Abstract. Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin or a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase 11, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm/mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.T IlE organization of eukaryotic nuclei is designed for the storage and expression of the genetic material that consists of a set of linear chromosomes surrounded by the nuclear membrane . Eukaryotic chromosomal DNAs are highly folded (the 2 x 106-#m -long human genome DNA is stored in a 10-#m-diameter nucleus), even in micro-organisms with small genomes (total yeast DNA is 4,000 #m long and is packed in a 2-#m-diameter nucleus). A number of nuclear proteins may be involved in chromosomal DNA compaction (Gasser and Laemmli, 1987; Earnshaw and Bernat, 1991). Specific DNA sequences may also function in the organizational principles of chromosomal packaging. A question relevant to this problem is whether certain DNA sequences are essential for spatial arrangements of chromosomes in the nucleus. The fission yeast Schizosaccharomyces pombe is an ideal organism in which to address this question, since it has a small genome consisting of only three chromosomes and is amenable to fine genetic analysis (Yanagida, 1989;Nurse, 1990 We recently reported the application of the fluorescence in situ hybridization (FISH) ~ method to this organism (Uzawa and Yanagida, 1992) and suggested its exploitation in the localization of individual DNA sequences within the nucleus. As an initial step towards understanding the principles of nuclear organization in fission yeast, w...

Abstract. Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. . Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad/+ gene. We show that the sadl.1 mutation affected both spindle formation and function. The sadl + gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sadl.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sadl protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sadl association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sadl-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sadl led to association with the nuclear periphery. As Sadl was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sadl is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sadl may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDCS, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of this gene, plol +, indicates that it is essential. Loss of plol + function leads to a mitotic arrest in which condensed chromsomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plol + function is required high in the regulatory cascade that controls septation. The overexpression of plol + in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division. Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plol + in cdc25-22 or cdc2-33 cells arrested in G2; in GI cells arrested at Start by the cdclO-V50 mutation, or in cells lacking the cyclin B homolog cdcl3 that undergo repeated S phases in the absence of mitosis.[Key Words: S. pombe; polo homolog; bipolar spindle; actin ring; septum; plol kinase] Received January 20, 1995; revised version accepted March 16, 1995.The polo ~ mutation of Drosophila was first identified as a maternal-effect mutant giving rise to embryos that show abnormal networks of microtubules associated with condensed chromosomes, in which the centrosomal antigen CP190 {formerly called Bx63; Frasch et al. 1986;Whitfield et al. 1988) fails to become organized into centrosomes with microtubule-organizing capability (Sunkel and Glover 1988). The mutation results in multipolar spindles and nondisjunction in male meiosis leading to reduced fertility. There is a high mitotic index in larval neuroblasts, with a wide range of defects including monopolar spindles and spindles with broad poles (Sunkel and Glover 1988;Llamazares et al. 1991).polo encodes a 576-amino-acid protein that has an amino-terminal putative kinase domain and a 300-residue carboxy-terminal domain (Llamazares et al. 1991). The polo kinase is highly conserved. The budding yeast and murine genes, CDC5 (Kitada et al. 1993) and mouse Plk (polo-like kinase; Clay et al. 1993;Lake and Jelinek 1993;Hamanaka et al. 1994), for example, show 52% and 65% identity in the kinase domain and 33% and 43% in the carboxy-terminal domain, with the latter homolo3Corresponding author. gies lying in distinct blocks. The human Plk has also been identified (Golsteyn et al. 1994;Hamanaka et al. 1994;Holtrich et al. 1994), and studies in the mouse suggest that there is another closely related kinase, Snk (serum-induced kinase; Simmons et al. 1992), that falls into the same gene family. These mammalian genes are expressed only in proliferating cells or tissues suggesting a cell cycle role.In mutants of the budding yeast gene CDC5, nuclear division is arrested at a late stage and the spindle is elongated (Ha...

When the first mutant allele of the Drosophila gene polo was first characterized over 10 years ago, attention focused on the defects that centrosome behavior exhibited at various stages of development (Sunkel and Glover 1988). The subsequent realization that the serine-threonine kinase it encodes is highly conserved from yeasts to humans has provoked a flurry of investigation into the function of the enzyme. A role for the polo-like kinases (plks) in regulating centrosome behavior has been borne out in several organisms, and the enzymes have attracted further attention recently with the realization that they regulate multiple stages of mitotic progression. In this article we review the current status of our understanding of the functions of plks from the time of commitment to M phase in the activation of Cdc25, through the activation of the anaphase promoting complex (APC), to the regulation of late mitotic events essential for cytokinesis. We discuss how to reconcile the sometimes apparently disparate observations made upon plk function in different organisms.

The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.