非细胞体系重建核染色质组装、结构和DNA复制

用lambda DNA和蛙卵提取物进行非细胞体系核重建实验,结果显示,lambda DNA参与构成了重建核的染色质;染色质组装是逐步进行的;组装的染色质含有典型的核小体串珠状一级结构、300的螺线管和染色质的更高级结构;重建核能够复制DNA。     

一种原始真核细胞——寇氏隐甲藻(Crypthecodinium cohnii)染色体骨架的证实

甲藻(dinoflagellate)的染色体是现存真核生物中最原始的,与原核生物的类核体最为近似。我们将染色体骨架制备方法与非树脂包埋去包埋剂超薄切片电子显微镜方法结合起来,首次直观地显示在寇氏隐甲藻(Crypthecodinium cohnii)的原始染色体中存在精细发达的水不溶性纤维蛋白骨架,分离的染色体骨架保持了与正常染色体对应的形状与大小,染色体骨架纤维直径约10nm,编织成网,蛋白骨架纤维的分布贯穿于整个染色体中,双向电泳显示染色体骨架的主要成分是酸性蛋白、实验结果说明,在原始染色体中已经出现了染色体骨架,并提示在真核生物进化过程中,染色体骨架的出现可能先于典型染色质的出现。本文对染色体骨架与染色体构建,及染色体骨架与核骨架的关系作了讨论。     

THE ASSOCIATION OF VACCINIA VIRUS WITH INTERMEDIATE FILAMENT

The intermediate filament-lamina-nuclear matrix system (IF-lamina-Nm System) can be shown clearlyusing selective extraction, embedment-free electron microscopy and whole mount cell preparation. A del-icate intermediate filament network was demonstrated in vaccinia virus factory for the first time. It isa part of the cytoplasmic intermediate filament network. The components and substructures of vacciniavirus bind firmly to intermediate filaments. Both assembled and assembling vaccinia viruses are fixedin the intermediate filament network. Direct connections between viruses and intermediate filaments canbe observed. We propose that vaccinia virus assembly depends on intermediate filaments as support.     

Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.     

鸭瘟病毒DNA合成动态与部位的电子显微镜放射自显影研究

本文应用~3H-TdR掺入的电子显微镜放射自显影技术研究了鸭瘟病毒DNA在细胞内合成的动态与部位,证明鸭瘟病毒DNA合成的持续时间很长,当病毒核壳体大量装配、病毒粒子已经成熟与释放时,病毒DNA的合成仍继续进行。病毒DNA是在核内电子密度低的基质中合成的。核内毒浆是病毒DNA积聚与核壳体装配的场所,不是病毒DNA合成的部位。     

HeLa细胞端粒DNA与核骨架的特异性结合

本文首先应用电子能量损失谱成像技术通过对磷元素的分析,直观地显示核酸与HeLa细胞核骨架纤维的结合;再用~3H-Thymidine掺入实验说明有一部分DNA紧密结合在核骨架上;继而用Dot和Southern分子杂交分析证明染色体端粒DNA特异地与核骨架紧密结合;最后采用DNA与蛋白质的体外吸附杂交实验进一步说明端粒DNA序列与Lamin B,波形蛋白(Vimentin)以及某些核骨架蛋白质有较高的亲和性。从而逻辑地推测HeLa细胞核骨架,Lamina参与染色体的空间排布及其行为。     

THE KERATIN INTERMEDIATE FILAMENTLIKE SYSTEM IN THE PLANT MESOPHYLL CELLS

A delicate intermediate filament-like network of mesophyll cells was observed both in maize and tobacco, using selective extraction together with whole-mount cell preparation for electron microscopy. The filament of the network is about 10 nm in diameter. Further test using immuno-gold labeling with anti-keratin antibodies indicated that the component of the intermediate filament-like system was keratin-like protein. Such a keratin-like intermediate filament system existing in plant cells was demonstrated for the first time. Meanwhile, 3-nm size filaments and their connection with 10-nm filaments were also shown in maize and tobacco protoplasts.     

VISUALIZATION OF THE CHROMOSOME SCAFFOLD IN A PRIMITIVE EUKARYOTE DINOFLAGELLATE Crypthecoainium cohnii

The chromosome scaffolds in higher eukaryotic nuclei have been described elsewhere. Butit is unknown when they evolved. The dinoflagellates are the primitive organisms that maybe the intermediate between prokaryotes and eukaryotes. Combining chromosome scaffold prep-aration methods with embedment-free section microscopy,we demonstrate that the dino-flagellate Crypthecodinium cohnii chromosome retains a protein scaffold after the depletionof DNA and soluble proteins. This scaffold preserves the morphology characteristic of thechromosome. Two-dimensional electrophoreses show that the chromosome scaffolds are mainlycomposed of acidic proteins. Our results suggest that a framework similar to the chromosomescaffold in the mammalian cell appeared in the primitive eukaryote. We propose that thechromosome scaffold possibly originated from the early stages of eukaryote evolution.     

CYTOPLASMIC ORIGIN OF AN INTRANUCLEAR DNA VIRUS (DUCK PLAGUE VIRUS)

Electron microscopic and autoradiographic studies have revealed that the assembly andmaturation of duck piague virus (DPV, an intranuclear DNA virus) occur both in the nucleusand is the cytoplasm. A dense cytoplasmic matrix (tentatively called viroplast) is the basicorganelle in which the assembly of nucleocapsids occurs. At the early stage of virion matura-tion, dense particles, approximately 40 nm in size, appear and become assembled nucleocapsidstructures later on. As capsids become mature, they obtain a diameter of 95--105 nm. Theviroplast matrix gradually decreases in size, and a loose structure appears inside as thenucleocapsids increase in number. When viroplast has almost disappeared, nucleocapsidsbecome abundant, regularly arranged around the cytoplasmic vacuoles. They bud into thesevacuoles, thereby acquiring their envelopes. These nucleocapsids differ from those found inthe nucleus both in morphology and in distribution. Electron microscopic autoradiography has shown that there is a huge amount of DNA inviroplast. DNA for the assembly of the nucleocapsids in the cytoplasm is derived from thevery viroplast.