Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR‐based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope‐tagged proteins. Copyright © 1999 John Wiley & Sons, Ltd.

Cell proliferation depends on the duplication of chromosomes followed by the segregation of duplicates (sister chromatids) to opposite poles of the cell prior to cell division (cytokinesis). How cells ensure that chromosome duplication, chromosome segregation, and cell division occur in the correct order and form an immortal reproductive cycle is one of the most fundamental questions in cell biology. Without such coordination, cells would not maintain a constant chromosome number and sexual reproduction as we know and love it would not be possible. A halfhearted cell cycleThe discovery of cyclin-dependent kinases (CDKs) went some way to answering this question. Successive waves of S-and M-phase-promoting CDKs first trigger chromosome duplication (S phase)-then the attachment of the replicated chromosomes to a bipolar spindle (M phase). In animal cells, S phase is induced by Cdk2 bound to S-phase cyclins (E-and A-type) whereas M phase is triggered by Cdk1 associated with mitotic cyclins (A-and B-type). In both fission yeast and budding yeast, S and M phase are induced by a single CDK (Cdk1) bound to Sphase-and M-phase-specific B-type cyclins, respectively. We now understand many of the regulatory mechanisms that activate S-and M-CDKs in the correct order. We also have a robust hypothesis for how cells ensure that no genomic sequence is duplicated more than once during the interval between the onset of S and M phases. Initiation of DNA replication requires two distinct steps: first, prereplicative complexes (pre-RCs) are assembled at future origins of replication, a process that can occur only in the absence of CDK activity. The second step, origin unwinding and the recruitment of replication enzymes, is triggered by CDK activation. Because pre-RC assembly is inhibited by CDK activity, chromosome rereplication requires a CDK cycle, a period of low CDK activity followed by a period of high CDK activity. Having activated S-CDKs in late G 1 , cells maintain high CDK activity until metaphase and this prevents refiring of replication origins.However, several crucial elements were missing from this CDK-dominated view of the cell cycle. Missing was the impetus that causes sister chromatids to separate at the metaphase-to-anaphase transition; the machinery that destroys mitotic cyclins during anaphase; a mechanism for ensuring that sister chromatid separation normally precedes cytokinesis and chromosome reduplication; and an understanding of how events that trigger sister chromatid separation and exit from mitosis also create the conditions that cause the chromosome cycle to be repeated. Insight into all these questions has recently stemmed from the identification of the machinery responsible for degrading mitotic cyclins, a ubiquitin-protein ligase called the anaphase-promoting complex or cyclosome (APC/C). By destroying anaphase inhibitory proteins, the APC/C triggers the separation of sister chromatids; by destroying mitotic cyclins, it creates the low CDK state necessary for cytokinesis and for reforming the pre-RCs c...

Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC). Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase. Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1). Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction. The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase.

Cohesion between sister chromatids during G2 and M phases depends on the "cohesin" protein Scc1p (Mcd1p). Loss of cohesion at the metaphase to anaphase transition is accompanied by Scc1p's dissociation from chromatids, which depends on proteolysis of Pds1p mediated by a ubiquitin protein ligase called the anaphase promoting complex (APC). We show that destruction of Pds1p is the APC's sole role in triggering Scc1p's dissociation from chromatids and that Pds1p forms a stable complex with a 180 kDa protein called Esp1p, which is essential for the dissociation of Scc1p from sister chromatids and for their separation. We propose that the APC promotes sister separation not by destroying cohesins but instead by liberating the "sister-separating" Esp1 protein from its inhibitor Pds1p.