In vivo characterization of the poly(ADP-ribosylation) of SV40 chromatin and large T antigen by immunofractionation.
In vivo characterization of the poly(ADP-ribosylation) of SV40 chromatin and large T antigen by immunofractionation.
复制标题
通过免疫分级对 SV40 染色质和大 T 抗原的聚（ADP-核糖基化）进行体内表征。
DOI：
10.1016/0014-4827(87)90098-x
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发表时间：
1987
影响因子：
3.7
通讯作者：
Smulson,ME
中科院分区：
文献类型：
--
作者：
Baksi,K;Alkhatib,H;Smulson,ME
我们通过使用大 T 抗原和聚 (ADP-核糖) 抗体证实了 SV40 大 T 抗原的聚 (ADP-核糖基化)，并因此开始表征病毒蛋白的这种翻译后核修饰如何调节其生物学功能。含有复制中间体 DNA 的 SV40 微型染色体亚群与含有成熟微型染色体 DNA 的病毒染色质级分相比，对抗聚 (ADP-Rib)-Sepharose 具有显着更高的亲和力。使用抗大T-琼脂糖柱通过两种不同的方法从粗提物中分离T抗原：(1)在体内用[35S]甲硫氨酸标记大T抗原，并对感染细胞提取物进行免疫分级以分离大T抗原；(2)对感染细胞提取物中的大T抗原进行免疫分级，然后进行免疫染色。使用这些技术，发现受感染细胞的总 T 抗原中有 1-10% 是聚（ADP-核糖基化）的。微染色体制剂也在抗大T-琼脂糖凝胶上进行免疫分级。该基质上多聚（ADP-核糖基化）微型染色体的高水平保留表明，病毒染色质的这种翻译后修饰可能与大T抗原调控下的病毒复制和转录步骤有关。
We have confirmed the poly(ADP-ribosylation) of large T antigen of SV40 by using antibodies to both large T antigen and poly(ADP-ribose) and consequently have begun to characterize how this post-translational nuclear modification of the viral protein modulates its biological functions. SV40 minichromosomal subpopulation containing replicative intermediate DNA was shown to have a significantly higher affinity for anti-poly(ADP-Rib)-Sepharose than viral chromatin fractions containing mature minichromosomal DNA. An anti-large T-Sepharose column was used to isolate T antigen from crude extracts by two different approaches: (1) large T antigen was labeled with [35S]methioninein vivoand the infected cell extract was immunofractionated to isolate large T antigen and (2) large T antigen from infected cell extracts was immunofractionated followed by immunostaining. Using these techniques, 1–10% of the total T antigen from infected cells was found to be poly(ADP-ribosylated). Minichromosome preparationsper sewere also subjected to immunofractionation on anti-large T-Sepharose. The high level of retention of poly(ADP-ribosylated) species of minichromosomes on this matrix suggested that this post-translational modification of viral chromatin may be related to those steps in viral replication and transcription under regulation by large T antigen.