ORIGIN OF ADENOVIRUS DNA-REPLICATION - ROLE OF THE NUCLEAR FACTOR-I BINDING-SITE INVIVO
ORIGIN OF ADENOVIRUS DNA-REPLICATION - ROLE OF THE NUCLEAR FACTOR-I BINDING-SITE INVIVO
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DOI：
10.1016/0022-2836(85)90263-3
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发表时间：
1985-01-01
影响因子：
5.6
通讯作者：
HAY, RT
中科院分区：
文献类型：
--
作者：
HAY, RT
本发明描述了一种检测装置，其在体内检测由含有暴露的单拷贝腺病毒（Ad）复制起点的线性分子指导的单轮起始和DNA合成。该测定和先前描述的测定测量多轮DNA复制，用于鉴定Ad2和Ad4复制起点内对ori功能重要的DNA序列。将含有来自Ad2或A4基因组末端的序列的线性DNA分子与同源和异源辅助病毒共转染，并比较DNA合成的净量。含有Ad4反向末端重复序列的线性分子在同源辅助物的存在下复制得更好20倍，而Ad2和Ad4反向末端重复序列均被Ad4有效利用。对Ad2 ori和AD4相应区域的DNA序列分析表明，虽然只有10个变异碱基对，但其中8个位于细胞蛋白核因子I识别的Ad2 DNA序列内。这种蛋白质是实现体外Ad2 DNA复制的最大速率所必需的，因此这些差异鉴定了对Ad2 ori功能至关重要的DNA序列。Ad4 ITR不包含功能性核因子I结合位点，并且缺失分析已经证明Ad4基因组的His区域不需要ori功能。与Ad2相反，Ad4 DNA复制起始所需的DNA序列完全位于Ad4反向末端重复序列的末端18个碱基对内。
An assya is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and previously described assay, which measured multiple round of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or A4 genome termini were cotransfected with homologous and heterologous helper virus and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in AD4 indicated that,although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular proteinnuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that his region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.