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Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.

Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin–dependent kinases (CDKs) and their associated cyclins, (ii) kinases and phosphatases that regulate CDK activity, and (iii) stoichiometric inhibitors that sequester cyclin–CDK dimers. Presumably regulation of cell division in the earliest ancestors of eukaryotes was a considerably simpler affair. Nasmyth (1995) recently proposed a mechanism for control of a putative, primordial, eukaryotic cell cycle, based on antagonistic interactions between a cyclin–CDK and the anaphase promoting complex (APC) that labels the cyclin subunit for proteolysis. We recast this idea in mathematical form and show that the model exhibits hysteretic behaviour between alternative steady states: a G1–like state (APC on, CDK activity low, DNA unreplicated and replication complexes assembled) and an S/M–like state (APC off, CDK activity high, DNA replicated and replication complexes disassembled). In our model, the transition from G1 to S/M (‘Start’) is driven by cell growth, and the reverse transition (‘Finish’) is driven by completion of DNA synthesis and proper alignment of chromosomes on the metaphase plate. This simple and effective mechanism for coupling growth and division and for accurately copying and partitioning a genome consisting of numerous chromosomes, each with multiple origins of replication, could represent the core of the eukaryotic cell cycle. Furthermore, we show how other controls could be added to this core and speculate on the reasons why stoichiometric inhibitors and CDK inhibitory phosphorylation might have been appended to the primitive alternation between cyclin accumulation and degradation.

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase --> anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin's association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.

In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies. Activation of the Cdc28 protein kinase by G1‐specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events. Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln‐Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop. We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start‐related events, they are not required for the punctual activation of SBF‐ or MBF‐driven transcription. Cln3, on the other hand, is essential. By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B‐Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B‐Cdc28 kinases and thereby trigger S phase. Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF‐ and MBF‐mediated transcription by Cln3‐Cdc28 kinase. This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle.

S phase entry depends on cyclin‐dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits. We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication. The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1. The former is due to Swi5, while the latter is due to MBF or SBF transcription factors. Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly. Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents. This ‘reductional’ anaphase occurs with almost wild‐type kinetics and depends on the activity of G2 cyclins. Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects. We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the ‘reductional’ anaphase.

It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B‐type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.

Cyclin‐dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras‐associated GTPase‐activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.

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.

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.

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.

In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies. Activation of the Cdc28 protein kinase by G1‐specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events. Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln‐Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop. We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start‐related events, they are not required for the punctual activation of SBF‐ or MBF‐driven transcription. Cln3, on the other hand, is essential. By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B‐Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B‐Cdc28 kinases and thereby trigger S phase. Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF‐ and MBF‐mediated transcription by Cln3‐Cdc28 kinase. This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle.

S phase entry depends on cyclin‐dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits. We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication. The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1. The former is due to Swi5, while the latter is due to MBF or SBF transcription factors. Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly. Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents. This ‘reductional’ anaphase occurs with almost wild‐type kinetics and depends on the activity of G2 cyclins. Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects. We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the ‘reductional’ anaphase.

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.

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

cdc28, one of several genes required for cell division in the yeast Saccharomyces cerevisiae, has been isolated on recombinant plasmids. A recombinant plasmid pool containing the entire yeast genome was constructed by partial digestion of yeast DNA with the four-base recognition restriction endonuclease Sau3A to give the equivalent of random fragments, size selection on sucrose gradients, and introduction of the fragments into the yeast vector YRp7 by use of the homology of Sau3A ends with those generated in the vector by cleavage with BamHI. Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus, confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants.

In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle. Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner. Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2. Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner. Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle. However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and 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.

Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.

Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G1. beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro- Arg-Lys655) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G1 or G2/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser646 and Ser664) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.

DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.

It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B‐type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.

Cyclin‐dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras‐associated GTPase‐activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.

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.

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 ceils 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.