Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.

Atomic force microscopy observation on nuclear reassembly in a cell-free system

Cell-free system is interesting and useful for studying nuclear assembly in mitosis. Atomic force micro- scopy (AFM), which is a simple way for imaging fixed reas-semble nuclei with high resolution, has not been used in the cell-free system. In this paper, we put forward an air-drying sample preparation for AFM. Using AFM, we observed nu-clear reassembly process within 100 nm resolution in a cell-free system. As a result, we found that the images were artifact-free, and with higher resolution compared with fluo-rescent optical microscope images. Furthermore, the mor-phology of membrane vesicles was obtained clearly, and a dynamic change of morphology during the vesicles?ap-proaching to nuclear envelope was also observed, which is enlightened to understand the mechanism of nuclear enve-lope assembly.

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

It was shown that nuclear reassembly was induced by small pieces of DNA fragments in cell free extracts of Xenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM Cellulose are used to eliminate the capacity of the egg extract S 150 to assemble chromatin, while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S 150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell free system of Xenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.

Biological activity assays of cell-free reassembled nuclei——Injecting cell-free reassembled nuclei into unfertilized eggs can induce the eggs to cleave and reconstitute asters,and the injected nuclei undergo cell cycle changes

Nucleus may reassemble spontaneously in cell-free mixture of HeLa metaphase chromosomes,Xenopus egg extracts and ATP-regenerating system,and the nucleus shows some biological activities.It isfound that,after being injected into unfertilized mature eggs,the cell-free reassembled nuclei can cause theeggs to cleave and reconstitute asters in their cytoplasm,and the injected nuclei undergo changes in response tocell cycle regulators stored in the eggs,and that reinjecting cytostatic factors(CSF)into the eggs can stabilizethe eggs in mitotic phase,cause the nuclei disassembly and chromatin condensation to chromosomes.

NUCLEAR RECONSTITUTION IN THE Xenopus laevis EGG EXTRACTS CELL-FREE SYSTEM

Ⅰ. INTRODUCTIONNuclear reconstitution (nuclear assembly )was reported by several laboratories in recent years and regarded as an important advance in cell biology. In vitro nuclear assembly system provides a good model to study the organization and function of nucleus, chromatin, nuclear envelope and nuclear matrix. Although it is too early to evaluate its significance in theory and in bioengineering, there is no doubt that the prospect in this field is inspiring.

Nicotiana ovule extracts induce nuclear reconstitution of demembranated Xenopus sperm in cell-free system

Nicotiana tabaccum ovule extracts induced nuclear reconstitution of demembranated Xenopus leavis sperm in a cell-free system. Demembranated Xenopus sperm began to swell after 15 min of incubation with Nicotiana ovule extracts. Accompanying the process of incubation, Xenopus sperm decondensed and their shapes changed gradually from long and ellipse to round. The completely decondensed chromatin was surrounded with membrane structure, which was a mixture envelope of a double membrane and a single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion to re-constituted nuclei and DNA electrophoresis. Nicotiana ovule extracts supplied one more experimental model and system. The new system could promote powerfully the research on mechanisms of cell division and cell cycle regulation.

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾（Ｘｅｎｏｐｕｓ ｌａｅｖｉｓ）去膜精子染色质和卵提取物成功地进行了细胞核体外重建。当精子染色质加入卵提取物后，首先发生染色质去浓缩作用，染色质整体结构膨胀；膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜；核孔复合体以某种未知方式组装入双层膜而形成核膜结构，并逐渐完全覆盖膨大的染色质，最终形成典型的间期核结构。

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。

角蛋白纤维的新功能──在爪蟾卵提取物核膜重组装中的作用

激活的非洲爪蟾（Xenopuslaevis）卵提取物温育过程中，直径200nm的膜泡附着在一种直径10nm纤维上，形成“珠链”结构。用透射电镜整装制样技术观察了温育中“珠链”结构的形成过程，发现10nm纤维可抗Triton抽提，免疫荧光和蛋白免疫印迹试验表明10nm纤维可能是由56kD的碱性角蛋白与42kD的酸性角蛋白构成。向提取物中加入碱性用蛋白抗体AE3则可抑制环形片层的形成，而核膜的组装也受到很大影响。这些结果显示角蛋白纤维在核股重新形成的过程中起到重要的作用。

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts

Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin in Xenopus egg ex-tracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to dis-rupt the microtubule nucleation, nuclear envelope reassem-bly was seriously inhibited. If the microtubules were stabi-lized by taxol, another microtubule drug, the nuclear enve-lope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an impor-tant role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chroma-tin surfaces.

Chromatin and Nucleosome Organizations and DNA Replication of Nucleus Reassembled in vitro Using Purified Exogenous DNA and Xenopus Egg Extracts

It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell ? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of 'beads-on-a-string', 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles. Prolonging digestion of the 165-bp particle DNA fragment will yield a 146-bp fragment, which may be wounded in the nucleosome core. Experiments also provide evidence that the chromatin reconstitution in the cell-free reassembled nucleus is a progressive process, and that the nucleus can replicate its DNA. Based on these observations, we suppose that the chromatin of the cell-free reassembled nucleus may be structurally and functionally similar to the chromatin of the intact cells.

Nuclear Matrix in the Nucleus Assembled in vitro

1 Introduction Nuclear assembly occurs universally in cell life, such as postmitotic or postmeiotic nuclear assembly. It is a good model for studying the organization and function of nucleus. The organism or even single cell, however, is a complex system in which many factors interact on each other, which is difficult to manipulate, in vitro cell-free nuclear assembly system can provide a good tool. Nuclear matrix is an insoluble proteinaceous filament network in nucleus. Much research work has demonstrated that many imoortant life actiyities such as

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.