scholarly journals Characterizing Sterol Defect Suppressors Uncovers a Novel Transcriptional Signaling Pathway Regulating Zymosterol Biosynthesis

2005 ◽  
Vol 280 (43) ◽  
pp. 35904-35913 ◽  
Author(s):  
Melody Germann ◽  
Christina Gallo ◽  
Timothy Donahue ◽  
Reza Shirzadi ◽  
Joseph Stukey ◽  
...  
Keyword(s):  
High Temperature ◽  
Gas Chromatography Mass Spectrometry ◽  
Growth Defect ◽  
Squalene Epoxidase ◽  
Temperature Sensitive ◽  
Enzymatic Assays ◽  
Oxidosqualene Cyclase ◽  
Recessive Mutations ◽  
Complementation Groups

erg26-1ts cells harbor defects in the 4α-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1ts ets1-1 and erg26-1ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1ERG7 at their loci in erg26-1ts ets1-1 and erg26-1ts and ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.

scholarly journals Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 677-691 ◽  
Author(s):  
C S Chan ◽  
D Botstein
Keyword(s):  
Spindle Pole Body ◽  
Temperature Shift ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Chromosome Gain ◽  
Recessive Mutations ◽  
Isolation And Characterization ◽  
Complementation Groups ◽  
Bud Growth

Abstract We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

scholarly journals Characterization of mutations that suppress the temperature-sensitive growth of the hpr1 delta mutant of Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 945-956 ◽  
Author(s):  
H Y Fan ◽  
H L Klein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Lethality ◽  
Direct Repeat ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Rad Genes ◽  
Complementation Groups ◽  
Rad Mutants ◽  
Extragenic Suppressors

Abstract The hpr1 delta 3 mutant of Saccharomyces cerevisiae is temperature-sensitive for growth at 37 degrees and has a 1000-fold increase in deletion of tandem direct repeats. The hyperrecombination phenotype, measured by deletion of a leu2 direct repeat, is partially dependent on the RAD1 and RAD52 gene products, but mutations in these RAD genes do not suppress the temperature-sensitive growth phenotype. Extragenic suppressors of the temperature-sensitive growth have been isolated and characterized. The 14 soh (suppressor of hpr1) mutants recovered represent eight complementation groups, with both dominant and recessive soh alleles. Some of the soh mutants suppress hpr1 hyperrecombination and are distinct from the rad mutants that suppress hpr1 hyperrecombination. Comparisons between the SOH genes and the RAD genes are presented as well as the requirement of RAD genes for the Soh phenotypes. Double soh mutants have been analyzed and reveal three classes of interactions: epistatic suppression of hpr1 hyperrecombination, synergistic suppression of hpr1 hyperrecombination and synthetic lethality. The SOH1 gene has been cloned and sequenced. The null allele is 10-fold increased for recombination as measured by deletion of a leu2 direct repeat.

scholarly journals Dominant suppressors of yeast actin mutations that are reciprocally suppressed.

Genetics ◽  
1989 ◽  
Vol 121 (4) ◽  
pp. 675-683
Author(s):  
A E Adams ◽  
D Botstein
Keyword(s):  
High Temperature ◽  
Linkage Group ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Mutant Library ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
New Gene ◽  
Allele Specificity

Abstract A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.

scholarly journals Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype.

1985 ◽  
Vol 5 (5) ◽  
pp. 907-915 ◽  
Author(s):  
N S Atkinson ◽  
R W Dunst ◽  
A K Hopper
Keyword(s):  
Rna Processing ◽  
Elevated Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Recessive Lethal ◽  
Recessive Lethal Mutation ◽  
Sensitive Allele

The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1. We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript. On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect. This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature. A chromosomal disruption of RNA1 behaves as a recessive lethal mutation. Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene. Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination. Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5%. It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII. Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

scholarly journals A General Method for Identifying Recessive Diploid-Specific Mutations in Saccharomyces cerevisiae, Its Application to the Isolation of Mutants Blocked at Intermediate Stages of Meiotic Prophase and Characterization of a New Gene SAE2

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 797-816 ◽  
Author(s):  
Andrew H Z McKee ◽  
Nancy Kleckner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Prophase ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
New Approach ◽  
Strand Breaks ◽  
Recessive Mutations ◽  
New Genes ◽  
New Gene

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11). Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S, and mutations in three new genes designated SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S). Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks. These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.

scholarly journals Cycloheximide-resistant temperature-sensitive lethal mutations of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (2) ◽  
pp. 303-315
Author(s):  
J H McCusker ◽  
J E Haber
Keyword(s):  
Inhibitory Concentration ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Lethal Mutations ◽  
New Methods ◽  
Similar Temperature ◽  
Complementation Groups ◽  
Wild Type Yeast

Abstract We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37 degrees), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5 degrees). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37 degrees but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.

TEMPERATURE-SENSITIVE LETHAL MUTATIONS ON YEAST CHROMOSOME I APPEAR TO DEFINE ONLY A SMALL NUMBER OF GENES

Genetics ◽  
1984 ◽  
Vol 108 (1) ◽  
pp. 67-90
Author(s):  
David B Kaback ◽  
Paul W Oeller ◽  
H Yde Steensma ◽  
Janet Hirschman ◽  
Diane Ruezinsky ◽  
...  
Keyword(s):  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recessive Mutations ◽  
Large Numbers ◽  
Complementation Groups ◽  
Lethal Allele ◽  
Available Information ◽  
Chromosome I ◽  
Ts Mutations

ABSTRACT A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts-) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60-100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.

Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype

1985 ◽  
Vol 5 (5) ◽  
pp. 907-915
Author(s):  
N S Atkinson ◽  
R W Dunst ◽  
A K Hopper
Keyword(s):  
Rna Processing ◽  
Elevated Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Recessive Lethal ◽  
Recessive Lethal Mutation ◽  
Sensitive Allele

The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1. We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript. On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect. This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature. A chromosomal disruption of RNA1 behaves as a recessive lethal mutation. Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene. Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination. Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5%. It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII. Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

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