No abstract

The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APCdependent proteolysis.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34Cdc2 is phosphorylated on two sites, one of which has been identified as Tyrl5. Dephosphorylation of Tyrl5 regulates the initiation of mitosis. To understand more completely the regulation of p34""'2 kinase activity, we have identified the second site of phosphorylation as Thrl67, a residue conserved amongst all p34Cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thrl67 phosphorylation is required for p34"""2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thrl67 might also play a role in the exit from mitosis.

Human (HeLa) cells contain a host-cell-encoded transcription factor, Sp1, which is required for transcription of simian virus 40 (SV40) promoters. Since the discovery of Sp1 we have been interested in learning what role this factor plays in uninfected cells. A monkey cellular gene promoter interacts with Sp1, but no gene products linked to this promoter have yet been identified. The finding that the sequence of the 5'-flanking DNA of the mouse dihydrofolate reductase (DHFR) gene contains several regions showing strong homology to the Sp1 binding region of simian virus 40 (SV40) prompted us to undertake experiments with dhfr. We report here that Sp1 binds to these regions in the dhfr promoter, and that Sp1-containing preparations stimulate transcription from the dhfr promoter in an in vitro reaction. Our results suggest that in addition to its interactions with the SV40 viral promoter, one function of Sp1 is to direct the expression of the cellular DHFR gene.

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.