scholarly journals Tagging Morphogenetic Genes by Insertional Mutagenesis in the Yeast Yarrowia lipolytica

2001 ◽  
Vol 183 (10) ◽  
pp. 3098-3107 ◽  
Author(s):  
Mathias Richard ◽  
Raymundo Rosas Quijano ◽  
Samira Bezzate ◽  
Florence Bordon-Pallier ◽  
Claude Gaillardin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yarrowia Lipolytica ◽  
Cell Walls ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Wild Type ◽  
Calcofluor White ◽  
Hyphal Formation ◽  
Yeast Yarrowia Lipolytica

ABSTRACT The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the STEand RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y. lipolytica morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of Y. lipolytica with a Tn3 derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous toS. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5, andPPH21. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y. lipolytica and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

scholarly journals Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 535-542 ◽  
Author(s):  
B A Kunz ◽  
M G Peters ◽  
S E Kohalmi ◽  
J D Armstrong ◽  
M Glattke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutator Phenotype ◽  
In The Wild ◽  
Single Base Pair ◽  
Almost All

Abstract Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity

2020 ◽  
Vol 42 (5) ◽  
pp. 773-785 ◽  
Author(s):  
Macarena Larroude ◽  
Heykel Trabelsi ◽  
Jean-Marc Nicaud ◽  
Tristan Rossignol
Keyword(s):  
Yarrowia Lipolytica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Diversity

scholarly journals Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Rui Yao ◽  
Pei Zhou ◽  
Chengjin Wu ◽  
Liming Liu ◽  
Jing Wu
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Survival Rate ◽  
Type Strain ◽  
Mutation Frequency ◽  
Wild Type Strain ◽  
Yeast Cells ◽  
Wild Type ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

scholarly journals Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

2009 ◽  
Vol 76 (3) ◽  
pp. 670-679 ◽  
Author(s):  
Eva González ◽  
M. Rosario Fernández ◽  
Didac Marco ◽  
Eduard Calam ◽  
Lauro Sumoy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Conditions ◽  
Wild Type ◽  
Genome Expression ◽  
Whole Genome Expression ◽  
Histidine Tag ◽  
Butanediol Dehydrogenase

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

scholarly journals A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth.

1984 ◽  
Vol 99 (4) ◽  
pp. 1441-1450 ◽  
Author(s):  
H Iida ◽  
I Yahara
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Control ◽  
Protein S ◽  
Resistant Mutant ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sulfur Starvation

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced. These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56). Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.

scholarly journals From Genes to Proteins to Behavior: A Laboratory Project That Enhances Student Understanding in Cell and Molecular Biology

2009 ◽  
Vol 8 (4) ◽  
pp. 291-308 ◽  
Author(s):  
Benjamin D. Aronson ◽  
Linda A. Silveira
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Biology ◽  
Type Strain ◽  
Wild Type Strain ◽  
Student Understanding ◽  
Student Survey ◽  
Wild Type ◽  
College Biology ◽  
Introductory College Biology ◽  
Introductory College

In the laboratory, students can actively explore concepts and experience the nature of scientific research. We have devised a 5-wk laboratory project in our introductory college biology course whose aim was to improve understanding in five major concepts that are central to basic cellular, molecular biology, and genetics while teaching molecular biology techniques. The project was focused on the production of adenine in Saccharomyces cerevisiae and investigated the nature of mutant red colonies of this yeast. Students created red mutants from a wild-type strain, amplified the two genes capable of giving rise to the red phenotype, and then analyzed the nucleotide sequences. A quiz assessing student understanding in the five areas was given at the start and the end of the course. Analysis of the quiz showed significant improvement in each of the areas. These areas were taught in the laboratory and the classroom; therefore, students were surveyed to determine whether the laboratory played a role in their improved understanding of the five areas. Student survey data demonstrated that the laboratory did have an important role in their learning of the concepts. This project simulated steps in a research project and could be adapted for an advanced course in genetics.

scholarly journals Mutation of waaC, Encoding Heptosyltransferase I in Campylobacter jejuni 81-176, Affects the Structure of both Lipooligosaccharide and Capsular Carbohydrate

2006 ◽  
Vol 188 (9) ◽  
pp. 3273-3279 ◽  
Author(s):  
Margaret I. Kanipes ◽  
Erzsebet Papp-Szabo ◽  
Patricia Guerry ◽  
Mario A. Monteiro
Keyword(s):  
Campylobacter Jejuni ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Lipid A ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Gas Liquid Chromatography ◽  
Wild Type ◽  
Isolation And Characterization

ABSTRACT Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first l-glycero-d-manno-heptose residue to 3-deoxy-d-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

scholarly journals Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (2) ◽  
pp. 1278-1292 ◽  
Author(s):  
C Mezard ◽  
A Nicolas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Type Strain ◽  
Wild Type Strain ◽  
Double Strand Breaks ◽  
Linear Plasmid ◽  
Wild Type ◽  
Dsb Repair ◽  
Strand Breaks ◽  
In The Wild

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

Sign in / Sign up

Export Citation Format

Share Document