Ser28磷酸化组蛋白H3在哺乳动物有丝分裂细胞中的动态分布

利用特异性抗Ser28磷酸化组蛋白H3抗体,应用间接免疫荧光标记技术,标记人乳腺癌细胞 (MCF-7)和小鼠成纤维细胞(NIH 3T3),用激光共聚焦显微术研究这两种哺乳动物细胞中Ser28磷酸化组蛋白 H3在有丝分裂过程中的动态分布,以研究Ser28磷酸化组蛋白在细胞有丝分裂过程中的作用.结果表明,Ser28 的磷酸化作用是这两种细胞有丝分裂期的特有事件.组蛋白H3的Ser28磷酸化信号首先出现在早期的核外周, Ser28磷酸化在中期达到高峰,并扩展到染色体的所有部分,后期和末期逐渐减退,随着胞质分裂的完成而消失. 实验结果表明,组蛋白H3 Ser28的磷酸化与有丝分裂染色体的凝集和解凝集过程有着时间和空间上的相关性. Ser28磷酸化使得组蛋白H3氨基末端的正电荷数降低,这可能是导致染色质变构凝集的原因之一.有丝分裂期 间组蛋白H3在Ser28位置磷酸化过程与Ser10相比有明显的差异,因此在动物细胞中,组蛋白H3氨基末端这 两个不同丝氨酸残基的磷酸化可能有不同的生物学功能.     

Thr3磷酸化的组蛋白H3在MCF-7细胞有丝分裂中的时空分布

利用SDS-PAGE和Western blotting证明了MCF-7细胞中组蛋白H3 Thr 3磷酸化是有丝分裂期特异性的.间接免疫荧光标记和激光共聚焦显微镜显示H3 Thr 3磷酸化起始于MCF-7细胞早前期的整个核中.事实表明,这种整体性的磷酸化是有丝分裂期间染色体整体性的变构凝集所必需的.中期,以点状分布的Th...     

洋葱根尖分生区细胞中的微管

用间接免疫荧光法在荧光显微镜下可以观察到植物根尖细胞分裂周期中微管捧列的变化.本文通过这一免疫细胞化学的方法研究了洋葱根尖分生区细胞中的微管周期.结果表明洋葱根尖细胞周期中有四种微管排列的方式,且这四种排列方式是一个循序渐进变化的过程,即周质微管、早前期带微管、纺锤体微管和成膜体微管.这四种微管的出现与细胞分裂周期密切相关,构成了典型的细胞微管周期.     

小麦根尖细胞有丝分裂同步化诱导与中期染色体分离

本研究以小麦根尖分生组织为材料，用Ｈｕ和ＡＰＭ双阻断法对细胞有丝分裂中期同步化和前期、后期、末期和部分同步化诱导作了探讨，并对中期染色体分离也作了研究，所得结构表明，用１．２５ｍ ｍｏｌ／Ｌ Ｈｕ、４μｍ ｍｏｌ／Ｌ ＡＰＭ处理，可使细胞有丝分裂中期指数（Ｍｅｔ．Ｉ）提高至５０％，经１８ｈ单独处理，去除Ｈｕ作用，水培使期恢复生长至８ｈ时，可检测到１１．５％的前期分裂细胞。Ｈｕ单独处理也可诱导后期和     

大蒜根尖细胞在观察有丝分裂中的应用

介绍大蒜根尖细胞有丝分裂标本的制备方法。该方法适用于课堂教学中对正常植物组织有丝分裂过程的观察。由于细胞中的微核、染色体畸变极易识别,也可用于检测环境诱变剂。     

黄瓜根尖细胞中微管冷稳定性和微管蛋白与其抗寒性的关系

以不同抗寒性黄瓜品种为试材,研究了经不同时间低温处理后黄瓜根尖细胞中微管的动态变化及抗寒锻炼后微管蛋白的变化,结果表明黄瓜根尖细胞中不同部位的微管冷稳定性有差异,质膜下的周质微管具有最强的冷稳定性,抗寒品种黄瓜根尖细胞中微管冷稳定性明显高于不抗寒品种,同时抗寒锻炼后微管蛋白的含量有所增加.     

EB1蛋白在调节微管动态、纺锤体定位和染色体稳定性中的作用

EB1(the end-binding protein 1)蛋白家族是一群广泛存在且高度保守的微管相关蛋白,存在于从酵母到人类的广泛的生物体中.它与微管正极和中心体结合,参与了绝大部分基于微管的生理过程,包括:维持细胞极性,调节染色体稳定性,有丝分裂纺锤体的定位,将微管锚定到成核位点.自从1995年,Su等人在人细胞中发现了EB1基因,各种生物体中EB1的同源物质被相继报道.十年来,人们通过对不同生物体的研究,试图揭开EB1在细胞中的分布以及它的生理功能.然而到目前为止,对于EB1的了解还非常有限.本文结合国外的研究成果,对EB1蛋白在调节微管动态、纺锤体定位和染色体的稳定性方面以及它与APC(the adenomatous polyposis coli)之间的相互作用作以综述.     

预处理对辣椒根尖细胞染色体制片的影响

该实验以中椒四号辣椒为实验材料,用常规压片法制备辣椒根尖细胞染色体标本．研究了短时间温度预处理对辣椒根尖细胞有丝分裂活性的影响,并比较了不同的预处理剂和冰水预处理时间对中期分裂相的影响．结果表明,经28℃非离体升温处理3h,冰水预处理24h、辣椒根尖可以获得数目较多、分散均匀的辣椒根尖细胞染色体早中期分裂相．     

~(60)Coγ—射线照射小麦种子对有丝分裂和减数分裂的影响

本文报道了不同剂量(1万仑、1.5万仑、2万仑、2.5万仑和3万仑)~(60)C_oγ—射线照射小麦干种子后,对发茅势、发芽率、初生根长度和根尖分生组织细胞有丝分裂和花粉母细胞减数分裂的影响. 电离辐射损伤引起根尖分生组织细胞有丝分裂的抑制作用随着剂量的增大而增强.并借助细胞学的方法观察了根尖分生组织细胞有丝分裂和花粉母细胞减数分裂中由于电离辐射所引起的染色体畸变,如小核(微核)、断片、桥、落后染色体、不均等分裂、染色质团缩、染色体环等细胞形态学变化.     

三极细胞有丝分裂中期纺锤体的激光共聚焦显微镜观察

用松胞素 Ｂ（ Ｃｙｔｏｃｈａｌａｓｉｎ Ｂ, Ｃ Ｂ）处理培养的 Ｈｅｌａ 细胞,抑制胞质分裂,引起 Ｈｅｌａ 细胞发生不正常分裂,可形成多极细胞（三极、四极等）．通过荧光免疫染色法显示多极细胞有丝分裂中期的微管,使用激光共聚焦显微系统观察三极细胞纺锤体和中期染色体的空间相对关系,推测了纺锤体微管的分布与有丝分裂后期染色体分离的相关性．本方法还可用于研究有丝分裂期纺锤体微管对胞质分裂分裂沟形成的影响．     

Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h (refs 2-6). During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. Here we show that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin.     

Regulation of cell cycle progression and gene expression by H2A deubiquitination

Post-translational histone modifications have important regulatory roles in chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on histone H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X-chromosome inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. Here we report the identification and functional characterization of the major deubiquitinase for histone H2A, Ubp-M (also called USP16). Ubp-M prefers nucleosomal substrates in vitro, and specifically deubiquitinates histone H2A but not H2B in vitro and in vivo. Notably, knockdown of Ubp-M in HeLa cells results in slow cell growth rates owing to defects in the mitotic phase of the cell cycle. Further studies reveal that H2A deubiquitination by Ubp-M is a prerequisite for subsequent phosphorylation of Ser 10 of H3 and chromosome segregation when cells enter mitosis. Furthermore, we demonstrate that Ubp-M regulates Hox gene expression through H2A deubiquitination and that blocking the function of Ubp-M results in defective posterior development in Xenopus laevis. This study identifies the major deubiquitinase for histone H2A and demonstrates that H2A deubiquitination is critically involved in cell cycle progression and gene expression.     

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.     

Multiple intron gain and loss events occurred during the evolution of Cenp-A gene

Centromere protein A(CENP-A) is a histone H3 like protein,and it plays a very important role in chromosomal segregation during mitosis and meiosis.The analyses on the exon-intron organization of the Cenp-A gene in representative genomes revealed that multiple intron gain and loss events have occurred during the evolution of Cenp-A gene in opisthokonta(common ancestor of fungi and animals).Moreover,our results revealed that at least two positions were conserved in the intron gain and loss events during the evolution of the Cenp-A gene.     

Crystal structure of the human centromeric nucleosome containing CENP-A

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg?80 and Gly?81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.     

Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.