Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.     

Toxicological Assessment of Arsenic-Induced Hematological Alterations and Chromosomal Aberrations in Tilapia Oreochromis mossambicus

Arsenic is an environmental contaminant and potential carcinogen. Toxicological assessment of As, which causes hematological alterations and chromosomal aberrations, was studied in freshwater fish Oreochromis mossambicus. Fish were exposed to 3 ppm, 28 ppm, and 56 ppm concentrations of sodium arsenite (NaAsO₂) and blood samples were collected after 48 h, 96 h, and 192 h of exposure. Hematological assay of exposed fish revealed abnormal mature and immature erythrocytes, deformed erythrocytes (spindle-shaped and triangular erythrocytes) and erythrocytes with segmented nuclei in all treatments. Arsenic exposure induced chromosomal aberration in a concentration-dependent manner, whereas, a decreasing trend was found after 192 h exposure. Observations on blood cells of exposed fish revealed chromosome breaks, chromatid breaks, and chromatid gaps. The alterations and aberrations of these parameters can be effectively used to assess toxicological effects of As on fish in the aquatic environment and at the same time this study elucidates the potential risks to humans who live in arsenic-contaminated areas.     

DNA damage in the kidney tissue cells of the fish Rhamdia quelen after trophic contamination with aluminum sulfate

Even though aluminum is the third most common element present in the earth''s crust, information regarding its toxicity remains scarce. It is known that in certain cases, aluminum is neurotoxic, but its effect in other tissues is unknown. The aim of this work was to analyze the genotoxic potential of aluminum sulfate in kidney tissue of the fish Rhamdia quelen after trophic contamination for 60 days. Sixty four fish were subdivided into the following groups: negative control, 5 mg, 50 mg and 500 mg of aluminum sulfate per kg of fish. Samples of the posterior kidney were taken and prepared to obtain mitotic metaphase, as well as the comet assay. The three types of chromosomal abnormalities (CA) found were categorized as chromatid breaks, decondensation of telomeric region, and early separation of sister chromatids. The tests for CA showed that the 5 mg/kg and 50 mg/kg doses of aluminum sulfate had genotoxic potential. Under these treatments, early separation of the sister chromatids was observed more frequently and decondensation of the telomeric region tended to increase in frequency. We suggest that structural changes in the proteins involved in DNA compaction may have led to the decondensation of the telomeric region, making the DNA susceptible to breaks. Moreover, early separation of the sister chromatids may have occurred due to changes in the mobility of chromosomes or proteins that keep the sister chromatids together. The comet assay confirmed the genotoxicity of aluminum sulfate in the kidney tissue of Rhamdia quelen at the three doses of exposure.     

DNA损伤反应性蛋白参与中心体调控

中心体是动物细胞有丝分裂期微管组织中心,对于细胞有丝分裂期形成纺锤体、正常分裂及染色体精确分离至关重要. 中心体失调控常造成遗传物质错误分配,最终诱发肿瘤形成.因此,对中心体结构及数量的精密调控将对细胞命运起着决定 作用.目前发现,中心体至少包含100多种调节蛋白,这些蛋白在细胞内的功能各异.最近很多研究显示,多种DNA损伤修复及 应答通路的激酶或磷酸酶定位于中心体,并且参与中心体调控.本文将对中心体结构、中心体复制、中心体分离、中心体扩 增、DNA损伤与中心体异常及DNA损伤反应性蛋白在中心体调控中的功能作一综述.     

Influence of physical environmental characteristics and anthropogenic factors on the position and structure of a contact zone between two chromosomal races of the house mouse on the island of Madeira (North Atlantic, Portugal)

Aim This study aimed to investigate if and how environmental characteristics (physical factors of the natural environment and the human impact on the landscape) influence the position and structure of a contact zone between two chromosomal races of the house mouse (Mus musculus domesticus Rutty 1712) from the island of Madeira. Location The western part of Madeira, a volcanic island in the North Atlantic. Methods Mice were sampled along a south/north‐western transect following the main road, in human‐modified outdoor habitats. Karyotypes of mice were determined using the yeast‐stimulated bone marrow cell method. Trapping sites were characterized in terms of their physical (altitude, temperature, precipitation and soil type) and habitat (human landscape use and occupancy) features. Demographic parameters of mouse populations, based on trapping‐with‐removal techniques, were also analysed (relative abundance, sex‐ratio, juvenile ratio and female fertility ratio), as well as body size (weight and length). Results Four chromosomal zones were identified on the basis of the frequency of two diagnostic rearrangements (Rb(6.7) in race E. Calheta and Rb(7.15) in race A. Cruz). E. Calheta was present in the two southern‐most zones, followed by the contact zone characterized by the presence of two inter‐racial hybrids and the co‐occurrence of mice belonging to the two races. The northern‐most part of the transect was occupied by A. Cruz. Environmental features differed leading us to split the transect into two parts. The southern part is characterized by lower altitude and precipitation, milder temperature, better soil quality supporting vegetable crops and vineyards, and more abundant and evenly distributed human habitats. This southern part is occupied by E. Calheta mice. The north‐western part presents characteristics opposite to those described above with cereals as the main cultivated crop, and it includes the contact zone as well as the zone inhabited by A. Cruz mice. The demographic parameters evaluated in this study did not differ significantly between chromosomal zones. Main conclusions This ecological survey highlights differences in climatic and edaphic features that have moulded the agricultural activities of humans, contributing to a differentiation of their spatial development, and hence the structure of potential habitats for mice. Results are interpreted within the source–sink framework of population dynamics, following which E. Calheta may function as a source and the areas where A. Cruz and the contact zone are located may function as a sink. Our study suggests that the position and chromosomal composition of the contact zone is influenced by the human component underlying broader environmental features. Similar characteristics were most likely present during the historical settlement of Madeira. They may have favoured the independent divergence of the two races and influenced the dynamics of the contact zone.     

Non-metric morphological divergence in the western house mouse, Mus musculus domesticus, from the Barcelona chromosomal hybrid zone

The effect of hybridization on morphological variation was investigated in 120 western house mice, Mus musculus domesticus , from the hybrid zone between the Barcelona and standard chromosomal races. The incidence of 37 non-metric cranial traits was calculated for standard mice (2 n  = 40) and Barcelona-standard hybrids (2 n  = 27–39). Subsequent analyses were conducted on several karyological subgroups, established by grouping the animals according to either their diploid number or their degree of chromosomal heterozygosity. Results revealed no significant difference by sex, asymmetry, or geographical distance. Significant phenetic divergences were found between the karyotypes studied in relation to several variants. Differences were especially substantial between the standard race and hybrid mice, even with respect to those hybrids with karyotypes close to that of the standard race. Within the hybrids, the maximum divergence corresponded to the 28-chromosome homozygotes, chromosomally close to the Barcelona race, and to the heterozygotes with more than two fusions. Since differences in non-metric trait frequencies are generally considered a measure of genetic divergence, the results suggest the occurrence of a barrier to gene flow in the Barcelona hybrid zone. The decrease of genetic exchange between the chromosomally differentiated mice might be due to reduced fertility in hybrids, associated with chromosomal heterozygosity.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 313–322.     

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: evidence for the organizing role of the nuclear matrix

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%-80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin-depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.     

Broadening the insecticidal spectrum of Lepidoptera-specific Bacillus thuringiensis strains by chromosomal integration of cry3A

A TnpI-TnpIA-mediated and thermosensitive recombination system was developed to construct genetically modified Bacillus thuringiensis strains encoding a crystal protein particularly active against Coleopteran species. Based on B. thuringiensis transposon Tn4430, an integrative vector, pBMB-R14E, was constructed, by which the cry3A delta-endotoxin gene highly toxic to Lepidoptera was delivered into a wildtype B. thuringiensis subsp. kurstaki strain YBT1520. The cry3A gene was integrated into the chromosome of the host strain. Then the integrative vector was eliminated by moving recombinant cultures to 46 degrees C. Two recombinant B. thuringiensis strains, BMB1520-S and BMB1520-T, were obtained. In recombinant strains, the cry3A gene was stably expressed in measurable amounts and did not reduce the expression of endogenous crystal protein genes. Bioassay results showed that BMB1520-S and BMB1520-T, in addition to the activity against lepidopteran Plutella xylostella third-instar larvae present in the parental strains, exhibited a high level of activity against coleopteran Rhyllodecta vulgatissima third-instar larvae, absent from the parental strains.