Inorganic Polyphosphates and Exopolyphosphatases in Cell Compartments of the Yeast Saccharomyces cerevisiae Under Inactivation of PPX1 and PPN1 Genes

2006 ◽  
Vol 26 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Lidiya Lichko ◽  
Tatyana Kulakovskaya ◽  
Nikolai Pestov ◽  
Igor Kulaev
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Parent Strain ◽  
Mitochondrial Fraction ◽  
Chain Elongation ◽  
Inorganic Polyphosphates ◽  
Considerable Decrease ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Compartments ◽  
Chain Lengths

Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80–90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ∼40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.

scholarly journals Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2583-2592 ◽  
Author(s):  
C C Dykstra ◽  
K Kitada ◽  
A B Clark ◽  
R K Hamatake ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intragenic Recombination ◽  
Temperature Sensitive ◽  
Strand Transfer ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Prolonged Incubation ◽  
Transfer Protein ◽  
Dna Strand

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

scholarly journals Progression Into the First Meiotic Division Is Sensitive to Histone HN-HZB Dimer Concentration in Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 647-659
Author(s):  
Kochung Tsui ◽  
Lee Simon ◽  
David Norris
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Division ◽  
Expression Patterns ◽  
Spindle Pole ◽  
Chain Elongation ◽  
Histone H2a ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Pole Bodies ◽  
Dna Chain ◽  
Mitosis And Meiosis

The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2. Diploid strains lacking HTA1-HTB1 (hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate. This sporulation phenotype results from a partial depletion of H2A-H2B dimers. Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers. After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division. The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules. The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50Δ, or rad9Δ mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation. We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions.

scholarly journals Participation of the HIM1 gene of yeast Saccharomyces cerevisiae in the error-free branch of post-replicative repair and role Polη in him1-dependent mutagenesis

2020 ◽  
Author(s):  
E. A. Alekseeva ◽  
T. A. Evstyukhina ◽  
V. T. Peshekhonov ◽  
V. G. Korolev
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Damage Tolerance ◽  
Dna Damage Tolerance ◽  
Uv Mutagenesis ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recombination Repair ◽  
Repair Pathways

Abstract In eukaryotes, DNA damage tolerance (DDT) is determined by two repair pathways, homologous repair recombination (HRR) and a pathway controlled by the RAD6-epistatic group of genes. Monoubiquitylation of PCNA mediates an error-prone pathway, whereas polyubiquitylation stimulates an error-free pathway. The error-free pathway involves components of recombination repair; however, the factors that act in this pathway remain largely unknown. Here, we report that the HIM1 gene participates in error-free DDT. Notably, inactivation RAD30 gene encoding Polη completely suppresses him1-dependent UV mutagenesis. Furthermore, data obtained show a significant role of Polη in him1-dependent mutagenesis, especially at non-bipyrimidine sites (NBP sites). We demonstrate that him1 mutation significantly reduces the efficiency of the induction expression of RNR genes after UV irradiation. Besides, this paper presents evidence that significant increase in the dNTP levels suppress him1-dependent mutagenesis. Our findings show that Polη responsible for him1-dependent mutagenesis.

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein

1988 ◽  
Vol 8 (3) ◽  
pp. 1282-1289
Author(s):  
W Haggren ◽  
D Kolodrubetz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Mobility ◽  
Hmg Proteins ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Library ◽  
Associated Proteins ◽  
Genomic Dna Library

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal

1990 ◽  
Vol 10 (4) ◽  
pp. 1399-1405
Author(s):  
A S Lewin ◽  
V Hines ◽  
G M Small
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Citrate Synthase ◽  
Glyoxylate Cycle ◽  
Mitochondrial Fraction ◽  
Yeast Cells ◽  
Mitochondrial Activity ◽  
Major Peak ◽  
Gene Encoding ◽  
Peroxisomal Targeting

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

scholarly journals Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.

1990 ◽  
Vol 10 (6) ◽  
pp. 2458-2467 ◽  
Author(s):  
R E Baker ◽  
D C Masison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Disruption ◽  
Mitotic Chromosome ◽  
Binding Protein ◽  
Chromosome Loss ◽  
Chromosome X ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unexpected Consequence ◽  
Cell Doubling Time

CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CP1. The gene codes for a protein of molecular weight 39,400. When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1. We have given the CP1 gene the genetic designation CEP1 (centromere protein 1). CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed in which most of CEP1 was deleted. Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss. An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D. Thomas, H. Cherest, and Y. Surdin-Kerjan, J. Mol. Biol. 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.

scholarly journals A Role for Ubiquitination in Mitochondrial Inheritance in Saccharomyces cerevisiae

1999 ◽  
Vol 145 (6) ◽  
pp. 1199-1208 ◽  
Author(s):  
Harold A. Fisk ◽  
Michael P. Yaffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Mitochondrial Inheritance ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Ubiquitination ◽  
Ubiquitin Ligase Activity ◽  
Mitochondrial Distribution

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

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