ABSTRACT
The organization of the genome into functional units, such as enhancers and active or repressed promoters, is associated with distinct patterns of DNA and histone modifications. The Encyclopedia of DNA Elements (ENCODE) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, identifying hundreds of thousands of potential regulatory regions and transcription factor binding sites. Part of the ENCODE consortium, GENCODE, has annotated the human genome with novel transcripts including new noncoding RNAs and pseudogenes, highlighting transcriptional complexity. Many disease variants identified in genome-wide association studies are located within putative enhancer regions defined by the ENCODE project. Understanding the principles of chromatin and epigenome organization will help to identify new disease mechanisms, biomarkers and drug targets, particularly as ongoing epigenome mapping projects generate data for primary human cell types that play important roles in disease.
Subject(s)
Chromatin/physiology , Epigenesis, Genetic/genetics , Genome, Human/genetics , Chromosome Mapping/methods , DNA Methylation/genetics , Forecasting , Histones/genetics , Human Genome Project , Humans , Regulatory Elements, Transcriptional/genetics , Transcription Factors/metabolism , Transcription, Genetic/geneticsSubject(s)
Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , DNA Methylation/drug effects , Histones/metabolism , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Acetylation/drug effects , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cells, Cultured , Epigenesis, Genetic/drug effects , Epigenomics , HumansABSTRACT
Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes
Subject(s)
Humans , Animals , Centromere , Histones , Base Sequence , Centromere , DNA, Fungal , Drosophila melanogaster , Drosophila Proteins/ultrastructure , Fungal Proteins , Histones , Saccharomyces cerevisiae , SchizosaccharomycesABSTRACT
The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.
Subject(s)
Cell Cycle Proteins , Centromere/chemistry , Centromere/ultrastructure , Evolution, Molecular , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/cytology , Centromere/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/chemistry , Chromosomes, Fungal/genetics , Chromosomes, Fungal/ultrastructure , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Heterochromatin/chemistry , Heterochromatin/genetics , Heterochromatin/ultrastructure , Humans , Kinetochores , Microscopy, Electron , Microscopy, Fluorescence , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Repetitive Sequences, Nucleic Acid/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Hrp1 of Schizosaccharomyces pombe is a member of the CHD protein family, characterized by a chromodomain, a Myb-like telobox-related DNA-binding domain and a SNF2-related helicase/ATPase domain. CHD proteins are thought to be required for modification of the chromatin structure in transcription, but the exact roles of CHD proteins are not known. Here we examine the sub-cellular localization and biochemical activity of Hrp1 and the phenotypes of hrp1 Delta and Hrp1-overexpressing strains. Fluorescence microscopy revealed that Hrp1 protein is targeted to the nucleus. We found that Hrp1 exhibited DNA-dependent ATPase activity, stimulated by both single- and double-stranded DNA. Overexpression of Hrp1 caused slow cell growth accompanied by defective chromosome condensation in anaphase resulting in a 'cut' (celluntimelytorn) phenotype and chromosome loss. The hrp1 Delta mutation also caused abnormal anaphase and mini-chromosome loss phenotypes. Electron micrographs demonstrated that aberrantly shaped nucleoli appeared in Hrp1-overexpressing cells. Therefore, these results suggest that Hrp1 may play a role in mitotic chromosome segregation and maintenance of chromatin structure by utilizing the energy from ATP hydrolysis.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin/metabolism , Chromosome Segregation , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/metabolism , mRNA Cleavage and Polyadenylation Factors , Adenosine Triphosphate/metabolism , Anaphase , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cell Nucleolus/ultrastructure , Cell Nucleus/chemistry , Chromatin/genetics , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , DNA/physiology , Fluorescent Antibody Technique, Indirect , Gene Deletion , Gene Expression Regulation, Fungal , Hydrolysis , Mitosis , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/ultrastructureABSTRACT
In the fission yeast Schizosaccharomyces pombe genes are transcriptionally silenced when placed within centromeres, within or close to the silent mating-type loci or adjacent to telomeres. Factors required to maintain mating-type silencing also affect centromeric silencing and chromosome segregation. We isolated mutations that alleviate repression of marker genes in the inverted repeats flanking the central core of centromere I. Mutations csp1 to 13 (centromere: suppressor of position effect) defined 12 loci. Ten of the csp mutants have no effect on mat2/3 or telomere silencing. All csp mutants allow some expression of genes in the centromeric flanking repeat, but expression in the central core is undetectable. Consistent with defective centromere structure and function, chromosome loss rates are elevated in all csp mutants. Mutants csp1 to 6 are temperature-sensitive lethal and csp3 and csp6 cells are defective in mitosis at 36 degrees. csp7 to 13 display a high incidence of lagging chromosomes on late anaphase spindles. Thus, by screening for mutations that disrupt silencing in the flanking region of a fission yeast centromere a novel collection of mutants affecting centromere architecture and chromosome segregation has been isolated.
Subject(s)
Centromere/genetics , Chromosomes, Fungal/genetics , Schizosaccharomyces/genetics , Transcription, Genetic , DNA, Fungal/genetics , Electrophoresis, Gel, Pulsed-Field , Gene Expression Regulation, Fungal , Genetic Markers , Genotype , Mutagenesis , RNA, Fungal/genetics , Repetitive Sequences, Nucleic Acid , Schizosaccharomyces/cytology , Schizosaccharomyces/growth & development , Suppression, GeneticABSTRACT
We have investigated the effects of inhibition of histone de-acetylase activity on silencing at the silent mating-type loci in fission yeast. Treatment of exponentially growing cells with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in derepression of a marker gene inserted 150 bp distal from the silent mat3-M locus. The natural targets for the silencing mechanism in this region were only partially derepressed and the activation appeared to be asymmetric, i.e. the mat2-P cassette remained silent at concentrations that clearly partially derepressed the mat3-M cassette. We further noted that treatment of wild-type h90 cells resulted in the generation of altered sporulation phenotypes, indicating that the treatment affected the expression of mating-type genes and/or mating-type switching. The results are discussed in the light of recent accumulated data regarding the role of deacetylation for silencing in other species.
Subject(s)
Genes, Fungal/genetics , Genes, Mating Type, Fungal , Histone Deacetylase Inhibitors , Schizosaccharomyces/genetics , Enzyme Inhibitors/pharmacology , Fungal Proteins/genetics , Gene Expression Regulation , Gene Expression Regulation, Fungal/drug effects , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Recombinant Fusion Proteins/geneticsABSTRACT
Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles.
Subject(s)
Fungal Proteins/genetics , Repressor Proteins/genetics , Retroelements , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/genetics , Transcription Factors/genetics , Amino Acid Sequence , Enzyme Inhibitors/pharmacology , Fungal Proteins/metabolism , Hemagglutinins , Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Molecular Sequence Data , Mutagenesis , Repressor Proteins/metabolism , Saccharomyces cerevisiae/genetics , Schizosaccharomyces pombe Proteins , Transcription Factors/metabolismABSTRACT
hda1+ (histone deacetylase 1) is a fission yeast gene which is highly similar in sequence to known histone deacetylase genes in humans and budding yeast. We have investigated if this putative histone deacetylase contributes to transcriptional silencing in the fission yeast Schizosaccharomyces pombe. A precise deletion allele of the hda1+ open reading frame was created. Cells lacking the hda1+ gene are viable. However, genetic analysis reveals that cells without hda1 + display enhanced gene repression/silencing of marker genes, residing adjacent to telomeres, close to the silent mating-type loci and within centromere I. This phenotype is very similar to that recently reported for rpd3 mutants both in Drosophila and budding yeast. No defects in chromosome segregation or changes in telomere length were detected. Cells lacking the hda1+ gene display reduced sporulation. Growth of hda1 cells is partially inhibited by low concentrations of Trichostatin A (TSA), a known inhibitor of histone deacetylase enzymes. TSA treatment is also able to overcome the enhanced silencing found in heterochromatic regions of hda1 cells. These results indicate a genetic redundancy with respect to deacetylase genes and partially overlapping functions of these in fission yeast. The significance of these results is discussed in the light of recent discoveries from other eukaryotes.
Subject(s)
Histone Deacetylases/genetics , Schizosaccharomyces/enzymology , Amino Acid Sequence , Animals , Base Sequence , Centromere , DNA Primers , Humans , Molecular Sequence Data , Open Reading Frames , Schizosaccharomyces/genetics , Sequence Homology, Amino Acid , TelomereABSTRACT
Histone acetylation may act to mark and maintain transcriptionally active or inactive chromosomal domains through the cell cycle and in different lineages. A novel role for histone acetylation in centromere regulation has been identified. Exposure of fission yeast cells to TSA, a specific inhibitor of histone deacetylase, interferes with repression of marker genes in centromeric heterochromatin, causes chromosome loss, and disrupts the localization of Swi6p, a component of centromeric heterochromatin. Transient TSA treatment induces a heritable hyperacetylated state in centromeric chromatin that is propagated in lineages in the absence of drug. This state is linked in cis to the treated centromere locus and correlates with inheritance of functionally defective centromeres and persistent chromosome segregation problems. Thus, assembly of fully functional centromeres is partly imprinted in the underacetylated or transcriptionally silent state of centromeric chromatin.
Subject(s)
Centromere/physiology , Histone Deacetylase Inhibitors , Schizosaccharomyces/genetics , Acetylation , Centromere/chemistry , Centromere/drug effects , Centromere/genetics , Chromatin/drug effects , Chromatin/metabolism , Chromosomes, Fungal , Enzyme Inhibitors/pharmacology , Genetic Markers , Hydroxamic Acids/pharmacology , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Folding , Schizosaccharomyces/drug effects , Schizosaccharomyces/enzymologyABSTRACT
Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown also to affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localise with the three silent chromosomal regions. Here the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated in further detail. Fluorescence in situ hybridisation (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalised from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents which destabilise microtubules and show a synergistic interaction with a mutation in the beta-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres which is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation.
Subject(s)
Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/metabolism , Transcription Factors/metabolism , Anaphase , Centromere , Fungal Proteins/genetics , In Situ Hybridization, Fluorescence , Mutagenesis , Schizosaccharomyces/genetics , Telomere , Transcription Factors/genetics , Tubulin/genetics , Tubulin/metabolismABSTRACT
Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribution of chromosomes into daughter cells. Transcriptionally silent heterochromatin of unknown function is associated with centromeres in many organisms. In the fission yeast Schizosaccharomyces pombe, the silent mating-type loci, centromeres, and telomeres are assembled into silent heterochromatin-like domains. The Swi6 chromodomain protein affects this silencing, and now it is shown that Swi6p localizes with these three chromosomal regions. In cells lacking Swi6p, centromeres lag on the spindle during anaphase and chromosomes are lost at high rates. Thus, Swi6p is located at fission yeast centromeres and is required for their proper function.
Subject(s)
Centromere/physiology , Fungal Proteins/physiology , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/physiology , Transcription Factors/physiology , Centromere/chemistry , Chromosomes, Fungal , Fluorescent Antibody Technique , Fungal Proteins/analysis , Fungal Proteins/genetics , Genes, Fungal , Genes, Mating Type, Fungal , Heterochromatin/chemistry , In Situ Hybridization, Fluorescence , Interphase , Mitosis , Mutation , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Telomere/chemistry , Transcription Factors/analysis , Transcription Factors/geneticsABSTRACT
The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.
Subject(s)
Centromere/physiology , Chromosomes, Fungal/physiology , Gene Expression Regulation, Fungal/genetics , Mutation/physiology , Schizosaccharomyces/genetics , Base Sequence , Crosses, Genetic , Genes, Fungal/genetics , Heterochromatin , Meiosis/physiology , Models, Genetic , Molecular Sequence Data , RNA, Fungal/biosynthesis , RNA, Messenger/biosynthesis , Schizosaccharomyces/metabolism , Telomere/physiologyABSTRACT
The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikI genes on chromosome III. The three H4-H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4-H3 gene pairs. H4.1-H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2-H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3-H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.
Subject(s)
Chromosomes, Fungal , Genes, Fungal , Histones/genetics , Schizosaccharomyces/genetics , Blotting, Southern , Chromosome Mapping , DNA, Fungal/analysis , Genomic Library , Restriction MappingABSTRACT
In Schizosaccharomyces pombe the mating-type information is stored at two transcriptionally silent loci (mat2 and mat3). The region between these sites (K region) is inert for meiotic crossing over. The mating-type genes (M or P) are expressed only when present at a third, active locus (mat1). We have earlier shown that the positional regulation of P genes is based on repression at the silent site, caused by elements in the flanking DNA sequences. In this study we have mutagenized a sterile mat1 deleted strain and selected for cells that are able to conjugate. Recessive mutations of this type should define genes encoding trans-acting factors involved in repression of the silent mating-type loci. Before this work mutations in two genes, clr1 and swi6, had been shown to allow both expression of the silent loci and recombination in the K region. The sensitivity of the present selection is demonstrated by the isolation of new mutations that derepress one or both of the silent loci (M-mating or bi-mating). The frequency of M-mating mutants was almost two orders of magnitude higher than that of bi-mating mutants and in all mutants analyzed mat3-M expression was significantly higher than mat2-P expression. The mutations define three new genes, clr2, clr3 and clr4. In addition we show that the rik1 mutant previously known to allow recombination in the K region also depresses the silent loci.
Subject(s)
Conjugation, Genetic , Genes, Fungal , Genes, Mating Type, Fungal , Schizosaccharomyces/genetics , Alleles , Base Sequence , Chromosome Mapping , Chromosomes, Fungal , DNA Primers , DNA, Complementary/analysis , Genes, Dominant , Genes, Recessive , Genetic Complementation Test , Genotype , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Transcription, GeneticABSTRACT
We show that in fission yeast two DNA fragments at the silent P mating type locus provide plasmids with the capability of autonomous replication. Bacterial vectors containing these sequences replicate in a polymeric form in fission yeast very much like plasmids with the commonly used replication sequence ars1, do. There are, however, several differences between the two new ars sequences. The percentage of cells containing the plasmid during selection, the plasmid copy number and the plasmid segregation during mitosis are all dependent on the choice of the ars sequence. A DNA fragment with ars activity from the left side of the silent P cassette represses the expression of the marker gene, ura4+, at least three hundred fold compared to plasmids containing only the other new ars sequence or only ars1. The importance of replication in this promoter independent transcriptional regulation is further substantiated by the fact that the repression is partially released in the presence of ars1 on the same plasmid.
Subject(s)
Genes, Fungal/physiology , Genes, Mating Type, Fungal , Replicon/physiology , Schizosaccharomyces/genetics , Mitosis , Plasmids/physiology , Promoter Regions, Genetic , Suppression, Genetic , Transcription, Genetic , Transformation, GeneticABSTRACT
We have characterized the nuclear gene NAM8 in Saccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wild-type strains, its overexpression is a prerequisite for the suppressor action. Inactivation of the NAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. The NAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.
Subject(s)
Fungal Proteins/genetics , Genes, Fungal , Genes, Suppressor , Mitochondria/metabolism , RNA Splicing/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins, Small Nuclear , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Fungal Proteins/metabolism , Gene Amplification , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids , RNA, Fungal/metabolism , RNA-Binding Proteins/metabolism , Transcription, GeneticABSTRACT
Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences. The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region. The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression. Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants. These mutants can de-repress the resistance gene at other positions than the one used for their selection. Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette. These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.
Subject(s)
Gene Expression Regulation, Fungal/physiology , Genes, Fungal/genetics , Genes, Mating Type, Fungal , Schizosaccharomyces/genetics , Trans-Activators/genetics , Alleles , Chromatin/physiology , Kanamycin Resistance/genetics , Mutation , Plasmids , Regulatory Sequences, Nucleic Acid , Repressor Proteins/geneticsABSTRACT
The fission yeast, Schizosaccharomyces pombe, expresses one of two alternative mating types. They are specified by one of two determinants (M or P) present at the mat1 locus. In addition, silent copies of M and P are present on the same chromosome. In the present work we demonstrate that the difference between the active and the silent stage of the P determinant is controlled by four repressive elements that are located at the silent locus. There are two elements to the left and two to the right of the mating type cassette. Both elements to the left and either one of the two elements to the right are required for an effective blockage of transcription. When they are combined, the four elements define a highly efficient silencer functionally similar to the HMRE and HMLE and HMLI silencers in Saccharomyces cerevisiae. In addition, the DNA surrounding the silent P locus confers symmetric partitioning in mitosis to Schizosaccharomyces pombe ars plasmids.