Investigation of vaccinia virus DNA replication employing a conditional lethal mutant defective in DNA.
Investigation of vaccinia virus DNA replication employing a conditional lethal mutant defective in DNA.
复制标题
使用 DNA 缺陷的条件致死突变体研究痘苗病毒 DNA 复制。
DOI：
10.1016/0042-6822(84)90048-5
复制
发表时间：
1984
期刊：
影响因子：
3.7
通讯作者：
Dales,S
中科院分区：
文献类型：
--
作者：
Pogo,BG;Berkowitz,EM;Dales,S
牛痘病毒DNA缺陷型温度敏感（ts）突变体6389感染L细胞后，[3H]胸苷在39°时被抑制，但在温度降至32°（允许温度）时恢复。经过30分钟的滞后期，DNA合成是线性的，取决于连续的蛋白质合成。在10到60分钟的脉冲中标记的新生DNA的沉降分析显示，突变分子的产生速度较慢，但与在相同环境下合成的野生型牛痘分子大致相同。在超过60分钟的较长孵育期间，观察到标记的DNA分子比成熟的全长病毒基因组沉积得更快。突变型DNA与成熟病毒粒子的整合速度不如宽型DNA快。从病毒体中提取后，ts6389 DNA沉淀为基因组大小和更大、更快的沉淀DNA。用限制性内切酶处理后，ts6389病毒体DNA在琼脂糖凝胶上分离后显示出一个额外的片段，这可能是分子末端片段融合的结果。综上所述，这些观察结果表明，在牛痘DNA复制过程中形成了串联中间体。通过测量在第一轮复制中标记的分子片段和亚片段的放射性，可以将复制的起始位点定位在末端150bp内的一个区域。
After infection of L cells with the DNA-defective temperature-sensitive (ts) mutant 6389 of vaccinia virus, [3H]thymidine incorporation into cytoplasmic DNA is inhibited at 39°, but resumes upon shiftdown to 32°, the permissive temperature. Following a 30-min lag period DNA synthesis is linear and contingent upon continuous protein synthesis. Sedimentation analysis of nascent DNA labeled during 10 to 60-min pulses revealed that the mutant molecules are produced at a slower rate, but are approximately the same size as those of wild-type vaccinia, synthesized under the same circumstances. During more prolonged incubation beyond 60 min, labeled DNA molecules sedimenting more rapidly than mature, full-length virus genomes are observed. The integration of mutant DNA into mature virions is less rapid than that of the wide-type DNA. Upon extraction from the virosomes, the ts6389 DNA sediments as both genome-size and larger, faster sedimenting DNA. Upon treatment with restriction endonucleases, the ts6389 virosomal DNA exhibited an additional fragment after separation on agarose gels, perhaps as a consequence of fusion between the terminal fragments of the molecule. Taken together these observations suggest that concatemeric intermediates are formed during vaccinia DNA replication. By measuring the radioactivity incorporated into the fragments and subfragments of the molecules labeled during the first round of replication, the initiation site of replication can be localized to a region within the terminal 150 bp.