scholarly journals Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (4) ◽  
pp. 2013-2017 ◽  
Author(s):  
K J Dornfeld ◽  
D M Livingston
Keyword(s):  
Fold Increase ◽  
Mitotic Recombination ◽  
Protein Product ◽  
Wild Type ◽  
Recombination Rates ◽  
Plasmid Recombination ◽  
Gal1 Promoter ◽  
High Level ◽  
Rate Limiting ◽  
Mutant Cells

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.

Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (4) ◽  
pp. 2013-2017
Author(s):  
K J Dornfeld ◽  
D M Livingston
Keyword(s):  
Fold Increase ◽  
Mitotic Recombination ◽  
Protein Product ◽  
Wild Type ◽  
Recombination Rates ◽  
Plasmid Recombination ◽  
Gal1 Promoter ◽  
High Level ◽  
Rate Limiting ◽  
Mutant Cells

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.

Isolation of the Schizosaccharomyces pombe RAD54 homologue, rhp54+, a gene involved in the repair of radiation damage and replication fidelity

1996 ◽  
Vol 109 (1) ◽  
pp. 73-81 ◽  
Author(s):  
D.F. Muris ◽  
K. Vreeken ◽  
A.M. Carr ◽  
J.M. Murray ◽  
C. Smit ◽  
...  
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Deletion Mutant ◽  
Chromosome Loss ◽  
Recombinational Repair ◽  
Wild Type ◽  
Facs Analysis ◽  
High Level ◽  
Mutant Cells ◽  
Very High

The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homologue of the fission yeast Schizosaccharomyces pombe, rhp54+, was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67% with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells revealed that a large portion of the cells are elongated and occasionally contain aberrant nuclei. In addition, FACS analysis showed an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. Furthermore, the rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions.

Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1191-1199
Author(s):  
M Bernstein ◽  
F Kepes ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Product ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Partial Restoration ◽  
Alpha Factor ◽  
Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2435-2437 ◽  
Author(s):  
Linda M. Scott ◽  
Mike A. Scott ◽  
Peter J. Campbell ◽  
Anthony R. Green
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Mitotic Recombination ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Jak2 Mutation ◽  
Homozygous Mutant ◽  
Mixed Populations ◽  
V617f Mutation ◽  
Mutant Cells

Abstract An acquired V617F JAK2 mutation occurs in patients with polycythemia vera (PV) or essential thrombocythemia (ET). In a proportion of V617F-positive patients, mitotic recombination produces mutation-homozygous cells that come to predominate with time. However, the prevalence of homozygosity is unclear, as previous reports studied mixed populations of wild-type, V617F-heterozygous, and V617F-homozygous mutant cells. We therefore analyzed 1766 individual hematopoietic colonies from 34 patients with PV or ET in whom granulocyte sequencing demonstrated that the mutant peak did not predominate. V617F-positive erythroid burst-forming units (BFU-Es) were more frequent in patients with PV compared with patients with ET (P = .022) and, strikingly, V617F-homozygous BFU-Es were detected in all 17 patients with PV, but in none of the patients with ET (P < .001). Moreover, mutation-homozygous cells were present in 2 patients with ET after polycythemic transformation. These results demonstrate that V617F-homozygous erythroid progenitors are present in most patients with PV but occur rarely in those with ET.

scholarly journals Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (3) ◽  
pp. 1191-1199 ◽  
Author(s):  
M Bernstein ◽  
F Kepes ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Product ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Partial Restoration ◽  
Alpha Factor ◽  
Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

scholarly journals Transcriptomic analysis of astaxanthin hyper-producing Coelastrum sp. mutant obtained by chemical mutagenesis

2021 ◽  
Author(s):  
Ameerah Tharek ◽  
Shaza Eva Mohamad ◽  
Iwane Suzuki ◽  
Koji Iwamoto ◽  
Hirofumi Hara ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Beta Carotene ◽  
Transcriptomic Analysis ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Genomic Information ◽  
Green Microalga ◽  
High Level ◽  
Mutant Cells ◽  
Β Carotene

AbstractA newly isolated green microalga, Coelastrum sp. has the capability to produce and accumulate astaxanthin under various stress conditions. At present, a mutant G1-C1 of Coelastrum sp. obtained through chemical mutagenesis using ethyl methane sulfonate displayed an improvement in astaxanthin accumulation, which was 2-fold higher than that of the wild-type. However, lack of genomic information limits the understanding of the molecular mechanism that leads to a high level of astaxanthin in the mutant G1-C1. In this study, transcriptome sequencing was performed to compare the transcriptome of astaxanthin hyper-producing mutant G1-C1 and wild-type of Coelastrum sp. with respect to astaxanthin biosynthesis. This is to clarify why the mutant produced higher astaxanthin yield compared to the wild-type strain. Based on the transcriptomic analysis, the differentially expressed genes involved in astaxanthin biosynthesis were significantly upregulated in the mutant G1-C1 of Coelastrum sp. Genes coding phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase involved in β-carotene biosynthesis in the mutant cells were upregulated by 10-, 9.2-, 8.4-, and 8.7-fold, respectively. Genes coding beta-carotene ketolase and beta-carotene 3-hydroxylase involved in converting β-carotene into astaxanthin were upregulated by 7.8- and 8.0-fold, respectively. In contrast, the lycopene ε-cyclase gene was downregulated by 9.7-fold in mutant G1-C1. Together, these results contribute to higher astaxanthin accumulation in mutant G1-C1. Overall, the data in this study provided molecular insight for a better understanding of the differences in astaxanthin biosynthesis between the wild-type and mutant G1-C1 strains.

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene

1984 ◽  
Vol 4 (2) ◽  
pp. 268-275
Author(s):  
A Laughon ◽  
R Driscoll ◽  
N Wills ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory System ◽  
Wild Type ◽  
High Level Expression ◽  
Gal1 Promoter ◽  
Encoded Proteins ◽  
High Level

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.

scholarly journals Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

1984 ◽  
Vol 4 (2) ◽  
pp. 268-275 ◽  
Author(s):  
A Laughon ◽  
R Driscoll ◽  
N Wills ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory System ◽  
Wild Type ◽  
High Level Expression ◽  
Gal1 Promoter ◽  
Encoded Proteins ◽  
High Level

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.

Structure and development of ommatidia in Oncopeltus fasciatus

Development ◽  
1974 ◽  
Vol 32 (2) ◽  
pp. 337-353
Author(s):  
P. M. J. Shelton ◽  
P. A. Lawrence
Keyword(s):  
Fold Increase ◽  
Oncopeltus Fasciatus ◽  
Pigment Cells ◽  
Wild Type ◽  
Cell Recruitment ◽  
Larval Stages ◽  
Eye Growth ◽  
Cone Cells ◽  
Cell Position ◽  
Mutant Cells

The structure and development of ommatidia has been examined in Oncopeltus fasciatus (Lygaeidae). Each ommatidium is composed of 18 cells comprising four crystalline cone cells, two primary pigment cells, four secondary pigment cells and eight retinula cells. Ommatidia are of the apposition type and the retinula cells are arranged on the open rhabdomere plan. During the five larval stages the retina grows anteriorly to provide a 12-fold increase in numbers of ommatidia. Grafts of presumptive eye epidermis show that the proliferating anterior border of the developing retina is an area of epidermal cell recruitment rather than a special budding zone. Mosaic retinae were formed by grafting epidermis from near the eye of wild-type donors into the corresponding region of mutant hosts. Ommatidia which developed at the borders of the graft contained mixtures of wild-type and mutant cells in variable and unpredictable combinations. Similar mosaic structures were seen in retinae generated by irradiation at early stages of development. The suggestion that each ommatidium is clonally derived from a single epidermal stem cell is thus disproved. The frequent occurrence of mosaic ommatidia containing only one cell of a different genotype from the rest suggests that the formation of ommatidial clusters follows the main proliferative phase of eye growth. We conclude that cell determination within ommatidia is not connected with lineage but is dependant upon cell position within the developing ommatidium.

scholarly journals Geranylgeranylated Snares Are Dominant Inhibitors of Membrane Fusion

2000 ◽  
Vol 151 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Eric Grote ◽  
Misuzu Baba ◽  
Yoshinori Ohsumi ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Snare Proteins ◽  
Wild Type ◽  
Protein Receptor ◽  
High Level ◽  
Order Complexes ◽  
Mutant Cells

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide–sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone–shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.

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