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 assem...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.展开更多
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 ...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.展开更多
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 violace...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.展开更多
文摘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.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 19890380 and 39730240).
文摘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.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 19890380 & 30070388) the Special Funds for Major State Basic Research of China
文摘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.
