蒜有丝分裂前期和前中期染色体的螺旋结构

本文研究了蒜(Allium sativum)根端细胞有丝分裂前期和前中期染色体的螺旋结构及其形成过程。在光镜和电镜下都看到前期核内存在螺旋化的染色线。从早前期到晚前期染色线的螺旋化是逐渐进行的。开始只有部分染色线螺旋化,螺幅直径约为1.2-1.5微米,以后螺幅增大,达1.8～2微米,并且螺旋结构变得更为紧密。前中期染色体中可见由直径600 nm左右的染色线形成的螺旋结构,螺旋比晚前期更加紧密。本文对中期染色体的高层次结构进行了讨论。     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌PhysarumpolycophalumSchw的营养生长阶段为没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质是不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩成染色质,分散的核仁物质逐渐合并形成新的核仁。     

多头绒泡菌有丝分裂后细胞核重建过程的形态学研究

以进行自然同步核内有丝分裂的多头绒泡菌(Physarum polycephalum)原生质团为材料,应用常规制片和整体银染后制片的电镜技术研究了有丝分裂后细胞核的形态构建过程。形成新核仁的前体物质在有丝分裂中期时散在染色体区域的周围,末期时与染色体组一起到达两极。子细胞核刚形成时核仁物质与染色质混合,以后核仁物质相互汇合并同染色质逐渐分开,最后形成一个大核仁。染色质在有丝分裂后期开始解集缩,到两极后在新形成的子核中进一步松解。染色质在充分松解后又开始集缩活动,形成一些集缩比较紧密的染色质小块。随着细胞核的进一步发育在核膜和核仁之间形成许多大小不等,形状不规则的染色质团块。     

蚕豆染色体集缩和解集缩过程中的螺旋结构

运用常规电镜技术观察到,在有丝分裂前期的集缩过程中,蚕豆(Vicia faba)染色体横切面为直径约0.5μm的染色质纤维形成的环状结构;染色体纵切面上存在着平行排列的0.5μm染色质纤维,它们与染色体长轴所成的角度近似直角。通过立体电镜观察可清晰辨认出这些纤维盘绕成的螺旋结构。在有丝分裂末期至间期的解集缩过程中,染色体横切面由环形变为“C”形。这种“C”形构造显示了染色体螺旋结构的解螺旋过程。在染色体集缩和解集缩过程中均可观察到0.5μm染色质纤维和直径约0.2μm的染色质纤维。本文讨论了放射环模型和多级螺旋模型。     

小麦间期集缩染色质的超微结构研究

本文以普通小麦(Triticum aestivum L.)根端分生组织为材料,在透射电镜下对间期细胞核内的集缩染色质的高层次结构进行了研究。在其中观察到直径约为20—25nm、50nm及110—120nm 的不同等级染色线,并且发现直径110—120nm 的染色线是由50nm 的染色线组成的,而直径约50nm 的染色线是由20—25nm 的染色线组成的。对这三个层次染色质结构之间的集缩方式进行了讨论。     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌Ｐｈｙｓａｒｕｍ ｐｏｌｙｃｅｐｈａｌｕｍ Ｓｃｈｗ的营养生长 没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质呈不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩     

多头绒泡菌有丝分裂后细胞核重建过程的形态学研究

以进行自然同步核内有丝分裂的多头绒孢菌（Ｐｈｙｓａｒｕｍｐｏｌｙｃｅｐｈａｌｍ）原生质团为材料,应用常规制片和整体银染后制片的电镜技术研究了有丝分裂后细胞核的形态构建过程,形成新核仁的前体物质在有丝分裂中期时莠在染色体区域的周围,末期时与染色体组一起到达两极,子细胞核刚形成时核仁物质与染色质混合,以后核仁物质相互汇合并同染色质逐渐分开,最后形成一个大核仁,染色质在有丝分裂后期开始解集缩,到两极后在     

应用原子力显微镜对玉米染色质纤丝及染色体骨架的研究(英文)

应用原子力显微镜对经化学方法预处理的玉米染色体超微结构进行了研究。原子力显微镜观察的结果揭示,经30%醋酸处理的玉米染色体表面呈现出不均一的颗粒状结构。当缩小扫描范围后,在染色体表面发现直径分别为30和100 nm的两种染色质纤丝。100 nm的染色质纤丝由30 nm染色质纤丝螺旋缠绕而成。在经25%胰酶处理的玉米染色体上发现了两种类似的螺旋状染色质纤丝结构,其直径分别为30和100～150 nm。较细的染色质纤丝螺旋缠绕成较粗的纤丝,进而构成整个染色体。经低离子浓度溶液抽提,用原子力显微镜观察到了玉米染色体的染色体骨架结构。这种染色体骨架呈不规则的纤维网状,这些网状纤维在染色体中部显得较为紧密,在染色体的边缘则显得较松散。这一结果暗示染色体是由不同级别的染色质纤丝螺旋缠绕构成,为染色体的多级螺旋结构假说提供了新的证据。同时发现染色体骨架并不是呈轴样的结构存在,而是保留了染色体的基本形态,这种骨架形状也许是由分散在染色体中的染色体骨架蛋白在低离子浓度溶液抽提的过程中凝缩形成的。     

染色质集缩包装成染色体的过程和机制

染色质集缩包装形成染色体是有丝分裂的一个重要事件，它使母细胞的遗传物质得以平均地分配到两个子细胞中去． 本文综述了近年来在染色质集缩包装研究方面取得的进展， 参加集缩包装的几种蛋白质以及它们可能的作用方式．     

关于蚕豆根端分生组织细胞核中类胀泡结构的研究

本文对蚕豆根端分生组织细胞核中类胀泡结构的超微结构变化和细胞化学特点进行了研究。我们观察到,类胀泡结构是与集缩染色质紧密相连的核内结构。该结构是由直径30nm左右的纤维组成的较为疏松的网络,其中的30nm纤维可以进一步解集缩并释放出直径约10nm的颗粒,这些颗粒可能是核小体。Bernhard染色结果表明,类胀泡结构含有RNP。放射自显影结果表明,类胀泡结构具有转录活性。我们推测,蚕豆细胞核中的类胀泡结构可能是非核仁基因表达的一种形态。     

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G₂-early prophase. The 250–400 nm thick compact chromatin threads in G₂ nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1–1.5 m) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120–250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究（简报）

It remains unclear about the intermediate construction of chromosome due to its highly compact nature and the limitation in methods. The present study was designed to investigate the construction of chromatin and mitotic chromosome in situ with scanning electron microscopy. Mouse testes were selected as the material, because of in which the spermatogenic cells divide actively and successively to form the sperm. Such a feature would be able to study the structure of mammalian chromatin and chromosomes along with the change of nuclear cycle. The animal were perfused with 200 ml of 0.075 mol/L KCl hypotonic solution to remove blood and placed for 15-20 min on ice followed by 0.5% glutaraldehyde and 0.5% formaldehyde for fixing. Through treated by the routine process of fractured and freeze dried with t-butyl alcohol, the specimens were then coated with a 3 nm thick platinum and observed with Hitachi S-430 scanning electron microscopy. It was found that the hypotonic treatment with 0.075 mol/L KCl solution was suit for demonstrating the nuclear structure, when the organelles were well preserved. The chromatin fibers of 10-30 nm and 80-125 nm in diameter could be recognized in the interphase nuclei, which were arranged losely at the region of euchromatin, and folded with each other into chromatin masses at the region of heterochromatin, while the chromatin fibers with the diameter of 80-125 nm often could be viewed on the mitotic chromosomes. Since its presence in interphase nuclei and mitotic chromosomes, it was considered that the chromatin fibers with 80-125 nm in diameter might play a role in the condensation of chromosome, serve as a type of the intermediate structure.     

Experimental visualization of chromoneme as one of the higher levels of chromatin compactization in the mitotic chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究(简报)

正常情况下,染色质和染色体在细胞内呈高度致密状态,在光镜和透射电镜下常呈浓染的斑块状。由于方法学上的困难,至今对染色质乃至染色体的微细结构,仍缺乏清楚的了解。特别是关于染色质如何凝缩形成染色体方面,现仍存在有争论。扫描电镜的冷冻割断技术,曾被用于对游离细胞间期核染色质的观察,并取得了较好的     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha

Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinalpha were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importinalpha beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinalpha membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importinalpha membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-alpha or gamma-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.     

Chromonema and chromomere

A study of ultrathin sections of normal Chinese hamster cells and cells treated with decreasing concentrations of bivalent cations (Ca²⁺ and Mg²⁺) in situ revealed several discrete levels of compaction of DNA-nucleoprotein (DNP) fibrils in mitotic chromosomes and the chromatin of interphase nuclei. At concentrations ranging from 3 mM CaCl₂ and 1 mM MgCl₂ to ten times less, the chromosomes are found to contain fibrous elements (chromonemata) about 100 nm in diameter. As Ca²⁺ concentration is gradually decreased to 0.2–0.1 mM, the chromosomes decondense into a number of discrete chromatin structures, the chromomeres. As decondensation proceeds, these chromomeres acquire a rosettelike structure with DNP fibrils radiating from an electron-dense core. Upon complete decondensation of chromosomes, individual chromomeres persist only in the centromeric regions. The following levels of DNP compaction in mitotic chromosomes are suggested: a 10-nm nucleosomal fibril, a 25-nm nucleomeric fibril, and the chromonema, a fibrous structure, about 100 nm in diameter, composed of chromomeres. Interphase nuclei also contain structures which are morphologically similar to the chromomeres of mitotic chromosomes.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."