scholarly journals Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

1982 ◽  
Vol 2 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R K Chan ◽  
C A Otte
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Solid Medium ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Opposite Mating Type ◽  
Alpha Factor ◽  
Complementation Groups ◽  
Chromosome Iii ◽  
The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones

1982 ◽  
Vol 2 (1) ◽  
pp. 11-20
Author(s):  
R K Chan ◽  
C A Otte
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Solid Medium ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Opposite Mating Type ◽  
Alpha Factor ◽  
Complementation Groups ◽  
Chromosome Iii ◽  
The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

scholarly journals Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (2) ◽  
pp. 657-668 ◽  
Author(s):  
X Wu ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Strand Break ◽  
Normal Donor ◽  
Alpha Cells ◽  
Cis Acting ◽  
Mating Type Switching ◽  
Chromosome Iii ◽  
The Right ◽  
Donor Preference

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.

scholarly journals Mutations affecting donor preference during mating type interconversion in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 139 (4) ◽  
pp. 1495-1510 ◽  
Author(s):  
K S Weiler ◽  
L Szeto ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mating Type ◽  
Null Allele ◽  
Alpha Cells ◽  
Opposite Mating Type ◽  
Donor Preference ◽  
Donor Locus ◽  
Type Information ◽  
Selection Of

Abstract Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to alpha or alpha to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MAT alpha cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MAT alpha background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.

scholarly journals A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure

2019 ◽  
Author(s):  
Mingguang Li ◽  
Ryan D. Fine ◽  
Manikarna Dinda ◽  
Stefan Bekiranov ◽  
Jeffrey S. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Control Region ◽  
Mating Type ◽  
Locus Control Region ◽  
A Cells ◽  
Mating Type Switching ◽  
Chromosome Iii ◽  
The Right ◽  
Donor Preference ◽  
Recombination Enhancer

AbstractThe NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We go on to demonstrate that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses the transcription of RDT1 until it is redistributed to a dsDNA break at the MAT locus induced by the HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type switching efficiency and donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating type switching, thus providing a novel model for dissecting condensin function in vivo.Author summarySir2 is a highly conserved NAD+-dependent protein deacetylase and defining member of the sirtuin protein family. It was identified about 40 years ago in the budding yeast, Saccharomyces cerevisiae, as a gene required for silencing of the cryptic mating-type loci, HML and HMR. These heterochromatic cassettes are utilized as templates for mating-type switching, whereby a programmed DNA double-strand break at the MATa or MATα locus is repaired by gene conversion to the opposite mating type. The preference for switching to the opposite mating type is called donor preference, and in MATa cells, is driven by a cis-acting DNA element called the recombination enhancer (RE). It was believed that the only role for Sir2 in mating-type switching was silencing HML and HMR. However, in this study we show that Sir2 also regulates expression of a small gene (RDT1) in the RE that is activated during mating-type switching. The promoter of this gene is also bound by the condensin complex, and deleting this region of the RE drastically changes chromosome III structure and alters donor preference. The RE therefore appears to function as a complex locus control region (LCR) that links transcriptional control to chromatin architecture, and thus provides a new model for investigating the underlying mechanistic principles of programmed chromosome architectural dynamics.

scholarly journals PRECISE MAPPING OF THE HOMOTHALLISM GENES HML AND HMR IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1980 ◽  
Vol 96 (2) ◽  
pp. 315-320
Author(s):  
Amar J S Klar ◽  
Jean McIndoo ◽  
James B Hicks ◽  
Jeffrey N Strathern
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Control Mechanism ◽  
Negative Control ◽  
Switching Function ◽  
Novel Approach ◽  
Precise Mapping ◽  
Chromosome Iii ◽  
The Right ◽  
Saccharomyces Yeasts

ABSTRACT The HML and HMR loci carry unexpressed copies of MAT  a and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).

scholarly journals Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest.

1991 ◽  
Vol 11 (2) ◽  
pp. 1030-1039 ◽  
Author(s):  
S Marcus ◽  
C B Xue ◽  
F Naider ◽  
J M Becker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Morphological Alteration ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Phenylmethylsulfonyl Fluoride ◽  
Pheromone Response ◽  
Mating Response ◽  
Time Required ◽  
Energy Dependent

Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.

Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest

1991 ◽  
Vol 11 (2) ◽  
pp. 1030-1039 ◽  
Author(s):  
S Marcus ◽  
C B Xue ◽  
F Naider ◽  
J M Becker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Morphological Alteration ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Phenylmethylsulfonyl Fluoride ◽  
Pheromone Response ◽  
Mating Response ◽  
Time Required ◽  
Energy Dependent

Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae

1981 ◽  
Vol 1 (6) ◽  
pp. 522-534
Author(s):  
B Weiffenbach ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Deoxyribonucleic Acid ◽  
Type Locus ◽  
Mating Type Switching ◽  
Lethal Event ◽  
Bona Fide ◽  
Chromosome Iii ◽  
Mat Locus ◽  
Type Information

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.

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