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  • 1
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The centromeres of a genome separate in a sequential, nonrandom manner that is apparently dependent upon the quantity and quality of pericentric heterochromatin. It is becoming increasingly clear that the biological properties of a centromere depend upon its physicochemical makeup, such as its tertiary structure, and not necessarily on its particular nucleotide sequence. To test this idea we altered the physical state of the AT-rich pericentric heterochromatin of mouse with Hoechst 33258 (bis-benzimidazole) and studied a biological parameter, viz., sequence of separation. We report that an alteration in the physical state of heterochromatin, i.e., decondensation, is accompanied by aberrations in the pattern of centromere separation. The most dramatic effect seems to be on chromosomes with large blocks of heterochromatin. Many chromosomes with large blocks of heterochromatin that, in untreated cells, separate late tend to separate early. Decondensation with Hoechst 33258 does not seem to alter the sequence of separation of inactive centromeres relative to that of active centromeres. These data indicate that alteration in the physical parameters of the pericentric heterochromatin might dispose the centromeres to errors. It is likely that this aberration results from early replication of the pericentric heterochromatin associated with active centromeres.
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  • 2
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man. In vertebrates, Plks participate in centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex. Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machinery that controls exit from M-phase. Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells. We found that GFP-Plk1 associates with centrosomes, the equatorial spindle midzone and the postmitotic bridge of dividing cells, confirming and extending previous results obtained with conventional immunofluorescence microscopy. In addition, however, we observed fluorescence emanating from the midbody between dividing cells, and from discrete dots associated with mitotic chromosomes. This latter staining pattern being reminiscent of centromeres, we performed double-labeling experiments with antibodies against the centromeric marker CENP-B, and reexamined the subcellular localization of endogenous Plk1 using different fixation procedures. Our data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes. This novel localization of Plk1 suggests that substrates and/or regulators of Plks may be found among kinetochore-associated proteins with important functions in chromosome segregation and/or spindle checkpoint mechanisms.
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  • 3
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions.
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  • 4
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract.  We describe genetic interactions between mutations in mgr, asp, and polo, genes required for the correct behaviour of the spindle poles in Drosophila. The phenotype of a polo 1 mgr double mutant is more similar to mgr than polo 1 , but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The asp E3 mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. We discuss whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination. A polo 1 asp E3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo 1 phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. We speculate that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo.
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  • 5
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with α-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis.
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  • 6
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract.  TD-60 and INCENP are two members of the chromosome passenger protein family, and each has been suggested to play a role in the control of cytokinesis. Here we demonstrate by confocal immunofluorescence microscopy that TD-60 and INCENP distribute identically throughout the cell cycle. Both appear coordinately in G2-phase nuclei and become concentrated at centromeres during prophase. TD-60 and INCENP both then leave the chromosome together during anaphase and redistribute to the spindle midzone, as do other chromosome passenger proteins, and traverse the entire equatorial diameter from cortex to cortex. By image overlay and pixel count analysis we show that TD-60 and INCENP are distinct among known chromosome passenger proteins in extending beyond the spindle to the cortex. Further, we show that the cytokinesis-associated protein kinase AIM-1 also shares this distribution property. We suggest that this redistribution is required to signal cytokinesis. TD-60 and INCENP also show identical localization in cells that exit mitosis in the presence of dihydrocytochalasin B (DCB), an inhibitor of actin assembly. Such cells can resume cleavage upon removal of DCB and in a recovery subpopulation that cleaves only on one side, these proteins both colocalize to the cortex only where a cleavage furrow forms. Given the coincident distribution of TD-60 and INCENP during both interphase and mitosis, we suggest that these proteins may cooperate, perhaps within a protein complex, in signalling cytokinesis. Such a mechanism, using chromosome passenger proteins, may ensure that cytokinesis occurs only between the separated chromatids, and only after they have segregated.
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  • 7
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Genetic analysis of microtubule functions in the yeast Saccharomyces cerevisiae suggests that cells manage the levels and activities of the tubulin polypeptides. These reactions may be involved in protein folding, formation of the heterodimer, and maintenance of the appropriate balance between α- and β-tubulin. One protein involved in these functions is Rbl2p, which forms a complex with β-tubulin. Here we describe the identification of a novel yeast gene, RKI1, that interacts genetically with RBL2. Deletion of rki1 causes conditional defects in microtubule assembly and cell growth. Rki1p can be isolated in a complex containing Rbl2p. The results support the existence of cellular mechanisms for regulating microtubule function through the tubulin polypeptides.
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  • 8
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The fate of nuclear envelope proteins during the pachytene/metaphase I transition was investigated in rat spermatocytes cultured in vitro in the presence of the phosphatase inhibitor okadaic acid (OA). Under these experimental conditions lamin B1 and the lamina-associated proteins 2 (LAPs2) behave as already described in other cell types. In contrast to these results, meiotic lamin C2 appears to be degraded after addition of OA to the spermatocyte culture medium as this lamin was no longer detectable by immunofluorescence microscopy or by immunoblotting. Taking into account the peculiarities of the lamin C2 primary structure, it is tempting to speculate that degradation of this protein represents a critical step in the process of disassembly of the spermatocyte nuclear envelope.
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  • 9
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Several gene products involved in meiotic chromosome pairing and recombination in yeast have been identified in recent years. Two nuclear structures play key roles in the meiotic processes: the synaptonemal complex (SC), which is essential for the pairing of the chromosomes, and the recombination nodules (RNs), which mark the sites of recombination. Good morphological representation of the yeast SC and RNs is needed in order to show structural changes caused by specific mutations in protein-coding genes and for fine localization of proteins using immunoelectron microscopy (immuno-EM). This paper presents a newly developed preparation method for EM and immuno-EM that allows analysis of fine structural details and localization of proteins in the SC and RNs in yeast. Structural components of the SC are clearly seen and appear strikingly similar to those in the SC in other organisms. Antibodies against the SC protein Zip1, a transverse filament protein, label the central region of the SC strongly and specifically as expected. The improved method will be an important tool in high-resolution determination of the location of proteins in the meiotic yeast nucleus.
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  • 10
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes. The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase. In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process. Indeed, S. cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination. Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family. cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein. By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination. We mapped the mouse Spo11 gene to chromosome 2, band H2–H4.
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