scholarly journals Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating.

1985 ◽  
Vol 5 (4) ◽  
pp. 787-796 ◽  
Author(s):  
J Kurjan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Gene ◽  
Gene Mutations ◽  
Alpha Cells ◽  
Structural Genes ◽  
Coding Region ◽  
Considerable Decrease ◽  
Factor Production ◽  
Alpha Factor

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.

Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating

1985 ◽  
Vol 5 (4) ◽  
pp. 787-796
Author(s):  
J Kurjan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Gene ◽  
Gene Mutations ◽  
Alpha Cells ◽  
Structural Genes ◽  
Coding Region ◽  
Considerable Decrease ◽  
Factor Production ◽  
Alpha Factor

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.

scholarly journals Role of alpha-factor and the MF alpha 1 alpha-factor precursor in mating in yeast.

Genetics ◽  
1991 ◽  
Vol 127 (2) ◽  
pp. 299-307 ◽  
Author(s):  
S Caplan ◽  
J Kurjan
Keyword(s):  
Structural Gene ◽  
Insertion Mutation ◽  
Alpha Cells ◽  
Pheromone Production ◽  
Structural Genes ◽  
Wild Type ◽  
Alpha Factor ◽  
Precursor Structure ◽  
Pro Region

Abstract The peptide pheromones secreted by a and alpha cells (called a-factor and alpha-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major alpha-factor structural gene (MF alpha 1) that should result in an altered product inhibits alpha mating. These results suggested that the pheromone precursors (the MF alpha 1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of alpha-factor and the MF alpha 1 precursor in alpha mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MF alpha 1 pro region peptide but no alpha-factor could not be alleviated by addition of exogenous alpha-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce alpha-factor, but with a variety of alterations in MF alpha 1 precursor structure, mated at levels proportional to the levels of alpha-factor produced, suggesting that the only role of the alpha-factor precursor in mating is to produce alpha-factor. Both of these results argue against a role for the MF alpha 1 pro region separate from its role in alpha-factor production.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis

1986 ◽  
Vol 6 (8) ◽  
pp. 2757-2765
Author(s):  
R A Dubin ◽  
E L Perkins ◽  
R B Needleman ◽  
C A Michels
Keyword(s):  
Structural Gene ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Gene Mutations ◽  
Gene Products ◽  
Structural Genes ◽  
Maltose Permease ◽  
Maltose Fermentation ◽  
New Gene

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

scholarly journals Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

1986 ◽  
Vol 6 (8) ◽  
pp. 2757-2765 ◽  
Author(s):  
R A Dubin ◽  
E L Perkins ◽  
R B Needleman ◽  
C A Michels
Keyword(s):  
Structural Gene ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Gene Mutations ◽  
Gene Products ◽  
Structural Genes ◽  
Maltose Permease ◽  
Maltose Fermentation ◽  
New Gene

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

Protease B of Saccharomyces cerevisiae: Isolation and Regulation of the PRB1 Structural Gene

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Charles M Moehle ◽  
Martha W Aynardi ◽  
Michael R Kolodny ◽  
Frances J Park ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Structural Gene ◽  
Genomic Library ◽  
Time Lag ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Protease B ◽  
Endonuclease Mapping ◽  
Protein Molecular Weight

ABSTRACT We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (≃30,000 protein molecular weight) or the sole reported precursor (≃39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

scholarly journals Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612 ◽  
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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 Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes.

1989 ◽  
Vol 9 (10) ◽  
pp. 4507-4514 ◽  
Author(s):  
T Achstetter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Invertase Activity ◽  
Transcriptional Level ◽  
Alpha Cells ◽  
Mating Pheromone ◽  
Induced Expression ◽  
Alpha Factor ◽  
Suc2 Gene ◽  
Proteolytic Activities

Production of the mating pheromone alpha-factor was examined in Saccharomyces cerevisiae MAT alpha cells that had been exposed to the mating pheromone a-factor. A 2-h treatment with a-factor caused a significant increase in alpha-factor concentration in the medium as demonstrated by a halo assay. MF alpha 1 is one of the two genes coding for a precursor of alpha-factor. A Northern (RNA) analysis of total RNA from a-factor-treated MAT alpha cells revealed a rapid two- to threefold increase in MF alpha 1 transcript levels, reaching maximum within 60 min of exposure to the pheromone. Pheromone induction did not require ongoing protein synthesis. a-Factor-induced MF alpha 1 expression was quantitated by analysis of an MF alpha 1::SUC2 fusion gene whose product was assayed for invertase activity. Expression of the MF alpha 1::SUC2 gene in MAT alpha cells responded to the a-factor signal like the chromosomal version of MF alpha 1. Maturation of the alpha-factor precursor involves three proteolytic activities which are encoded by the KEX1, KEX2, and STE13 genes, respectively. Two of these genes, namely, KEX2 and STE13, were examined for pheromone-induced expression. Only the STE13 gene exhibited pheromone induction at the transcriptional level.

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