  
pp. 637-642 ◽  
Author(s):  
K. C. Thomas ◽  
Mary Spencer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Ethylene Production ◽  
Atp Synthesis ◽  
Yeast Cells ◽  
Yeast Culture ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Absolute Requirement ◽  
Wild Type Yeast

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.

scholarly journals Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

1990 ◽  
Vol 110 (1) ◽  
pp. 105-114 ◽  
Author(s):  
B K Haarer ◽  
S H Lillie ◽  
A E Adams ◽  
V Magdolen ◽  
W Bandlow ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Actin Polymerization ◽  
Yeast Cells ◽  
Predicted Amino Acid Sequence ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ellipsoidal Shape ◽  
Hydrolysis Of

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

scholarly journals Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 2878-2887 ◽  
Author(s):  
X Liu ◽  
J Bowen ◽  
M A Gorovsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Tetrahymena Thermophila ◽  
Yeast Species ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Ciliated Protozoan ◽  
Yeast Saccharomyces Cerevisiae ◽  
Evolutionarily Conserved ◽  
Cold Sensitive ◽  
Conserved Epitope

H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.

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 Molecular Basis for Telomere Repeat Divergence in Budding Yeast

2001 ◽  
Vol 21 (21) ◽  
pp. 7277-7286 ◽  
Author(s):  
Klaus Förstemann ◽  
Joachim Lingner
Keyword(s):  
Reverse Transcription ◽  
Budding Yeast ◽  
Repetitive Sequences ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomere Repeat ◽  
Telomere Binding Protein ◽  
Linear Chromosomes ◽  
Substrate Annealing

ABSTRACT Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3′ end. Although Saccharomyces cerevisiaecontains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3′ and 5′ regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.

scholarly journals Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle.

1990 ◽  
Vol 111 (3) ◽  
pp. 877-892 ◽  
Author(s):  
C K Raymond ◽  
P J O'Hara ◽  
G Eichinger ◽  
J H Rothman ◽  
T H Stevens
Keyword(s):  
Selection Procedure ◽  
Temperature Shift ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Strains ◽  
Type Pattern ◽  
Mother Cells ◽  
Vacuolar Protein

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins

1986 ◽  
Vol 6 (11) ◽  
pp. 3711-3721
Author(s):  
P J Schatz ◽  
L Pillus ◽  
P Grisafi ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Nucleotide Sequencing ◽  
Cell Fractionation ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Tubulin Genes

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae

1991 ◽  
Vol 11 (6) ◽  
pp. 3105-3114
Author(s):  
J Schnier ◽  
H G Schwelberger ◽  
Z Smit-McBride ◽  
H A Kang ◽  
J W Hershey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Peptide Bond ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

scholarly journals Protection and replication of telomeres in fission yeastThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2009 ◽  
Vol 87 (5) ◽  
pp. 747-758 ◽  
Author(s):  
Bettina A. Moser ◽  
Toru M. Nakamura
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Review Process ◽  
Peer Review Process ◽  
Yeast Cells ◽  
Special Issue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosomes ◽  
Selection Of ◽  
The Way

Telomeres, the natural ends of linear chromosomes, must be protected and completely replicated to guarantee genomic stability in eukaryotic cells. However, the protected state of telomeres is not compatible with recruitment of telomerase, an enzyme responsible for extending telomeric G-rich repeats during S-phase; thus, telomeres must undergo switches from a protected state to an accessible state during the cell cycle. In this minireview, we will summarize recent advances in our understanding of proteins involved in the protection and replication of telomeres, and the way these factors are dynamically recruited to telomeres during the cell cycle. We will focus mainly on recent results from fission yeast Schizosaccharomyces pombe , and compare them with results from budding yeast Saccharomyces cerevisiae and mammalian cell studies. In addition, a model for the way in which fission yeast cells replicate telomeres will be presented.

SOME FINE STRUCTURE FEATURES OF MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE

1971 ◽  
Vol 13 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Ellen Rapport
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fine Structure ◽  
Light Microscopy ◽  
Budding Yeast ◽  
Nuclear Organization ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Lipid Granules ◽  
Meiosis I

Fine structure features of yeast cells in meiosis are reported. Cytoplasmic and nuclear organization are compared during budding, meiosis I and meiosis II. Spindle plaques previously reported in budding yeast are identified during meiosis and the morphology of the plaques in meiosis I and II is illustrated. The dimunition of the vacuole and the increase in the number of lipid, granules in sporulating yeast, known from light microscopy, is confirmed.

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