Ch'ang L Y, Yang W K, Myer F E, Yang D M
Biology Division, Oak Ridge National Laboratory, Tennessee 37831-8077.
J Virol. 1989 Jun;63(6):2746-57. doi: 10.1128/JVI.63.6.2746-2757.1989.
Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.
构建了三个系列的重组DNA克隆，以细菌氯霉素乙酰转移酶（CAT）基因作为定量指标，来检测从小鼠基因组中分离出的鼠白血病病毒（MuLV）相关前病毒序列5'长末端重复序列（LTR）中启动子和增强子序列元件的活性。在转染了LTR-CAT构建体的小鼠NIH 3T3、人HT1080和貂CCL64培养细胞中测定瞬时CAT表达。三个多嗜性MuLV相关前病毒克隆的700碱基对（bp）LTR以及四个修饰的多嗜性前病毒克隆的750-bp LTR，无论有无相邻下游序列的完整结构，对CAT表达均显示出极低或可忽略不计的活性，而异嗜性MuLV LTR则具有高活性。将MuLV相关的LTR分为三个部分并分别进行检测。发现MuLV相关LTR的3'部分包含CCAAC和TATAA框，是一个功能性启动子，其活性约为异嗜性MuLV LTR相应部分的二分之一到三分之一。一个MboI-Bg/II片段，代表中间独特的190至200-bp插入片段，被发现是一个潜在的增强子，特别是在CCL64细胞中与猿猴病毒40启动子结合检测时。5'部分的PstI-MboI片段，包含增强子片段的蛋白质结合基序以及上游LTR序列，在CCL6细胞中显示出中等增强子活性，但在NIH 3T3细胞和HT1080细胞中几乎无活性；将该片段添加到异嗜性LTR-CAT构建体中会抑制CAT表达。使用嵌合LTR构建体的进一步分析确定，在包含MuLV相关LTR中增强子5'部分和紧邻上游序列的区域内存在一个强负调控元件。
