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K Nasmyth

Publications and source records attributed to K Nasmyth.

At least 127 records · Page 7Linked to original sources
PubMed1987

Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements.

A DNA binding protein (RAP1, previously called SBF-E) has been shown to bind to putative regulatory sites at both yeast mating-type silencers, yet is not the product of genetically identified regulators of the silent loci. Here, we report the purification of RAP1 by DNA affinity chromatography, and the isolation of its gene from a lambda gt11 genomic library using antibodies raised against the protein. Disruption of the chromosomal copy of this gene is lethal. We show that RAP1 protein also binds in vitro to the upstream activation site (UAS) of MAT alpha and ribosomal protein genes. In addition, we show that two different UAS-associated RAP1 binding sites can substitute in vivo for a silencer binding site. Our results suggest that RAP1 may be a transcriptional regulator that can play a role in either repression or activation of transcription, depending upon the context of its binding site.

Amino Acid Sequence↗
PubMed1987

Transcriptional regulation in the yeast life cycle.

The transition from haploid to diploid in homothallic yeast involves a defined sequence of events which are regulated at the level of transcription. Transcription factors encoded by SWI genes activate the HO endonuclease gene at a precise stage in the cell cycle of mother cells. The HO endonuclease initiates a transposition event which activates genes of the opposite mating type by causing them to move away from a silencer element. The activated mating type genes then regulate genes involved in cell signaling such as the mating type-specific pheromones and their receptors. Since HO is only activated in one of the sister cells after division (the mother), adjacent cells of opposite mating type are generated which respond to each others' secreted pheromones by inducing genes involved in conjugation. This leads to the formation of a diploid in which many of the genes involved in mating and mating-type switching become repressed due to the heterozygosity of the mating-type locus. This article summarizes what is known about these transcriptional controls and discusses possible parallels in higher eukaryotes.

Crosses, Genetic↗
PubMed1987

Cell cycle regulation of SW15 is required for mother-cell-specific HO transcription in yeast.

In haploid homothallic yeast, cell division gives rise to a mother cell that transiently transcribes the HO gene (as it undergoes START) and a daughter cell that does not. Consequently, only mother cells switch their mating types. Here, we test the proposition that a transcription factor called SWI5 is the "determinant" of mother-cell-specific HO transcription; that is, that SWI5 is the only factor missing in daughter cells. We show that SWI5 RNAs are cell-cycle regulated so that they are only produced after the post-START window of HO transcription. This regulation is vital for mother-cell specificity since constitutive transcription of SWI5 causes daughter cells to switch their mating types. We propose that SWI5 gene products are partitioned asymmetrically at cell division.

Base Sequence↗
PubMed1987

Both positive and negative regulators of HO transcription are required for mother-cell-specific mating-type switching in yeast.

The HO gene, which encodes an endonuclease responsible for initiating mating type switching in yeast, is transcribed at START during the cell cycle of mother cells but not at all during the cell cycle of daughter cells. At least six genes, called SWI1-6, are necessary for HO transcription. We describe the isolation and characterization of mutations in two new genes called SDI1 and SDI2, which partially suppress the requirement for SWI5 and which cause daughter cells to express HO. The analysis of mating type switching in swi5- sdi1- and SWI5+ sdi1- strains suggests that the mother cell specificity of HO transcription is due exclusively to the selective action of SWI5 in mother cells. SDI1 encodes (or regulates) a repressor protein that binds to the HO promoter and prevents HO transcription in daughter cells by causing HO to be fully SWI5 dependent.

Conjugation, Genetic↗
PubMed1987

Cell cycle control of the yeast HO gene: cis- and trans-acting regulators.

In this paper, we investigate the role of a short repeated sequence (CACGA4) in the cell-cycle regulation of HO. We show that this sequence activates transcription of a heterologous gene in a cell-cycle-dependent fashion indistinguishable from that of the wild-type HO promoter. We also show that, in addition to SWI1 through SWI5, at least five other genes (SWI6 through SWI10) are required for HO transcription. These genes fit into three distinct classes with respect to their targets within the HO promoter. SWI4 and SWI6 are specifically required for CACGA4-mediated activation of transcription. SWI1, SWI2, and SWI5 are required for transcription from sequences physically separate from and independent of the CACGA4 sequences. SWI3 may be required for both. Since all the SWI genes are required for HO transcription, the HO promoter must contain at least two essential upstream activation sequences, which are affected by different trans-acting factors and are subject to different types of control.

Cell Cycle↗
PubMed1987

The determination of mother cell-specific mating type switching in yeast by a specific regulator of HO transcription.

In haploid homothallic budding yeast, cell division gives rise to a mother cell which proceeds to switch its mating type and a daughter cell (the bud) which does not. Switching is initiated by a specific double strand cleavage of mating type DNA by an endonuclease encoded by the HO gene. Previous data suggest that the pattern of HO transcription is responsible for the mother cell specificity of switching. HO is transcribed transiently, at START, during the cell cycle of mother cells but not at all during the cell cycle of daughter cells. The HO promoter is complex. Sequences between -1000 and -1400 (called URS1) are essential for transcription, whereas sequences between -150 and -900 (called URS2) are necessary for cell cycle control. Moreover, 10 trans-acting gene products called SWI1-10 are necessary for maximum expression. In an attempt to identify the cis-acting DNA sequences which are responsible for mother cell specificity and to identify which SW1 genes are involved, a hybrid GAL/HO promoter was constructed in which the upstream activation region putatively involved in mother cell-specific activation (URS1) is replaced by the upstream activation region of the GAL1-10 promoter. The properties of this hybrid promoter show, for the first time, that: (i) the HO promoter is modular since mother cell specificity can be replaced by galactose dependence without compromising cell cycle control or a/alpha repression; (ii) transcription of HO is indeed the major rate-limiting event for switching which is absent in daughter cells; (iii) SWI1,2,3, 4,6,7,8.9 and 10 are unlikely to be involved in mother cell specificity but SW15 probably is.

Gene Expression Regulation, Fungal↗
PubMed1985

Characterization of a "silencer" in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer.

The mating type of yeast is determined by the allele, either a or alpha, at the MAT locus. Two other loci, HML and HMR, contain complete copies of the alpha and a genes, respectively, which are not expressed. The four SIR gene products are required in trans for repression of the silent loci, as are cis-acting sites on either side of HML and HMR, about 1000 bp from the mating-type promoters. We demonstrate that one of these cis-acting sequences, HMRE, is able to switch off at least two nonmating-type promoters. In common with enhancers, it is able to function in either orientation, relatively independently of its position with respect to the regulated promoter, and can act on promoters 2600 bp away. However since HMRE represses, rather than enhances, transcription we have called it a "silencer" sequence.

Base Sequence↗
PubMed1985

At least 1400 base pairs of 5'-flanking DNA is required for the correct expression of the HO gene in yeast.

Homothallic yeast cells undergo a specific pattern of mating-type switching initiated by an endonuclease encoded by the HO gene. HO transcription is affected by cell type (a, alpha, and a/alpha), by cell age (mother or daughter), and by the cell cycle. This paper investigates the sequences involved in HO transcription by replacing genomic DNA with copies mutated in vitro. A region between -1000 and 1400 (called URS1) is necessary for transcription in addition to a "TATA"-like region at -90. The 900 bp of DNA separating URS1 from the "TATA" box is not necessary for transcription nor for a/alpha repression and some measure of mother/daughter control, but it is necessary for correct cell cycle control.

Base Sequence↗
PubMed1985

A repetitive DNA sequence that confers cell-cycle START (CDC28)-dependent transcription of the HO gene in yeast.

The interconversion of mating types in yeast is initiated by an endonuclease encoded by the HO gene. HO is transcribed only transiently during the G1 phase of the cell cycle, as cells undergo START. A deletion analysis of HO 5'-flanking DNA suggests that there must be multiple copies of the sequence that confers START-specific transcription in the interval from -150 to -900. Analysis of this interval revealed 10 occurrences of sequences that closely match the consensus Pur N N Pyr C A C G A4. To test whether these sequences are the putative cell-cycle control elements, synthetically derived copies of the consensus were inserted at the break points of constitutive deletions and shown to restore START-dependent cell-cycle control.

Cell Cycle↗
PubMed1983

Molecular analysis of a cell lineage.

Mating type interconversion has a precise lineage which serves to minimize the time taken for yeast cells to achieve the diploid state. The HO gene either encodes or regulates an endonuclease which initiates the interconversion process. Expression of this gene is switched on during the G1 phase of mother cells and not at all during the cell cycle of their daughters. This behaviour can explain what is known about the lineage.

Alleles↗
PubMed1981

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe.

A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25 degrees C but become blocked at DNA replication or mitosis when incubated at 36 degrees C. Thirty-nine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambiguously assigned to either DNA replication or mitosis but were definitely required for one or the other. Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36 degrees C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons. A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.

Ascomycota↗
PubMed1979

The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast.

Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0.1 of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1--2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.

Ascomycota↗
PubMed1976

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe.

Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.

Ascomycota↗
PubMedDate not supplied

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion.

BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.

Animals↗