p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.

Conserved structural motifs in cyclins identified by sequence analysis.

Cyclins, as regulatory subunits of the ubiquitous p34cdc2 protein kinase, act as key controlling elements of the eukaryotic cell cycle. We have examined published sequences of A- and B-type cyclins for both amino acid and secondary structure homologies. In particular, we sought regions of homology outside the recognised area of sequence conservation known as the "cyclin box', as well as conserved features predicted to lie at the protein surface. Our analysis demonstrates the existence of a number of islands of homology outside the cyclin box, and indicates candidate residues for phosphorylation. One of these, a motif containing the amino acids SPXXXE/D is also present in fission yeast p13suc1, another protein known to interact with p34cdc2. This motif may define a possible p34cdc2 binding or phosphorylation site. A database search revealed that the CDC25 and SCD25 genes of the budding yeast Saccharomyces cerevisiae also contain some of the newly identified motifs, perhaps indicating a common regulatory or degradation pathway.

Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin.

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification. The implication of this novel finding for the biological role of these events is discussed.

Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast.

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.

Distribution of tubulin and actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. versatilis: a comparison with Schizosaccharomyces pombe.

Changes in the distribution of microtubules and F-actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. versatilis were investigated by fluorescence microscopy. The fluorescence images obtained with S. japonicus were markedly superior to those previously reported for S. pombe and revealed new details of cytoskeletal organization in this important genus. As in S. pombe, F-actin in S. japonicus was present as a concentration of 'dots' at the growing poles of interphase cells and as a filamentous equatorial ring directing the deposition of the cytokinetic septum. The transition between these two states occurred at late anaphase, in contrast to the situation in S. pombe where the appearance of the equatorial actin ring is tightly coupled to the early events of mitosis. During the course of cytokinesis in S. japonicus the actin ring constricted and broadened, suggesting that it is contractile. Microtubule organization in S. japonicus also revealed interesting differences from S. pombe. Whereas in S. pombe cytoplasmic microtubules are reinitiated from a pair of microtubule organizing centres (MTOCs) at the cell equator, in S. japonicus they arise by extensive microtubule growth from the spindle poles. Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography showed that, like S. pombe, S. japonicus contains two alpha-tubulins and a single beta-tubulin. Whilst the alpha 1- and beta-tubulins from the two species comigrated on one-dimensional polyacrylamide gels, the alpha 2 species were electrophoretically distinct. Although fundamental differences clearly exist between the two species, S. japonicus could prove to be a useful tool in basic studies of fission yeast cell biology.

The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization.

The products of the cdc13+ and cdc2+ genes form a stable complex that displays protein kinase activity in vitro. p63cdc13 is a substrate of p34cdc2, the catalytic subunit of the kinase. The histone H1 kinase activity of cdc2 oscillates during the cell cycle. Activation of the preformed cdc2/cdc13 complex at the G2/M transition requires cdc25+ gene function. Post-metaphase inactivation of the kinase is associated with loss of cdc13, which shares sequence homology with mitotic cyclins and, in common with these proteins, is degraded at each cell division. cdc13 and cdc2 co-localize in the cell nucleus. cdc2 is not degraded during mitosis, but in the absence of cdc13 it is not localized in the nucleus. These observations suggest that the cdc13+-encoded cyclin acts to regulate both the catalytic properties and the localization of the protein kinase of which it is a subunit.

Fission yeast cyclin: subcellular localisation and cell cycle regulation.

Entry into mitosis in the fission yeast Schizosaccharomyces pombe involves the interaction of a number of genes with the major cell cycle control gene, cdc2+. One of these, cdc13+, encodes a protein with homology to cyclin. By indirect immunofluorescence microscopy using antibodies to the appropriate bacterially-expressed protein, we have shown that both cdc13 and cdc2 are nuclear proteins in S. pombe. Both are localised to a nuclear domain distinct from that occupied by the DAPI-staining chromatin. The immunofluorescence signals of both proteins show a progressive increase during interphase but are undetectable at mitosis. Loss of cdc13 fluorescence at mitosis reflects the destruction of the protein. Thus, it behaves as a classic cyclin. This is not the case for cdc2, the level of which remains constant through the cell cycle. Cells carrying a disrupted copy of the cdc13+ gene fail to accumulate either cdc13 or cdc2 in the nucleus. Cells carrying a disrupted cdc2+ gene fail to accumulate cdc2 but reveal apparently normal levels of cdc13. cdc13 therefore appears to be required to localise cdc2 to the nucleus but not vice versa. The destruction of cdc13 at mitosis may allow cdc2 to redistribute to the cytoplasm.

Increased sensitivity to erythromycin in Escherichia coli associated with the presence of the ColV,I-K94 virulence plasmid.

Introduction of the virulence plasmid, ColV,I-K94, into Escherichia coli strains led to increased sensitivity to erythromycin. This was the result of increased passage of antibiotic into ColV,I-K94+ organisms because the plasmid effect was abolished in bacteria which had been made permeable by chemical treatment. Full sensitivity in ColV+ strains appears to depend on the simultaneous presence of transfer and colicin components. Increased erythromycin sensitivity associated with the plasmid was demonstrated in organisms grown at 37 degrees C; the sensitivity of ColV,I-K94+ organisms grown at 25 degrees C was similar to that of the parent strain. Added Mg++ or Ca++ ions reversed erythromycin inhibition in strains with the basal level of sensitivity (i.e., the Col- parent grown at 25 degrees C or 37 degrees C or the ColV,I-K94+ derivative grown at 25 degrees C) and in those with the plasmid-associated increase in sensitivity. Addition of phosphate or EDTA to broth increased erythromycin sensitivity in Col- and ColV,I-K94+ strains although the latter was affected most. Erythromycin was more inhibitory at pH 8.5 than at pH 7.4. This enhanced activity was more marked against the ColV,I-K94+ strain than against the Col- strain. The effects of growth in phosphate-containing medium and at alkaline pH were partially additive. We suggest that ColV,I-K94+ strains may be sensitive to erythromycin because ColV-specified proteins are extruded by a process of "self-promoted transfer" and that the effects of these proteins on the lipopolysaccharide component of the outer membrane facilitates antibiotic influx.