| 1. |
- Appelgren, Henrik, et al.
(author)
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Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells
- 2003
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:19, s. 4035-4042
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Journal article (peer-reviewed)abstract
- Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one Another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.
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| 2. |
- Kniola, Barbara
(author)
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Methods in electron microscopic quantitative immunocytochemistry using adenohypophysis as a model
- 1994
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Doctoral thesis (other academic/artistic)abstract
- Quantitative ultrastructural immunocytochemistry (QUIC) is based on the assessment of immunogold labeling over the surface of ultrathin resin-embedded sections. The density of specific labeling, expressed as the number of gold particles/mm2 of a specific area can only be used as an approximative estimation of antigen concentration in the object.The reliability of QUIC depends on the quality of the methods used to prepare the samples; therefore, to make QUIC reliable, the entire preparative protocol! has to be evaluated, step by step. Secretory granules in cells of the pituitary gland have been chosen as a model system. They constitute targets that are sensitive enough for analyses of exogenous factors, such as fixation, dehydration, embedding and immunostaining. Morphological parameters, such as diameter, area, roundness and gray values could be measured by computerized image analysis and the results related to the experimental factors.Certain combinations of processing for EM were found to give the highest immunolabeling. A further criteria was, however, added-reproducibility of immunostaining among series of ultrathin sections. The most consistent labeling densities were found after cryofixation followed by physical dehydration and embedding in epoxy resin. One of the explanations could be that the greater resin surface relief of the latter provides a larger accessible area for the attachment of immunogold reactants. The advantages of certain techniques, such as EM in STEM mode and X-ray microanalysis were demonstrated and facilitated the evaluation of techniques in QUIC.
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| 3. |
- Kniola, Barbara, et al.
(author)
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The domain structure of centromeres is conserved from fission yeast to humans
- 2001
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In: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 12:9, s. 2767-2775
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Journal article (peer-reviewed)abstract
- The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.
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| 4. |
- Provost, P, et al.
(author)
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Dicer is required for chromosome segregation and gene silencing in fission yeast cells
- 2002
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 99:26, s. 16648-16653
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Journal article (peer-reviewed)abstract
- RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1(+)) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximate to23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1(+) was required for silencing of two genes containing a conserved motif.
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