scholarly journals A simple, cheap, and robust protocol for the identification of mating type in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Samantha D. M. Arras ◽  
Lucy Mitsugi-McHattie ◽  
Matthew A. Woods ◽  
Charlotte E. Johnson ◽  
Sylvie Hermann-Le Denmat ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Genetic System ◽  
Rapid Identification ◽  
Culture Vessel ◽  
Diploid Strain ◽  
Liquid Media ◽  
Opposite Mating Type ◽  
Specialized Equipment ◽  
Cell Densities

AbstractSaccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward as long as the mating type and ploidy of the strains are known. Haploid S. cerevisiae cells are either MATa or MATα mating type. Several techniques can be used to determine mating type (or ploidy), but are typically time-consuming, require specialized components, and/or the results are inconsistent and transient. Here we validated a simple, cheap and robust method to enable rapid identification of S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they creep up culture vessel sides, a phenotype that can easily be detected visually. In contrast, mixtures of cells of the same mating type or with a diploid strain(s) simply settle out. The method does not require specialized equipment, and is robust to different media, cell densities, temperatures and strains. It can be performed in 96-well plates, and the phenotype is observable for several days. The simplicity and robustness of this method makes it ideal for routine verification of S. cerevisiae mating type, and it could be used to screen for genes underlying the creeping phenotype.

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 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 MUTANTS OF THE KILLER PLASMID OF SACCHAROMYCES CEREVISIAE DEPENDENT ON CHROMOSOMAL DIPLOIDY FOR EXPRESSION AND MAINTENANCE

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 273-285
Author(s):  
Reed B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Mating Type ◽  
Chromosomal Gene ◽  
Wild Type ◽  
Opposite Mating Type ◽  
Diploid Clone ◽  
Wild Type Phenotype ◽  
Chromosomal Genes ◽  
Killer Plasmid

ABSTRACT Mutants of the killer plasmid of Saccharomyecs cerevisiaehave been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.—Haploids carrying these mutant plasmids (called d for diploid-dependent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K+R+) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.—Sporulation of the phenotypically K+R+ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.

Physical monitoring of mating type switching in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2342-2349 ◽  
Author(s):  
B Connolly ◽  
C I White ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mating Type ◽  
G1 Phase ◽  
Opposite Mating Type ◽  
Synthesis Inhibitor ◽  
Mating Type Switching ◽  
G2 Phase ◽  
Kinetics Of ◽  
Ho Gene

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

scholarly journals Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones.

1979 ◽  
Vol 80 (2) ◽  
pp. 326-333 ◽  
Author(s):  
J S Tkacz ◽  
V L MacKay
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Mating Type ◽  
Mating Types ◽  
Opposite Mating Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Cell Surface ◽  
Cell Surface Changes ◽  
Surface Changes ◽  
In Cells

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.

scholarly journals Physical monitoring of mating type switching in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (6) ◽  
pp. 2342-2349 ◽  
Author(s):  
B Connolly ◽  
C I White ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mating Type ◽  
G1 Phase ◽  
Opposite Mating Type ◽  
Synthesis Inhibitor ◽  
Mating Type Switching ◽  
G2 Phase ◽  
Kinetics Of ◽  
Ho Gene

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

Improved media for testing the mating reaction and genetic complementation of Ustilago hordei

1992 ◽  
Vol 70 (4) ◽  
pp. 788-793 ◽  
Author(s):  
Alfredo D. Martinez-Espinoza ◽  
Karla J. Dugan ◽  
Michael E. Bjarko ◽  
John E. Sherwood
Keyword(s):  
Mating Type ◽  
Activated Charcoal ◽  
Rapid Identification ◽  
Sexual Cycle ◽  
Mating Test ◽  
Opposite Mating Type ◽  
Mating Reaction ◽  
Ustilago Hordei ◽  
Key Words Barley ◽  
The Stability

The sexual cycle of Ustilago hordei, which results in the formation of teliospores, requires growth on its barley host for completion. However, the early steps of mating, including conjugation and the formation of dikaryotic mycelium, can occur on artificial media. The addition of activated charcoal to a variety of media enhanced the stability and intensity of the mating reaction as measured by mycelium formation. The incubation time at which the strongest mating reaction occurred was also reduced. The dikaryotic nature of the mycelia that resulted from mating on charcoal-containing media was confirmed by fluorescence microscopy. Complementation assays using minimal medium containing activated charcoal demonstrated allelism of mutations in auxotrophic sporidial strains of opposite mating type. The ease and reliability of this mating test allow for rapid identification of the mating type of unknown isolates and progeny of crosses, as well as providing a dependable procedure for performing complementation tests. Key words: barley, covered smut, Hordeum vulgare, mating type.

Sign in / Sign up

Export Citation Format

Share Document