Brady J, Khoury G
Mol Cell Biol. 1985 Jun;5(6):1391-9. doi: 10.1128/mcb.5.6.1391-1399.1985.
We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.
我们研究了猴病毒40（SV40）T抗原在诱导晚期基因表达中的作用，该作用独立于其通过DNA复制扩增模板的功能。Northern印迹和S1核酸酶分析表明，刺激发生在转录水平。至少两个模板元件，即T抗原结合位点和72碱基对重复序列，似乎对这种诱导很重要。使用模板突变体，我们证明T抗原结合位点II内的缺失使T抗原介导的晚期基因表达降低了约10至20倍。此外，SV40 72碱基对重复序列的单个保留拷贝内的多个点突变降低了T抗原介导的晚期基因表达。使用体内竞争研究，我们证明含有SV40控制区（核苷酸5171至272）的竞争DNA片段在COS-1细胞中定量降低了SV40晚期基因表达。相比之下，与含有SV40核苷酸1至294（去除了所有T抗原结合位点I和一半位点II）的质粒竞争效率要低得多。最后，我们证明在晚期模板区域（SV40核苷酸180至2533）内使用T抗原结合位点远端的竞争片段进行体内竞争实验，导致COS-1细胞中晚期基因表达超诱导。这一发现表明，诸如阻遏物或衰减子等负性因子可能在诱导之前调节SV40晚期基因表达。我们的结果与一个模型一致，在该模型中，晚期基因表达的诱导涉及SV40起始区域与DNA结合蛋白的相互作用，其中之一可能是T抗原。SV40晚期转录单元的激活可能涉及SV40增强子的诱导或阻遏样蛋白的去除或两者兼有。
