Rasmussen J A, Gilboa E
J Virol. 1987 May;61(5):1368-74. doi: 10.1128/JVI.61.5.1368-1374.1987.
A Moloney murine leukemia virus-derived retroviral vector (N4) carrying the bacterial neomycin resistance gene (neo) was used to study the chromatin configuration of integrated proviral DNA in NIH 3T3-derived cell lines containing one copy of the vector DNA per cell. Three independently obtained cell lines were examined. In two of these cell lines, the vector was introduced by viral infection, while in the third the construct was introduced by DNA transfection. Such transfected cell lines (including the one examined) usually express 10- to 50-fold less virus-specific RNA than do cell lines obtained by viral infection. All three cell lines exhibited similar patterns of DNase I-hypersensitive (HS) sites. Two strong DNase I HS sites were detected in the 5' long terminal repeat, which contains signals required for proper and efficient initiation of viral transcription. One of these sites was found to overlap the viral enhancer sequences, while the other site mapped very close to the start site for viral transcription. A third HS site was detected in nearby internal viral sequences. Only one HS site was found in the 3' long terminal repeat, which contains the signal(s) required for proper addition of a poly(A) tail to viral transcripts. This HS site was located in the region of the viral enhancer. Several weak DNase I HS sites were also found in the cellular sequences adjacent to the integration sites, at different locations in each cell line analyzed. No common pattern of cellular DNase I HS sites was found. These observations suggest that the 5' and 3' long terminal repeats of integrated retroviral proviruses exhibit different chromatin conformations, possibly reflecting the different functions encoded by the otherwise identical sequences, and the DNase I HS sites detected in these studies reflect only a potential for transcription and are not a reflection of the high transcriptional activity characteristic of retroviruses.
携带细菌新霉素抗性基因（neo）的莫洛尼鼠白血病病毒衍生逆转录病毒载体（N4），用于研究在每个细胞含有一份载体DNA的源自NIH 3T3的细胞系中整合的原病毒DNA的染色质构型。检测了三个独立获得的细胞系。在其中两个细胞系中，载体通过病毒感染导入，而在第三个细胞系中，构建体通过DNA转染导入。这类转染的细胞系（包括所检测的这个）通常比通过病毒感染获得的细胞系表达的病毒特异性RNA少10至50倍。所有三个细胞系都表现出相似的脱氧核糖核酸酶I超敏（HS）位点模式。在5'长末端重复序列中检测到两个强脱氧核糖核酸酶I HS位点，该序列包含病毒转录正确和有效起始所需的信号。发现其中一个位点与病毒增强子序列重叠，而另一个位点定位在非常靠近病毒转录起始位点的位置。在附近的病毒内部序列中检测到第三个HS位点。在3'长末端重复序列中仅发现一个HS位点，该序列包含向病毒转录本正确添加聚（A）尾所需的信号。这个HS位点位于病毒增强子区域。在每个分析的细胞系中不同位置的整合位点相邻的细胞序列中也发现了几个弱脱氧核糖核酸酶I HS位点。未发现细胞脱氧核糖核酸酶I HS位点的共同模式。这些观察结果表明，整合的逆转录病毒原病毒的5'和3'长末端重复序列表现出不同的染色质构象，可能反映了由其他方面相同的序列编码的不同功能，并且在这些研究中检测到的脱氧核糖核酸酶I HS位点仅反映了转录潜力，而不是逆转录病毒特有的高转录活性的反映。
