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K Nasmyth

Publications and source records attributed to K Nasmyth.

At least 109 records · Page 6Linked to original sources
PubMed1992

Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae.

Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis.

CDC2 Protein Kinase↗
PubMed1992

A new method for the isolation of recombinant baculovirus.

An improved method for the isolation of baculovirus recombinants is described. The method involves the replication and maintenance of the baculovirus genome in the yeast Saccharomyces cerevisiae which was accomplished by the isolation of a baculovirus recombinant containing yeast ARS and CEN sequences ensuring stable replication in yeast and a URA3 selectable marker. The viral DNA maintained its ability to replicate in insect cells. An efficient and rapid selection system was set up, to isolate viral recombinants in yeast; DNA from selected yeast colonies was transfected into insect cells to obtain recombinant virus. We demonstrate the utility of this system by isolating recombinant viruses that express two different members of the CREB/ATF family of transcription factors.

Animals↗
PubMed1992

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.

Amino Acid Sequence↗
PubMed1992

Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1.

The FUS3 and KSS1 kinases are components of the pheromone-dependent signal transduction pathway in yeast. We show that FUS3 and KSS1 become rapidly phosphorylated after pheromone treatment. Similar to mammalian MAP kinases, this modification occurs at two amino acids of FUS3, threonine-180 and tyrosine-182. A mutation introduced at either position results in complete loss of function in vivo. Amino acid substitutions that destroy catalytic activity of the kinase do not prevent phosphorylation of the mutant products, a result that excludes an autocatalytic activation pathway. The modification of FUS3 is dependent on kinases encoded by the STE11 and STE7 genes. Furthermore, a hyperactive allele of STE11 causes increased phosphorylation of FUS3 in the absence of pheromone stimulation. Thus, either STE7 or STE11 could be the kinase responsible for the phosphorylation of FUS3.

Alleles↗
PubMed1992

SWI6 is a regulatory subunit of two different cell cycle START-dependent transcription factors in Saccharomyces cerevisiae.

Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells undergo START. Their promoters all contain one or more versions of an 8-base pair motif (ACGCGTNA) called the MluI cell cycle box (MCB). MCBs have been shown to be both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POLI DNA polymerase genes. A different late G1-specific transcription element called the SCB (CACGAAAA) is bound by a factor containing the SWI4 and SWI6 proteins. We describe here the formation in vitro of complexes on TMP1 MCBs that contain the SWI6 protein and, we suggest, a 120 kDa protein that is distinct from SWI4. Transcription due to SCBs and MCBs occurs in the absence of SWI6 but it is no longer correctly cell cycle regulated. We suggest that SWI6 is an essential regulatory subunit of two different START-dependent transcription factors. One factor (SBF) contains SWI4 and binds to SCBs whereas the other (MBF) contains p120 and binds MCBs.

Base Sequence↗
PubMed1991

The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast.

Entry into the mitotic cycle (START) requires a protein kinase encoded by the CDC28 gene and one of three redundant G1-specific cyclins encoded by CLN1, -2, and -3. SWI4 and SWI6 are transcription factors required for the START-dependent activation of the HO endonuclease gene. They also fulfill an overlapping but essential role for cell division since cells deleted for both genes are inviable. We show that the essential role of SWI4 and SWI6 is to ensure the activity of G1-specific cyclin genes. SWI4 and SWI6 appear necessary for the transcription of CLN1 and CLN2, but not for that of CLN3. CLN3 function is, however, also dependent on SWI4 and SWI6.

Blotting, Northern↗
PubMed1991

The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5.

The intracellular localization of the S. cerevisiae transcription factor SWI5 is cell cycle dependent. The protein is nuclear in G1 cells but cytoplasmic in S, G2, and M phase cells. We have identified SWI5's nuclear localization signal (NLS) and show that it can confer cell cycle-dependent nuclear entry to a heterologous protein. Located within or close to the NLS are three serine residues, mutation of which results in constitutive nuclear entry. These residues are phosphorylated in a cell cycle-dependent manner in vivo, being phosphorylated when SWI5 is in the cytoplasm and dephosphorylated when it is in the nucleus. As all three serines are phosphorylated by purified CDC28-dependent H1 kinase activity in vitro, we propose a model in which the CDC28 kinase acts directly to control nuclear entry of SWI5.

Amino Acid Sequence↗
PubMed1991

Positive feedback in the activation of G1 cyclins in yeast.

Yeast cells become committed to the mitotic cell cycle at a stage during G1 called Start. To enter Start, cells must grow to a critical size. They also require the CDC28 protein kinase and at least one of three G1-specific cyclins encoded by CLN1, 2, and 3. It is thought that Start is triggered by the accumulation of G1 cyclins that bind to the CDC28 kinase and activate it. So what determines the accumulation of G1 cyclins? For CLN1 and CLN2, transcriptional activation could be involved because their RNAs appear transiently during the cell cycle as cells undergo Start. Here we report that the appearance of CLN1 and CLN2 RNAs depends on an active CDC28 kinase and is stimulated by CLN3 activity. We propose that CDC28 kinase activity due to CLN1 and CLN2 proteins arises through a positive feedback loop which allows CLN proteins to promote their own synthesis.

Cyclins↗
PubMed1991

A general approach to the isolation of cell cycle-regulated genes in the budding yeast, Saccharomyces cerevisiae.

We describe a general approach to the isolation of cell cycle-dependently regulated transcripts in Saccharomyces cerevisiae. This approach is based on the physical identification of cell cycle-regulated transcripts by Northern hybridization using as probes yeast DNA isolated from an ordered S. cerevisiae genomic library. The purpose of this is twofold; first, to assess the importance of transcriptional regulation in cell cycle control; and second, to identify novel genes that may have important roles in the eukaryotic cell cycle. We report the isolation of two previously uncharacterized genes that are transcribed at points in the cell cycle to which specific transcriptional activation has not been assigned: namely, mitosis and early G1 phase. It is argued that these transcripts serve as important landmarks for cell cycle events that are not readily distinguished by either morphological or cytological criteria. The cell cycle-dependent transcription of the RNR1 and CLN1 genes is also described and the implications for cell cycle control, in G1, are discussed with reference to these two genes.

Blotting, Northern↗
PubMed1991

The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae.

cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.

Alleles↗
PubMed1991

Changes in a SWI4,6-DNA-binding complex occur at the time of HO gene activation in yeast.

The yeast HO gene is transcribed transiently during G1 as cells undergo START. START-specific HO activation requires two proteins, SWI4 and SWI6, which act via a motif (CACGA4) repeated up to 10 times within the URS2 region of the HO promoter. We identified a DNA-binding activity containing SWI4 and SWI6 that recognizes the CACGA4 sequences within URS2. Two forms of SWI4,6-DNA complexes called L and U can be distinguished by their electrophoretic mobility. L complexes can be detected at all stages of the cell cycle, but U complexes are only detected in cells that have undergone START. The formation of U complexes may be the trigger of HO activation. The SWI6 protein is concentrated in the nucleus throughout G1, but at some point in S or G2 significant amounts accumulate in the cytoplasm. This change in cellular location of the SWI6 protein might contribute to the turnoff of HO transcription after cells have undergone START.

Base Sequence↗
PubMed1991

A new role for MCM1 in yeast: cell cycle regulation of SW15 transcription.

In the yeast Saccharomyces cerevisiae cell cycle-regulated SW15 transcription is essential for ensuring that mother and not daughter cells switch mating type. We have identified a 55-bp promoter sequence that appears to be responsible for restricting transcription to the late S, G2, and M phases of the cell cycle. Two proteins, MCM1, a transcription factor described previously, and SFF (SWI five factor, a newly identified factor) bind this sequence in vitro. MCM1 binds the DNA tightly on its own, but SFF will only bind as part of a ternary complex with MCM1. We observe a strong correlation between the ability of mutated SWI5 promoter sequences to form a ternary MCM1-SFF-containing complex in vitro and to activate transcription in vivo, which suggests that efficient transcription requires that both proteins bind DNA. Through its interactions with cell type-specific coactivators and corepressors, MCM1 controls cell type-specific expression of pheromone and receptor genes. By analogy, we propose that SFF enables MCM1 to function as a part of a cell cycle-regulated transcription complex.

Base Composition↗
PubMed1990

The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry.

HO encodes a site-specific endonuclease that initiates mating type switching in S. cerevisiae. It is expressed only transiently during the cell cycle of mother cells, as they undergo Start, but not in daughter cells. Since SWI5 appears to be the only HO transcription factor missing when daughter cells undergo Start, we were interested in the intracellular distribution of SWI5 at cell division. We discovered that SWI5 is found equally concentrated in the nuclei of both mother and daughter cells at the end of anaphase, suggesting that its subsequent fate must somehow differ. Prior to the end of anaphase, SWI5 accumulates in the cytoplasm and only moves into the nucleus when cells enter G1. A version of the HO promoter that has lost its dependence on Start is nevertheless still strongly cell cycle regulated and is activated when SWI5 moves into the nucleus.

Cell Compartmentation↗
PubMed1989

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.

Amino Acid Sequence↗
PubMed1988

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA.

The short sequence motif named 'zinc finger', first recognized repeated in tandem in the Xenopus transcription factor IIIA (TFIIIA), is also found in the yeast transcriptional activator SWI5 (ref. 3) and many other regulator proteins. Embedded in the 709-amino-acid polypeptide chain of SWI5 are three tandemly repeated zinc-finger motifs. Because the zinc fingers of TFIIIA are known to bind to DNA, it is probable that in the case of SWI5 these finger motifs also play an important, but not necessarily exclusive, role in the sequence-specific binding of the protein to DNA. To test this prediction we have expressed the 89-amino-acid sequence of the domain containing the three zinc fingers of SWI5 in Escherichia coli as a cleavable fusion protein, purified under denaturing conditions and folded in vitro. This experimental approach allows us to study directly both the metal requirement and DNA-binding properties of the isolated polypeptide. We find that zinc is required for specific DNA recognition and, most significantly, DNaseI protection studies show that the isolated three-fingered domain is sufficient for sequence-specific binding to DNA.

Amino Acid Sequence↗
PubMed1987

A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation.

Repression of the yeast silent mating type loci requires cis-acting sequences located over 1 kb from the regulated promoters. One of these sites, a "silencer," exhibits enhancer-like distance- and orientation-independence. The silencer demonstrates both autonomous replication sequence (ARS) activity and a centromere-like segregation function, suggesting roles for DNA replication and segregation in transcriptional repression. Here we identify three sequences (A, E, and B) involved both in repression and in either ARS or segregation activity. The sequences are functionally redundant: no one is essential for complete transcriptional control, but mutations in any two inactivate the silencer. Surprisingly, elements E and B can each activate transcription from heterologous promoters, and E shows striking homology to several yeast upstream activation sequences. Therefore, sequences individually involved in replication, segregation, and transcriptional activation can, at the silencer, efficiently repress transcription.

Base Sequence↗