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首页> 外文期刊>European Journal of Cell Biology: Journal of Deutsche Gesellschaft fur Elektronenmikroskopie: Journal of Deutsche Gesellschaft fur Zellbiologie >In vitro nuclear assembly with affinity-purified nuclear envelopeprecursor vesicle fractions, PV1 and PV2
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In vitro nuclear assembly with affinity-purified nuclear envelopeprecursor vesicle fractions, PV1 and PV2

机译：亲和纯化的核被膜前体囊泡级分PV1和PV2的体外核大会

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Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation, PV1 contained larger vesicles, When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. an in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.
机译：通过染色质结合方法从非洲爪蟾卵提取物中亲和纯化核被膜前体囊泡。在4摄氏度下与染色质结合的囊泡用高盐缓冲液解离，并通过差速离心进一步分离成核被膜前体囊泡级分1（PV1）和2（PV2），PV1包含较大的囊泡。补充PV1的胞浆级分，与染色质结合的囊泡，彼此融合，形成双层核膜，并组装成球形小核。但是，如此组装的核没有生长到正常大小。在如此组装的核上未发现核孔复合物。另一方面，PV2包含较小的囊泡。 PV2囊泡与染色质结合，在非洲爪蟾卵细胞溶质级分中几乎不融合，并且没有细胞核。当将补充有PV2的PV1用于核组装反应时，组装核增长到正常大小。如此组装的核膜中存在核孔复合体。这些结果表明，1）两个小泡种群PV1和PV2是正常大小的原子核组装所必需的; 2）PV1包含染色质靶向分子和膜融合机制，3）PV2包含染色质靶向分子）和核孔复合物组装所必需的一个或多个分子，以及4）PV1具有组装核膜的能力，PV2对于核孔复合物的组装和核生长至正常大小是必需的。建立了由亲和纯化的囊泡组分PV1和PV2组成的体外核装配系统。

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