Tack L C, Beard P
J Virol. 1985 Apr;54(1):207-18. doi: 10.1128/JVI.54.1.207-218.1985.
We isolated simian virus 40 (SV40) chromosomes from lytically infected CV-1 cells at various times during the late phase and transcribed them in vitro with either whole-cell or nuclear extracts of HeLa cells. The late promoter was 3- to 10-fold more active than the early promoter. With bare SV40 DNA templates, the early promoter was up to 10-fold stronger than the late promoter. The relative strengths of the early and late promoters on SV40 chromosomes were essentially independent of template concentration or length of the replicative phase of the infection. When monoclonal antibodies or antisera against T antigen (T Ag) were added to SV40 chromosomes or when T Ag, both free and chromatin bound, was removed by immunoprecipitation with anti-T, the activity of the late promoter remained essentially unchanged. Washing with 0.4 M NaCl removed T Ag from more than 90% of the mature chromosomes associated with T Ag. Transcription from the late promoter still predominated in the salt-washed T Ag-depleted chromosomes, even though there was a marked increase in early promoter activity. The depression of the early promoter could be reversed by adding the T Ag-containing extract back to the depleted chromosomes. Extraction of SV40 chromosomes with 1.5 M NaCl resulted in a decrease in the activity of the late promoter and a further increase in the activity of the early promoter so that the relative amounts of early and late RNA synthesized were similar to those for bare SV40 DNA templates. Late RNA synthesis from bare SV40 DNA templates was stimulated by high-speed supernatants prepared from nuclear extracts of SV40-infected cells but not from those of uninfected cells. Pretreatment of the supernatants with anti-T did not alter the result. Our findings indicate that the activity of the early and late SV40 promoters is regulated by at least two different mechanisms at the chromosomal level. One is mediated by a subclass of T Ag bound to SV40 chromosomes which represses early SV40 transcription but has no effect on late transcription. A second level of regulation, involving a tightly bound trans-acting chromosomal factor and a stable nucleoprotein structure, favors the late promoter over the early promoter by up to 10-fold.
我们在裂解感染后期的不同时间从CV - 1细胞中分离出猴病毒40（SV40）染色体，并用HeLa细胞的全细胞提取物或核提取物进行体外转录。晚期启动子的活性比早期启动子高3至10倍。对于裸露的SV40 DNA模板，早期启动子比晚期启动子强达10倍。SV40染色体上早期和晚期启动子的相对强度基本上与模板浓度或感染复制阶段的长度无关。当将针对T抗原（T Ag）的单克隆抗体或抗血清添加到SV40染色体中时，或者当通过用抗T免疫沉淀去除游离的和与染色质结合的T Ag时，晚期启动子的活性基本保持不变。用0.4 M NaCl洗涤可从超过90%与T Ag相关的成熟染色体中去除T Ag。即使早期启动子活性有明显增加，晚期启动子的转录在经盐洗涤且T Ag缺失的染色体中仍占主导。通过将含T Ag的提取物重新添加到缺失染色体中，早期启动子的抑制作用可以被逆转。用1.5 M NaCl提取SV40染色体导致晚期启动子活性降低，早期启动子活性进一步增加，使得合成的早期和晚期RNA的相对量与裸露的SV40 DNA模板相似。裸露的SV40 DNA模板的晚期RNA合成受到SV40感染细胞的核提取物制备的高速上清液的刺激，但未受未感染细胞的核提取物制备的高速上清液的刺激。用抗T对上清液进行预处理不会改变结果。我们的研究结果表明，SV40早期和晚期启动子的活性在染色体水平上至少受两种不同机制的调控。一种机制由与SV40染色体结合的T Ag亚类介导，该亚类抑制SV40早期转录，但对晚期转录没有影响。第二种调控水平涉及紧密结合的反式作用染色体因子和稳定的核蛋白结构，使晚期启动子比早期启动子更具优势，优势高达10倍。
