Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.

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The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.409 ◽

1990 ◽

Vol 10 (1) ◽

pp. 409-412 ◽

Cited By ~ 12

Author(s):

G P Livi ◽

J B Hicks ◽

A J Klar

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Transcriptional Control ◽

Gene Mutations ◽

State Level ◽

Mating Type Gene ◽

Gene Transcripts ◽

Mating Type Genes ◽

Type Gene ◽

Type Information

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.

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Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4441-4452.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4441-4452

Author(s):

M Marshall ◽

D Mahoney ◽

A Rose ◽

J B Hicks ◽

J R Broach

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Position Effect ◽

Protein Activity ◽

Multicomponent Complex ◽

Mating Type Genes ◽

Chromosome Iii ◽

Covalently Linked ◽

High Level ◽

Mat Locus

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

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RAD52-INDEPENDENT MITOTIC GENE CONVERSION IN SACCHAROMYCES CEREVISIAE FREQUENTLY RESULTS IN CHROMOSOMAL LOSS

Genetics ◽

10.1093/genetics/111.1.7 ◽

1985 ◽

Vol 111 (1) ◽

pp. 7-22

Author(s):

James E Haber ◽

Mark Hearn

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Conversion ◽

Mitotic Recombination ◽

Gene Conversion Event ◽

Intragenic Recombination ◽

Wild Type ◽

Conversion Event ◽

Chromosomal Loss ◽

Mitotic Gene Conversion ◽

Striking Effect

ABSTRACT We have examined spontaneous, interchromosomal mitotic recombination events between his4 alleles in both Rad+ and rad52 strains of Saccharomyces cerevisiae. In Rad+ strains, 74% of the His+ prototrophs resulted from gene conversion events without exchange of flanking markers. In diploids homozygous for the rad52-1 mutation, the frequency of His+ prototroph formation was less than 5% of the wild-type value, and more than 80% of the gene conversion events were accompanied by an exchange of flanking markers. Most of the rad52 intragenic recombination events arose by gene conversion accompanied by an exchange of flanking markers and not by a simple reciprocal exchange between the his4A and his4C alleles. There were also profound effects on the kinds of recombinant products that were recovered. The most striking effect was that RAD52-independent mitotic recombination frequently results in the loss of one of the two chromosomes participating in the gene conversion event.

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The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.409-412.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 409-412

Author(s):

G P Livi ◽

J B Hicks ◽

A J Klar

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Transcriptional Control ◽

Gene Mutations ◽

State Level ◽

Mating Type Gene ◽

Gene Transcripts ◽

Mating Type Genes ◽

Type Gene ◽

Type Information

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.

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Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4441 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4441-4452 ◽

Cited By ~ 48

Author(s):

M Marshall ◽

D Mahoney ◽

A Rose ◽

J B Hicks ◽

J R Broach

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Position Effect ◽

Protein Activity ◽

Multicomponent Complex ◽

Mating Type Genes ◽

Chromosome Iii ◽

Covalently Linked ◽

High Level ◽

Mat Locus

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

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Structure and function of mating type genes in Cochliobolus spp. and asexual fungi

Canadian Journal of Botany ◽

10.1139/b95-322 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 778-783 ◽

Cited By ~ 19

Author(s):

B. Gillian Turgeon ◽

Amir Sharon ◽

Stefan Wirsel ◽

Kenichi Yamaguchi ◽

Solveig K. Christiansen ◽

...

Keyword(s):

Mating Type ◽

Opposite Mating Type ◽

Asexual Fungi ◽

Mating Type Genes ◽

Type Gene ◽

And Function ◽

Mat Locus ◽

Mat Genes ◽

Mat Gene ◽

Transgenic Strains

Mating type (MAT) genes of Cochliobolus heterostrophus have homologs in other heterothallic Cochliobolus spp., in homothallic Cochliobolus spp., and in asexual fungi thought to be taxonomically related to Cochliobolus (e.g., Bipolaris spp.). To examine the cause of asexuality in B. sacchari, its homolog of C. heterostrophus MAT-2 was cloned. The B. sacchari sequence was 98% identical to that of C. heterostrophus MAT-2, the gene conferred homothallism when expressed in a C. heterostrophus MAT-1 strain, and transgenic strains mated with C. heterostrophus MAT-1. Thus the cause of asexuality in B. sacchari is not absence or lack of a functional MAT gene. When the C. heterostrophus MAT genes were expressed in B. sacchari, however, no sexual development occurred, suggesting that this asexual fungus lacks an attribute, other than the mating type gene, which is required for mating. Although cloned MAT genes function upon transformation into recipient strains, they do not confer full fertility. When an homologous or heterologous (e.g., from C. carbonum, C. victoriae, or B. sacchari) MAT gene is transferred into a C. heterostrophus strain of opposite mating type, the strain can self and cross to tester strains of either mating type. However, any transgenic strain carrying both a resident MAT gene and an homologous or heterologous MAT transgene develops normal perithecia but few ascospores in a cross that requires function of the transgene. To determine if the resident MAT gene interferes with function of the transgene, the MAT locus was deleted from the genome of C. heterostrophus and then replaced with the MAT gene of C. heterostrophus, C. carbonum, C. victoriae, or B. sacchari. Interference was eliminated and abundant ascospores were formed when the four transgenic strains were crossed to C. heterostrophus strains of opposite mating type. Key words: asexual fungi, DNA-binding proteins, heterologous expression, transformation.

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Donor locus selection during Saccharomyces cerevisiae mating type interconversion responds to distant regulatory signals.

Genetics ◽

10.1093/genetics/132.4.929 ◽

1992 ◽

Vol 132 (4) ◽

pp. 929-942

Author(s):

K S Weiler ◽

J R Broach

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Positional Information ◽

Yeast Saccharomyces Cerevisiae ◽

Preferential Selection ◽

Locus Selection ◽

Chromosome Iii ◽

Mat Locus ◽

Donor Locus ◽

Selection Of

Abstract Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.

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Deletion of the Schizophyllum commune Aα Locus: The Roles of Aα Y and Z Mating-Type Genes

Genetics ◽

10.1093/genetics/144.4.1437 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1437-1444

Author(s):

C Ian Robertson ◽

Kirk A Bartholomew ◽

Charles P Novotny ◽

Robert C Ullrich

Keyword(s):

Mating Type ◽

Sexual Development ◽

Schizophyllum Commune ◽

Mating Types ◽

Developmental Pathway ◽

Mating Type Gene ◽

Mating Type Genes ◽

Regulatory Loci ◽

Type Gene

The Aα locus is one of four master regulatory loci that determine mating type and regulate sexual development in Schizophyllum commune. We have made a plasmid containing a URA1 gene disruption of the Aα Y1 gene. Y1 is the sole Aα gene in Aα1 strains. We used the plasmid construction to produce an Aα null (i.e., AαΔ) strain by replacing the genomic Y1 gene with URA1 in an Aα1 strain. To characterize the role of the Aα genes in the regulation of sexual development, we transformed various Aα Y and Z alleles into AαΔ strains and examined the acquired mating types and mating abilities of the transformants. These experiments demonstrate that the Aα Y gene is not essential for fungal viability and growth, that a solitary Z Aα mating-type gene does not itself activate development, that Aβ proteins are sufficient to activate the A developmental pathway in the absence of Aα proteins and confirm that Y and Z genes are the sole determinants of Aα mating type. The data from these experiments support and refine our model of the regulation of A-pathway events by Y and Z proteins.

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Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.688 ◽

1986 ◽

Vol 6 (2) ◽

pp. 688-702 ◽

Cited By ~ 144

Author(s):

J M Ivy ◽

A J Klar ◽

J B Hicks

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Blot Analysis ◽

Transcriptional Control ◽

Genomic Library ◽

Yeast Saccharomyces Cerevisiae ◽

Mating Type Genes ◽

Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

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