Spatial distribution of GC- and AT-rich DNA sequences within human chromosome territories

Previous topological analyses of DNA sequence organization in the interphase chromosome mainly focused on the spatial distribution of individual gene copies within chromosome territories. In order to achieve a more comprehensive view into the subchromosomal arrangement of DNA, we isolated the GC-richest/gene-richest fraction (H3 isochores) as well as AT-richest/gene-poorest fraction of human genomic DNA (L1+L2 isochores) and visualized the respective DNA within individual chromosome territories by means of dual-color FISH. Application of confocal laser scanning microscopy and dedicated 3D image analysis software, which differentiated territory subvolumes by peeling shells one voxel in width, revealed a significant difference in the intraterritorial distribution of these two DNA sequence classes. While the H3 isochores were found localized in all subvolumes of the territories at similar frequency, simultaneously detected L1+L2 isochores were observed more to the interior of the same chromosome territories. Thus the GC-rich sequences display a much higher variability in their intraterritorial localization than AT-rich DNA fragments.     

Identification of Chromosomal Bands Replicating Early in the S Phase of Normal Human Fibroblasts

Normal human fibroblasts (NHF1) were released from confluence arrest (G₀) and replated in medium containing bromodeoxyuridine (BrdU) and aphidicolin. Despite severe reduction in the rate of DNA synthesis by aphidicolin, cells reentering the cell cycle incorporated BrdU at regions of the human genome that replicated very early in S phase. After removal of aphidicolin and BrdU from the tissue culture medium, cells were collected in mitosis. Q-banding with 4′,6-diamidino-2-phenylindole/actinomycin D was used to identify metaphase chromosomes. A monoclonal anti-BrdU antibody and a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody were used to identify the BrdU-labeled sites. The criterion for scoring DNA replication sites was the detection of FITC fluorescence at homologous regions of both sister chromatids. Early replicating regions mapped within R-bands, but not all R-bands incorporated BrdU. Chromosomal bands 1p36.1, 8q24.1, 12q13, 15q15, 15q22, and 22q13 were labeled in 53% or more of the copies of these chromosomes in the data set, suggesting that these sites replicated very early in S phase. Chromosomal band 15q22 was the most frequently labeled site (64%), which indicates that it contains some of the earliest replicating sequences in normal human fibroblasts.     

Enhancer Dependent Repositioning of TCRb Locus with Respect to the Chromosome Territory

Intranuclear position of several genes is dynamically altered during development concordant with their activation. To understand this dynamic, but non-random, nuclear organization, it is important to identify the relevant regulatory elements and trans acting factors. Murine TCRb locus gets activated during thymic development. Enhancer Eb is important for VDJ recombination at TCRb locus as it is critically required for establishment of recombination center. Our analysis revealed that TCRb locus gets located out of the chromosome territory specifically in developing thymocytes. Further, CRISPR/Cas9 based deletion mutagenesis established an unambiguous role of enhancer Eb in defining TCRb location relative to chromosome territory. The ability to reposition the target locus relative to chromosome territory highlights a novel aspect pertaining to activity of enhancers which may contribute to their ability to regulate gene expression. Additionally, our observations have implications for understanding the role of enhancers in three-dimensional genome organization and function.     

Homologous Chromosome Pairing in Drosophila melanogaster Proceeds through Multiple Independent Initiations

The dynamics by which homologous chromosomes pair is currently unknown. Here, we use fluorescence in situ hybridization in combination with three-dimensional optical microscopy to show that homologous pairing of the somatic chromosome arm 2L in Drosophila occurs by independent initiation of pairing at discrete loci rather than by a processive zippering of sites along the length of chromosome. By evaluating the pairing frequencies of 11 loci on chromosome arm 2L over several timepoints during Drosophila embryonic development, we show that all 11 loci are paired very early in Drosophila development, within 13 h after egg deposition. To elucidate whether such pairing occurs by directed or undirected motion, we analyzed the pairing kinetics of histone loci during nuclear cycle 14. By measuring changes of nuclear length and correlating these changes with progression of time during cycle 14, we were able to express the pairing frequency and distance between homologous loci as a function of time. Comparing the experimentally determined dynamics of pairing to simulations based on previously proposed models of pairing motion, we show that the observed pairing kinetics are most consistent with a constrained random walk model and not consistent with a directed motion model. Thus, we conclude that simple random contacts through diffusion could suffice to allow pairing of homologous sites.     

The Implication of Early Chromatin Changes in X Chromosome Inactivation

1. Download high-res image (238KB)
2. Download full-size image

     

Gene dynamics and nuclear architecture during differentiation

Abstract Recent advances have demonstrated that placing genes in a specific nuclear context plays an important role in the regulation of coordinated gene expression, thus adding an additional level of complexity to the mechanisms of gene regulation. Differentiation processes are characterized by dynamic changes in gene activation and silencing. These alterations are often accompanied by gene relocations in relation to other genomic regions or to nuclear compartments. Unraveling of mechanisms and dynamics of chromatin positioning will thus expand our knowledge about cellular differentiation.     

染色体展片法观察拟南芥雄性减数分裂过程中的染色体形态

减数分裂指DNA复制1次, 细胞核分裂2次, 产生染色体数目减半的单倍体配子, 是真核生物有性生殖所必需的环节。拟南芥(Arabidopsis thaliana)是分子遗传学研究的传统模式生物。近年来, 随着显微镜技术的快速发展, 利用细胞学方法观察拟南芥减数分裂过程中的染色体形态和同源染色体互作事件, 将有助于深入认识减数分裂的分子遗传机制。该文详细描述了染色体展片法观察拟南芥雄性减数分裂细胞中的染色体形态。     

Chromosome choreography during the non-binary cell cycle of a predatory bacterium

1. Download : Download high-res image (97KB)
2. Download : Download full-size image

     

恶性血液病中着丝粒和着丝粒周染色体重排的研究进展

用染色体特异的卫星DNA探针染色体荧光原位杂交（FISH）分析恶性血液病,发现着丝粒和着丝粒周染色体的重排并非罕见。分子生物学技术和基因组计划的发展,促进了对异染色质分子本质的研究。本就着丝粒和着丝粒周异染色质的分子结构及其重排机制作一综述。     

染色体展片法观察拟南芥雄性减数分裂过程中的染色体形态

减数分裂指DNA复制1次, 细胞核分裂2次, 产生染色体数目减半的单倍体配子, 是真核生物有性生殖所必需的环节。拟南芥(Arabidopsis thaliana)是分子遗传学研究的传统模式生物。近年来, 随着显微镜技术的快速发展, 利用细胞学方法观察拟南芥减数分裂过程中的染色体形态和同源染色体互作事件, 将有助于深入认识减数分裂的分子遗传机制。该文详细描述了染色体展片法观察拟南芥雄性减数分裂细胞中的染色体形态。     

植物染色体原位杂交技术的发展与现状

本文主要介绍植物染色体原位杂交技术的发展历史,评述适用于不同研究目的的各种主要原位杂交技术的基本原理和方法,并介绍该技术在植物细胞遗传学领域的应用和发展。 Abstract：In this paper,we briefly introduce the development of in situ hybridization of plant chromosome. The fundamental principle and method of many main kinds of in situ hybridization have been reviewed for different research purposes,and their application and development on plant cytogenetics also been recommended.     

中国遗传学会科普工作会议在京召开

培养温度是花药培养的一个十分重要的条 件。但是有关这方面研究的报道是不多的。特 别是早期的一些报道,不但内容比较简单,而且 试验的温度范围都在28℃ 以下^[7-9.14.16]近年 来甘肃农科院等一些单位开始试验用高温培 养,得到良好的效果^[4.5.10.15不过陈英等在水 稻上初步看到在高温培养下花粉愈伤组织的诱 导率虽然提高,但愈伤组织的分化能力有降低 的趋势,特别是当愈伤组织转分化培养基的时 间偏晚时更是如此^[5]。我们过去在小麦上也见 到过类似现象^[1]。因此近年来我们探索了在高 温下诱导的小麦花粉愈伤组织的分化能力的保 持间题。但在这一研究中又看到不同基因型对 培养温度有不同的反应,从而又就这种对培养 温度反应的基因型差异做了初步的遗传学分 析。本文先报道关于基因型差异方面的结果。 其他结果将另文报道。     

Structural Integrity of Centromeric Chromatin and Faithful Chromosome Segregation Requires Pat1

The kinetochore (centromeric DNA and associated protein complex) is essential for faithful chromosome segregation and maintenance of genome stability. Here we report that an evolutionarily conserved protein Pat1 is a structural component of Saccharomyces cerevisiae kinetochore and associates with centromeres in a NDC10-dependent manner. Consistent with a role for Pat1 in kinetochore structure and function, a deletion of PAT1 results in delay in sister chromatid separation, errors in chromosome segregation, and defects in structural integrity of centromeric chromatin. Pat1 is involved in topological regulation of minichromosomes as altered patterns of DNA supercoiling were observed in pat1Δ cells. Studies with pat1 alleles uncovered an evolutionarily conserved region within the central domain of Pat1 that is required for its association with centromeres, sister chromatid separation, and faithful chromosome segregation. Taken together, our data have uncovered a novel role for Pat1 in maintaining the structural integrity of centromeric chromatin to facilitate faithful chromosome segregation and proper kinetochore function.