scholarly journals GENETIC AND MOLECULAR ANALYSIS OF THE GAL3 GENE IN THE EXPRESSION OF THE GALACTOSE/MELIBIOSE REGULON OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1986 ◽  
Vol 113 (2) ◽  
pp. 229-246
Author(s):  
Timothy E Torchia ◽  
James E Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Null Allele ◽  
Northern Blot Analysis ◽  
Wild Type Strain ◽  
Adaptation Period ◽  
Wild Type ◽  
Naturally Occurring ◽  
Type Strains ◽  
Rate Of Accumulation

ABSTRACT During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.

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.

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.

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 Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 321-326 ◽  
Author(s):  
H Mitsuzawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Slow Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Triple Mutant ◽  
Apparent Absence ◽  
Camp Pathway ◽  
Naturally Occurring ◽  
Elevated Activity ◽  
Growth Phenotype

Abstract The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

scholarly journals Influences of Histone Stoichiometry on the Target Site Preference of Retrotransposons Ty1 and Ty2 in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 761-776 ◽  
Author(s):  
Lori A Rinckel ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Target Site ◽  
Site Preference ◽  
Wild Type ◽  
Histone Proteins ◽  
Protein Levels ◽  
In The Wild ◽  
Type Strains ◽  
Orientation Bias

Abstract In Saccharomyces cerevisiae, the target site specificity of the retrotransposon Ty1 appears to involve the Ty integration complex recognizing chromatin structures. To determine whether changes in chromatin structure affect Ty1 and Ty2 target site preference, we analyzed Ty transposition at the CAN1 locus in mutants containing altered levels of histone proteins. A Δhta1-htb1 mutant with decreased levels of H2A and H2B histone proteins showed a pattern of Ty1 and Ty2 insertions at CAN1 that was significantly different from that of both the wild-type and a Δhta2-htb2 mutant, which does not have altered histone protein levels. Altered levels of H2A and H2B proteins disrupted a dramatic orientation bias in the CAN1 promoter region. In the wild-type strains, few Ty1 and Ty2 insertions in the promoter region were oriented opposite to the direction of CAN1 transcription. In the Δhta1-htb1 background, however, numerous Ty1 and Ty2 insertions were in the opposite orientation clustered within the TATA region. This altered insertion pattern does not appear to be due to a bias caused by selecting canavanine resistant isolates in the different HTA1-HTB1 backgrounds. Our results suggest that reduced levels of histone proteins alter Ty target site preference and disrupt an asymmetric Ty insertion pattern.

Development of mutants of Gliocladium virens tolerant to benomyl

1990 ◽  
Vol 36 (7) ◽  
pp. 484-489 ◽  
Author(s):  
G. C. Papavizas ◽  
D. P. Roberts ◽  
K. K. Kim
Keyword(s):  
Carbon Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Sclerotium Rolfsii ◽  
Wild Type ◽  
Gliocladium Virens ◽  
Rhizosphere Competence ◽  
Filter Paper Cellulase ◽  
Type Strains

Aqueous suspensions of conidia of Gliocladium virens strains Gl-3 and Gl-21 were exposed to both ultraviolet radiation and ethyl methanesulfonate. Two mutants of Gl-3 and three of Gl-21 were selected for tolerance to benomyl at 10 μg∙mL−1, as indicated by growth and conidial germination on benomyl-amended potato dextrose agar. The mutants differed considerably from their respective wild-type strains in appearance, growth habit, sporulation, carbon-source utilization, and enzyme activity profiles. Of 10 carbon sources tested, cellobiose, xylose, and xylan were the best for growth, galactose and glucose were intermediate, and arabinose, ribose, and rhamnose were poor sources of carbon. The wild-type strains and the mutants did not utilize cellulose as the sole carbon source for growth. Two benomyl-tolerant mutants of Gl-3 produced less cellulase (β-1,4-glucosidase, carboxymethylcellulase, filter-paper cellulase) than Gl-3. In contrast, mutants of Gl-21 produced more cellulase than the wild-type strain. Only Gl-3 provided control of blight on snapbean caused by Sclerotium rolfsii. Wild-type strain Gl-21 and all mutants from both strains were ineffective biocontrol agents. Key words: Gliocladium, benomyl tolerance, Sclerotium, rhizosphere competence.

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 Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Najme Gord Noshahri ◽  
Jamshid Fooladi ◽  
Ulrike Engel ◽  
Delphine Muller ◽  
Michaela Kugel ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Colorimetric Assay ◽  
Protein Band ◽  
Wild Type ◽  
Direct Identification ◽  
Native Page ◽  
Crude Extracts ◽  
Amino Donor ◽  
Bioreactor System ◽  
Type Strains

Abstractω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation

1989 ◽  
Vol 9 (9) ◽  
pp. 3869-3877
Author(s):  
P A Bricmont ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activation ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Product Analysis ◽  
Cis Acting ◽  
Induction Sequence ◽  
Basal Level Expression ◽  
Activation Sequence

The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid. Induced expression requires a functional DAL81 gene product. Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer. The UIS element mediates inhibition of URS-mediated function when inducer is present. We cloned and characterized the DAL81 gene and identified the element with which it was associated. The gene was found to encode a rare 3.2-kilobase-pair mRNA. The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression. Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions. These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available.

scholarly journals Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1805-1812 ◽  
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.

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