Fractionation of newly replicated nucleosomes by density labeling and rate zonal centrifugation for the analysis of the deposition sites of newly synthesized nucleosomal core histones.
Fractionation of newly replicated nucleosomes by density labeling and rate zonal centrifugation for the analysis of the deposition sites of newly synthesized nucleosomal core histones.
复制标题
通过密度标记和速率区带离心对新复制的核小体进行分级，以分析新合成的核小体核心组蛋白的沉积位点。
DOI：
10.1111/j.1432-1033.1985.tb09059.x
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
T. Ohsawa
中科院分区：
文献类型：
--
作者：
T. Senshu;K. Yamasu;T. Ohsawa
我们已经发现，新复制的核小体的部分分辨率可以通过在重水中预先形成的蔗糖密度梯度的速率区带离心来实现。在合适的同位素前体存在下，从用5-碘脱氧尿苷密度标记的MH-134 SC细胞获得核小体样品。该方法简单，并且可以在不使核小体结构不稳定的条件下进行。这给了我们一个令人兴奋的机会来研究新合成的组蛋白的沉积位点。从细胞中获得核小体，同时用5-碘脱氧尿苷和[3 H]赖氨酸脉冲标记，用于速率带分析。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳解析所得级分中的蛋白质，并通过银染色和荧光照相法可视化。新合成的H2 A和H2 B的分布与体核小体的分布非常一致。新合成的H3和H4的分布被转移到散装核小体峰的底部，但没有到新复制的（致密的）核小体的推定峰。这意味着新合成的组蛋白以不成比例的量沉积在DNA上，并且它们的沉积位点不限于新复制的DNA。
We have found that partial resolution of newly replicated nucleosomes can be achieved by rate zonal centrifugation through sucrose density gradients preformed in heavy water. Nucleosome samples were obtained from MH-134SC cells density labeled with 5-iododeoxyuridine in the presence of suitable isotopic precursors. The method is simple and can be performed under conditions that do not destabilize the nucleosome structure. This gave us an exciting opportunity to study the deposition sites of newly synthesized histones. Nucleosomes were obtained from cells pulse-labeled simultaneously with 5-iododeoxyuridine and [3H]lysine for the rate zonal analysis. Proteins in the resulting fractions were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and visualized by silver staining and fluorography. The distribution of newly synthesized H2A and H2B coincided closely with that of bulk nucleosomes. The distribution of newly synthesized H3 and H4 was shifted to the bottom sides of the bulk nucleosome peaks, but not so far as to the putative peaks of newly replicated (dense) nucleosomes. This means that newly synthesized histones are deposited on DNA in disproportionate amounts and that their sites of deposition are not restricted to newly replicated DNA.
3,5,3-三碘-L-甲状腺氨酸调节 GH1 细胞中甲状腺激素核受体水平。
DOI：
--
发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Raaka,BM;Samuels,HH
通讯作者：
Samuels,HH
新复制的染色质的两阶段成熟过程。
影响因子：
2.9
作者：
Smith,PA;Jackson,V;Chalkley,R
通讯作者：
Chalkley,R