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K A Nasmyth

Publications and source records attributed to K A Nasmyth.

At least 19 recordsLinked to original sources
PubMed1996

The Saccharomyces cerevisiae Start-specific transcription factor Swi4 interacts through the ankyrin repeats with the mitotic Clb2/Cdc28 kinase and through its conserved carboxy terminus with Swi6.

At a point in late G1 termed Start, yeast cells enter S phase, duplicate their spindle pole bodies, and form buds. These events require activation of Cdc28 kinase by G1 cyclins. Swi4 associates with Swi6 to form the SCB-binding factor complex which activates G1 cyclin genes CLN1 and CLN2 in late G1. In G2 and M phases, the transcriptional activity of SCB-binding factor is repressed by the mitotic Clb2/Cdc28 kinase. Mbp1, a transcription factor related to Swi4, forms the MCB-binding factor complex with Swi6, which activates DNA synthesis genes and S-phase cyclin genes CLB5 and CLB6 in late G1. Clb2/Cdc28 kinase is not required for the repression of MCB-binding factor transcriptional activity in G2 and M phase. We show here that the Swi4 carboxy terminus is sufficient for interaction with Swi6 in vitro. A carboxy-terminal domain of Swi6 is required and sufficient for interaction with Swi4. The carboxy terminus of Mbp1 is sufficient for interaction with Swi6, and the carboxy terminus of Swi6 is required for interaction with Mbp1. By coimmunoprecipitation, we show that Swi4 but not Mbp1 interacts with Clb2/Cdc28 kinase in vivo during the G2 and M phases of the cell cycle. We demonstrate that the ankyrin repeats of Swi4 mediate the interaction with Clb2/Cdc28 kinase. The ankyrin repeats constitute a domain by which a cell cycle-specific transcription factor can interact with cyclin-dependent kinase complexes, thus enabling it to link its transcriptional activity to cell cycle progression.

Amino Acid Sequence↗
PubMed1995

S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.

BACKGROUND: DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk). It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur. The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion. RESULTS: S. cerevisiae strains lacking C1b1-C1b4 undergo DNA replication once but fail to enter mitosis. We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis. The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sim mutants. Increased expression of the CLB5 gene prevents re-replication. Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can prevent re-replication in G2-phase cells. Inactivation of C1b kinases by expression of the specific C1b-Cdk1 inhibitor p40SIC1 is sufficient to induce a prereplicative state at origins of replication in cells blocked in G2/M phase by nocodazole. Re-activation of C1b-Cdk1 kinases induces a second round of DNA replication. CONCLUSIONS: We propose that S-phase-promoting cyclin B--Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state. This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase.

Cell Cycle↗
PubMed1988

Characterization of a transcription factor involved in mother cell specific transcription of the yeast HO gene.

The yeast HO gene, which encodes an endonuclease involved in initiating mating type interconversion, is expressed in mother cells but not in daughters. It has been demonstrated that the SWI5 gene, which is an activator of HO expression, plays a critical role in this differential mother/daughter expression of HO. In this paper we describe the cloning and sequencing of the SWI5 gene. The predicted amino acid sequence derived from the cloned SWI5 gene shows homology with the repeated DNA-binding domains ('zinc fingers') of Xenopus transcription factor TFIIIA. A region of the HO promoter involved in the SWI5-dependent transcriptional activation of HO was identified by deletion analysis of the HO promoter in the chromosome, and by testing the ability of HO DNA fragments to activate transcription in the context of a heterologous promoter. The SWI5 gene product was overproduced in yeast from the GAL1-10 promoter, since the SWI5 protein is made at very low levels in wild-type strains, and protein extracts were used to demonstrate that the SWI5 protein binds in vitro to a segment of the HO promoter required for transcriptional activation in vivo.

Amino Acid Sequence↗
PubMed1987

Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication.

The 'silent' yeast mating-type loci (HML and HMR) are repressed by sequences (HMLE and HMRE) located over 1 kb from their promoters which have properties opposite those of enhancers, and are called 'silencers'. Both silencers contain autonomously replicating sequences (ARS). Silencer activity requires four trans-acting genes called SIR (silent information regulator). We have identified two DNA binding factors , SBF-B and SBF-E, which bind to known regulatory elements at HMRE. SBF-B binds to a region involved in both the silencer and ARS functions of HMRE, but doesn not bind to HMLE. This factor also binds to the unlinked ARS1 element. SBF-E recognizes a sequence found at both silencers. These results suggest that the two silencers may be composed of different combinations of regulatory elements at least one of which is common to both. Neither factor appears to be a SIR gene product. Hence the SIR proteins may not directly interact with the silencer control sites.

DNA Replication↗
PubMed1984

Characterization of two genes required for the position-effect control of yeast mating-type genes.

The mating type of haploid yeast (a or alpha) is determined by information present at the MAT locus. Identical copies of a and alpha information are present at distal loci (HMR and HML), but transcription of these copies is repressed by the action, in trans, of four unlinked genes called SIR (silent information regulator). Repression by SIR also requires, in cis, DNA sequences called E which are found to the left of HML and HMR (but not MAT) and are greater than 1 kb from the mating-type gene promoters. SIR control can act on other promoters when they are brought near the E sequence, and thus the SIR gene products act in some general manner to repress transcription. We have determined the DNA sequence of two fragments which complement mutations in the SIR2 and SIR3 genes and show that these contain the structural genes by mapping the cloned sequences onto the yeast chromosome. The SIR2 and SIR3 coding sequences were identified by constructing gene disruptions and using these mutations to replace the normal chromosomal copies. Such null mutants of both SIR2 and SIR3 are defective in the position-effect control of the silent loci but have no other detectable phenotype. We have mapped the 5' and 3' ends of the SIR2 and SIR3 mRNAs and show that their level is unaffected by mutations in any of the four known SIR complementation groups.

Amino Acid Sequence↗
PubMed1984

Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region.

The genome of the yeast Saccharomyces cerevisiae contains three complete copies of the genetic information governing cell mating type. Normally, only the information in one of the copies (the MAT locus) is expressed; the other two copies (HML and HMR) are repressed and serve as donors of mating-type sequences that can be transposed to MAT in cells capable of switching mating type. We have mutagenized the silent HMR locus and have found that the repression of this locus requires two sites, one lying on each side of the mating-type sequences at HMR. The regulatory sites are positioned outside of the sequences that are included in the pair of divergent transcripts coded for by HMR, and lie about 1000 base-pairs to either side of the central promoter region of the locus. Deletion of one of the regulatory sites results phenotypically in complete loss of repression, whereas deletion of the other site gives only partial loss of control. Both of the sites are associated with an autonomous replication activity, though the relationship between this activity and the process of repression is unclear.

Base Sequence↗
PubMed1982

The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition.

The two yeast mating-type alleles MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed, except in sir- strains. Since SIR represses not only transcription from a silent copy but also its ability to act as a recipient in a mating-type interconversion, we have investigated whether it might act by regulating the entire chromatin structure of a silent locus. We have therefore compared the profile of DNAase I and micrococcal nuclease cleavage at HML alpha with MAT alpha and HMRa with MATa in sir- and SIR+ strains. We find that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT. One particularly striking change that SIR induces provides a simple explanation for one of its biological properties: control of directionality of switching. SIR causes the disappearance of a DNAase I-hypersensitive site at Y-Z boundary (found at MAT or HM sir-) that is coincident with a double-strand cleavage possibly created by HO in the initiation of a mating-type switch.

Base Sequence↗
PubMed1982

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus.

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.

Chromosome Mapping↗
PubMed1981

A position effect in the control of transcription at yeast mating type loci.

The two mating type loci MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed. It is concluded that sequences to the left of HMRa (and probably HML alpha) must somehow affect transcription initiated at the centre of each locus 700 to 1,400 base pairs away. A possible mechanism for this position effect is discussed.

Base Sequence↗
PubMed1981

Isolation of a gene from Drosophila by complementation in yeast.

Transformation of mutant yeast cells by cloned genomic DNA from a higher eukaryote has made it possible to isolate a Drosophila DNA sequence that complements a yeast adenine-8-mutation. A 0.8-kilobase poly(A)-containing RNA is transcribed from the cloned Drosophila segment in transformed yeast cells and can account for functional expression of the gene.

Adenine↗
PubMed1981

The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae.

The complete sequences of the yeast a mating-type locus, MATa, and of the silent alpha cassette, HML alpha, have been determined. A segment of 642 nucleotides is unique to MATa, and a corresponding segment of 747 nucleotides is unique to MAT alpha. The major mRNAs (a1, a2, alpha 1 and alpha 2) encoded by MATa and MAT alpha have been aligned with the DNA sequence. The a1 mRNA is encoded entirely within the a-specific DNA sequence. The a2 mRNA, which is transcribed divergently from a1 mRNA, is encoded in a region common to both Mata and Mat alpha. The alpha 1 and alpha 2 mRNAs are also transcribed divergently and have their 5' starts about 240 nucleotides apart within the alpha-specific sequence. The amino acid sequences of the MAT proteins have been predicted from the DNA sequences. An unanticipated conclusion is that the a1 protein, containing 148 amino acids, results from readthrough of a UGA at codon 45. Polymorphic forms of the homologous outer segments of the HML alpha, MAT alpha, MATa and HMRa sequences suggest that the boundaries of the segments involved in mating-type switching are immediately adjacent to the a-specific and alpha-specific sequences.

Base Sequence↗
PubMed1981

In vitro mutation analysis of the mating-type locus in yeast.

The mating-type locus (MAT) of Saccharomyces cerevisiae is a complex locus that codes for the regulators of cell type. Two unique messages are transcribed from each MAT allele. Using the in vitro mutagenesis technique whereby synthetic oligonucleotides containing restriction sites (linkers) were inserted into plasmids, we have constructed a series of mutations in cloned DNA containing either the MATa or MAT alpha locus. The new restriction site associated with each "linker" mutation has allowed the mutation to be mapped and sequenced. We have complemented genetically defined mutations (mata1, mat alpha 1 and mat alpha 2) with plasmids containing these in vitro mutations by yeast transformation, thereby mapping the genes onto the DNA sequence. MATa1 has been localized to the MATa unique region (Ya) from which the a1 message is transcribed. We find no function for the other MATa message by using our complementation assay. MAT alpha 1 maps to the MAT alpha unique (Y alpha) and adjacent (Z) region from which the alpha 1 message is transcribed. MAT alpha 2 maps to the other major message found in the common (X) region of the MAT alpha loci. Although most linker mutations that have a mutant phenotype appear to disrupt the translated portion of each gene, two mutations may disrupt transcription.

Chromosome Mapping↗