Polymer Models of Meiotic and Mitotic Chromosomes

Polymers tied together by constraints exhibit an internal pressure; this idea is used to analyze physical properties of the bottle-brush–like chromosomes of meiotic prophase that consist of polymer-like flexible chromatin loops, attached to a central axis. Using a minimal number of experimental parameters, semiquantitative predictions are made for the bending rigidity, radius, and axial tension of such brushes, and the repulsion acting between brushes whose bristles are forced to overlap. The retraction of lampbrush loops when the nascent transcripts are stripped away, the oval shape of diplotene bivalents between chiasmata, and the rigidity of pachytene chromosomes are all manifestations of chromatin pressure. This two-phase (chromatin plus buffer) picture that suffices for meiotic chromosomes has to be supplemented by a third constituent, a chromatin glue to understand mitotic chromosomes, and explain how condensation can drive the resolution of entanglements. This process resembles a thermal annealing in that a parameter (the affinity of the glue for chromatin and/or the affinity of the chromatin for buffer) has to be tuned to achieve optimal results. Mechanical measurements to characterize this protein–chromatin matrix are proposed. Finally, the propensity for even slightly chemically dissimilar polymers to phase separate (cluster like with like) can explain the apparent segregation of the chromatin into A+T- and G+C-rich regions revealed by chromosome banding.

The human SWI/SNF-B chromatin-remodeling complex is related to yeast Rsc and localizes at kinetochores of mitotic chromosomes

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

Studies on the structure of the chromosomes - I. Allium cepa-Stain fixatives and their utility

The structure of the mitotic chromosomes of Allium cepa has been elucidated by controlling the temperature and time of exposure of fresh roots to stain fixatives. The details seen in material stained in N HCl-orcein for 8 min. at 60° C. and squashed after varying intervals of storage at room temperature were essentially similar to pictures obtained with 1% aceto-orcein and 1% aceto-orcein-N HCl (10:1) under identical conditions of handling. The chromosomes appear quadri-partite at metaphase and bi-partite at anaphase. A rare instance of the precocious assumption of a quadri-partite condition by two anaphase chromosomes is illustrated. Caduceus coiling of chromonemata was seen in chromosome bridges also. Chromosomes have material easily dissociable from the chromonemata and their removal does not affect the structural integrity of the chromosome.

Application of C-banding in two Arachis wild species, Arachis pintoi Krapov. and W.C. Gregory and A-villosulicarpa Hoehne to mitotic chromosome analyses

Two wild diploid (2n = 20 chromosomes) and self-pollinating Arachis species, Arachis Pintoi Krapov and W.C.Gregory and A. villosulicarpa Hoehne were submmited to C-band technique to karyotype analyses. Root tips were employed in the analyses. Morphometric data chose that chromosome lengths varied from 3.12 in A. villosulicarpa to 1.45 in A. Pintoi. Karyotype formula obtained was 10sm to A. Pintoi and 9sm + 1m to A. villosulicarpa. There was a predominance of pericentromeric C-band in all mitotic metaphasic chromosomes in both species. Besides C-band values, both species still did not differ in respect to chromosome absolute and relative lengths, centromeric index, symmetry index and total karyotype haploid length. C-band and morphometric data did not show strong or significant differences which could separate these two species of peanut which belong to evolutive different sections.

Molecular cytogenetic analysis of heterochromatin in the chromosomes of tilapia, Oreochromis niloticus (Teleostei : Cichlidae)

The structure of the heterochromatic bands in mitotic chromosomes of the important tropical aquaculture species of tilapia, Oreochromis niloticus, was investigated by the combination of the C-banding technique, chromosomal digestion with two restriction endonucleases and fluorescence in situ hybridization (FISH) of two satellite DNAs (SATA and SATB). The tilapia chromosomes presented heterochromatic bands in the centromeres and in the short arms of almost all chromosomes that were differentially digested by the restriction endonucleases HaeIII and EcoRI. FISH of SATA showed that the satellite sequence is distributed in the centromeric region of all chromosomes of tilapia. FISH also revealed an intense hybridization signal for SATB in only one chromosome pair, but less intense signals were also present in several other pairs. The digestion of tilapia chromosomes by HaeIII and EcoRI was positively correlated with the position of SATA and SATB in chromosomes as revealed by FISH. The results obtained may be useful in future molecular and genetic studies of tilapias.

Phosphorylation of Histone H3S10 in Animal Chromosomes: Is There a Uniform Pattern?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Application of C-banding in two Arachis wild species, Arachis Pintoi Krapov. and W.C. Gregory and A. villosulicarpa Hoehne to mitotic chromosome analyses

Two wild diploid (2n = 20 chromosomes) and self-pollinating Arachis species, Arachis Pintoi Krapov and W.C. Gregory and A. villosulicarpa Hoehne were submmited to C-band technique to karyotype analyses. Root tips were employed in the analyses. Morphometric data chose that chromosome lengths varied from 3.12 in A. villosulicarpa to 1.45 in A. Pintoi. Karyotype formula obtained was 10sm to A. Pintoi and 9sm + 1m to A. villosulicarpa. There was a predominance of pericentromeric C-band in all mitotic metaphasic chromosomes in both species. Besides C-band values, both species still did not differ in respect to chromosome absolute and relative lengths, centromeric index, symmetry index and total karyotype haploid length. C-band and morphometric data did not show strong or significant differences which could separate these two species of peanut which belong to evolutive different sections.

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein–protein interactions involved in kinetochore assembly and function.

Triploidy induction in Heterobranchus longifilis (Family: Clariidae) by cold shock

Triploid was induced in African Catfish (Heterobranchus longifilis) by cold shocking activated eggs at 5 degree C for forty minutes starting 3-4 minutes after fertilization. Triploidy was confirmed from mitotic chromosomes prepared from embryo which showed 100% triploidy in the cold shocking treatment and 100% diploidy in the control treatment

Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo

Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosome’s shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Molecular mechanisms for the regulation of skin homeostasis

L'ubiquitination est une modification des protéines conservée, consistant en l'addition de résidus « ubiquitine » et régulant le destin cellulaire des protéines. La protéine « TRAF-interacting protein » TRAIP (ou TRIP) est une ligase E3 qui catalyse l'étape finale de l'ubiquitination. TRAIP est conservé dans l'évolution et est nécessaire au développement des organismes puisque l'ablation de TRAIP conduit à la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la réduction de l'expression de TRAIP dans des kératinocytes épidermiques humains réprime la prolifération cellulaire et induit un arrêt du cycle cellulaire en phase Gl, soulignant le lien étroit entre TRAIP et la prolifération cellulaire. Comme les mécanismes de régulation de la prolifération jouent un rôle majeur dans l'homéostasie de la peau, il est important de caractériser la fonction de TRAIP dans ces mécanismes. En utilisant des approches in vitro, nous avons déterminé que la protéine TRAIP est instable, modifiée par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont également révélé que l'expression de TRAIP est dépendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifié le facteur de transcription E2F1 comme en étant le responsable, en régulant directement le promoteur de TRAIP. Aussi, TRAIP endogène ou surexprimée est surtout localisée au niveau du nucléole, une organelle nucléaire qui est désassemblée pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imagé la protéine TRAIP fusionnée à une protéine fluorescente, à l'intérieur de cellules vivantes nommées HeLa, à l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localisée autour des chromosomes en début de mitose, puis est arrangée au niveau de l'ADN chromosomique en fin de mitose. La détection de TRAIP endogène à l'aide d'un anticorps spécifique a confirmé cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interférence ARN a inhibé leur capacité à s'arrêter en milieu de mitose. Nos résultats suggèrent que le mécanisme sous-jacent peut être lié au point de contrôle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock¬out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

Assessment of chromatin activity in mouse and human tissues

Pancreatic deoxyribonuclease preferentially digests active genes during all phases of the cell cycle including mitosis. Recently, a DNAse I-directed in ~ nick translation technique has been used to demonstrate differences in the DNAse I sensitivity of euchromatic and heterochromatic regions of mitotic chromosomes. This ill ~ technique has been used in this study to ask whether facultative heterochromatin of the inactive X chromosome can be distinguished from the active X chromosome in mouse and human tissues. In addition to this, in ~ nick translation has been used to distinguish constitutive heterochromatin in mouse and human mitotic chromosomes. Based on relative levels of DNAse I sensitivity, the inactive X chromosome could not be distinguished from the active X chromosome in either mouse or human tissues but regions of constitutive heterochromatin could be distinguished by their relative DNAse I insensitivity. The use of !D situ nick translation was also applied to tissue sections of 7.5 day mouse embryos to ask whether differing levels of DNAse I sensitivity could be detected between different tissue types. Differences in DNAse I sensitivities were detected in three tissues examined; embryonic ectoderm, an embryo-derived tissue, and two extraembryonic tissues, extraembryonic ectoderm and ectoplacental cone. Embryonic ectoderm and extraembryonic ectoderm nuclei possessed comparable levels of DNAse I sensitivity while ectoplacental cone was significantly less DNAse I sensitive. This suggests that tissue-specific mechanisms such as chromatin structure may be involved in the regulation of gene activity in certain tissue types. This may also shed some light on possible tissue specific mechanisms regulating X chromosome activity in the developing mouse embryo.

Elucidating the crosstalk between condensin subunits and its relevance in chromosome condensation

ADN subit une série de transformations structurelles complexes au cours de la division cellulaire, ce qui entraîne dans son compactage chromosomes mitotiques par un processus appelé la condensation des chromosomes. Le complexe de condensine pentamérique est fortement impliqué comme un effecteur majeur de ce phénomène. Il s'agit d'un complexe protéine de sous-unités multiples avec deux sous-unités catalytiques [SMC- Structural Maintenance of Chromosomes] et de trois sous-unités de régulation, hautement conservés de la levure à l'homme. Le complexe de condensine dans Saccharomyces cerevisiae est constitué de deux sous-unités de SMC [Smc2 et Smc4] et trois protéines non réglementaires [Brn1, Ycs4, Ycg1]. Malgré son importance, le mécanisme d'action de condensine reste largement inconnu. Par conséquent, l'objectif de cette recherche est de comprendre le mécanisme d'action de condensine et comment elle est affectée par l'interaction entre ses sous-unités réglementaires et non-réglementaires. Cette thèse identifie quatre morphologies dépendants du cycle cellulaire distincts du locus d'ADNr. Cette transformation du phénotype ADNr de G1 à la mitose dépend condensine. Afin de déterminer le rôle de l'interaction entre les sous-unités catalytiques et réglementaires de condensine dans la régulation du complexe condensine, nous avons identifié six résidus positifs sur l'extrémité C-terminale de BRN1 qui affectent la formation du complexe condensine, l'activité de la condensation et l'interaction avec tubuline, ce qui suggère que ces résidus ont un rôle dans la régulation de condensine. Ensemble, nos résultats suggèrent un modèle de règlement du condensine par l'interaction entre les sous-unités de condensine.