Abstract.A mutant, ndclO-1, was isolated by antitubulin staining of temperature-sensitive mutant banks of budding yeast, ndclO-1 has a defect in chromosome segregation since chromosomes remain at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPB). NDCIO was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechner, and J. Carbon. 1993. J. Cell Biol. 121:513-519) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 64:717-725). NDCIO is an essential gene. Antibodies to Ndcl0p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndclO-1 are consistent with Cbf2p/Ndcl0p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.

Chromosome separation during the cell-cycle transition from metaphase to anaphase requires the proteolytic destruction of anaphase inhibitors such as Pds1 [1-3]. Proteolysis of Pds1 is mediated by a ubiquitin-protein ligase, the anaphase-promoting complex (APC) or cyclosome [4,5]. The APC is also necessary for the ubiquitin-dependent degradation of mitotic cyclins in late telophase as cells exit mitosis [6-9]. Although phosphorylation seems to be involved [10], it is not clear what activates the APC at the onset of anaphase. In Saccharomyces cerevisiae, chromosome segregation also requires the CDC20 gene, whose product contains WD40 repeats [11,12]. We have investigated the functional relationship between the APC and the Cdc20 protein. We present evidence that strongly suggests that Cdc20 is an essential regulator of APC-dependent proteolysis such that in the absence of Cdc20, cells are unable to degrade either Pds1 at the onset of anaphase or the mitotic cyclin Clb2 during telophase. This notion is consistent with our observations that Cdc20 is localized in the nucleus and co-immunoprecipitates with an APC component, Cdc23.

Abstract. We have been investigating a set of genes, collectively called mups, that are essential to striated body wall mu...__scle cell _positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat-sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(upl), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail.The mup-2 mutations lie within these cDNA sequences: mup-2(upl) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intraceUular Ca 2÷. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles. STUDIES of many organisms, including vertebrates, indicate that the stable attachment of muscle to the skeleton requires muscle sarcomere assembly, muscle contraction and extracellular matrix formation. Caenorhabditis elegans offers unique advantages for investigations of the cellular mechanisms influencing the establishment and maintenance of muscle attachment. C. elegans has a simple cellular anatomy of only 959 adult somatic ceils and contains a small number of muscle types (for review see Waterston, 1988). The cell divisions and migrations giving rise to these muscle types have been fully described at the cellular level (Sulston et al., 1983). Since the spatial relationships of individual muscle cells are easily visualized and are invariant, the attachments of muscle cells can be precisely determined in wild-type and mutant worms. Given these attributes, it is possible to study specific gene mutations to

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.