Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus.
Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus.
复制标题
痘苗病毒感染去核、同步化的小鼠 L 细胞后，I 型拓扑异构酶活性。
DOI：
10.1128/jvi.57.2.433-437.1986
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发表时间：
1986
影响因子：
5.4
通讯作者：
Bauer,WR
中科院分区：
文献类型：
--
作者：
Poddar,SK;Bauer,WR
测定痘苗病毒感染小鼠L细胞质后I型拓扑异构酶活性出现的时间过程。当用不同步的细胞培养物进行去核过程时，发现高水平的宿主细胞特异性I型拓扑异构酶活性与所得细胞质相关。如果细胞首先通过两循环胸苷封闭方法同步化，然后在释放后去核，则对于S期去核细胞来说，宿主I型拓扑异构酶活性水平也很高，但对于从之前在G1或G2期期间同步化和去核的细胞制备的细胞质来说，其活性水平非常低。痘苗病毒感染G1期去核细胞质后，新合成的I型拓扑异构酶活性在感染后约3小时首次出现。从受感染的同步化细胞质中分离出病毒体，并测定 I 型拓扑异构酶活性的存在。在病毒体纯化过程中通常使用的低盐水平（0.01 M KCl，0.01 M Tris 盐酸盐，pH 8.0）下，活性保持在蔗糖梯度的顶部，与病毒体级分很好地分离。如果沉淀是在酶显示最佳活性的较高盐浓度 (0.15 M KCl) 下进行，则 I 型拓扑异构酶与病毒颗粒共沉淀到蔗糖梯度垫上。这些结果表明，可以在G1期去核细胞质中以高灵敏度检测依赖于痘苗病毒感染的I型拓扑异构酶活性。与病毒体的关联与拓扑异构酶活性参与 DNA 复制或晚期转录是一致的。
The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.