Tsukiyama T, Niwa O, Yokoro K
Department of Pathology, Hiroshima University, Japan.
J Virol. 1991 Jun;65(6):2979-86. doi: 10.1128/JVI.65.6.2979-2986.1991.
Mouse embryonal carcinoma (EC) cell lines were established which carry the stably integrated chloramphenicol acetyltransferase (CAT) gene under the control of the transcriptional elements of the long terminal repeat (LTR) of Moloney murine leukemia virus. The activity of three elements of the stably integrated LTR was analyzed in undifferentiated EC cells (stable CAT assay). Results of the study are summarized as follows. (i) In the stable assay, the promoter region of the LTR was inactive in undifferentiated ECA2 and F9 cells, and the level of the activity was 10(-4) of that in NIH 3T3 cells. (ii) In contrast to the results of the transient assay, the enhancer was active in undifferentiated ECA2 cells and in F9 cells. It activated CAT activity more than 60-fold and about 8-fold in ECA2 cells and F9 cells, respectively. (iii) Suppression by ELP, the embryonal LTR-binding protein, was more pronounced in the stable assay than in the transient assay. These data suggest that, when compared with NIH 3T3 cells, a major factor for the inactivity of the LTR in EC cells is the inefficiency of the promoter in this assay. Transcriptional activity of the LTR was analyzed during the differentiation of EC cells. In the case of ECA2 cells, the magnitude of activation by the enhancer did not change during differentiation. The activity of the promoter increased about 10-fold, and the suppression by ELP became negligible 4 days after the induction of differentiation. Upon differentiation of F9 cells, the activity of the enhancer increased more than 300-fold, but the promoter remained inactive. The pattern of LTR-binding proteins also varied during the differentiation of EC cells. Our present data suggest that the activity of LTR elements as assayed by the stable assay differs from the activity as assayed by the transient assay. It also indicates that the activity of these elements exhibits cell-type-specific changes during the differentiation of EC cells.
建立了携带在莫洛尼鼠白血病病毒长末端重复序列（LTR）转录元件控制下稳定整合的氯霉素乙酰转移酶（CAT）基因的小鼠胚胎癌细胞系。在未分化的胚胎癌细胞中分析了稳定整合的LTR三个元件的活性（稳定CAT测定）。研究结果总结如下。（i）在稳定测定中，LTR的启动子区域在未分化的ECA2和F9细胞中无活性，其活性水平是NIH 3T3细胞中的10^(-4)。（ii）与瞬时测定结果相反，增强子在未分化的ECA2细胞和F9细胞中具有活性。它在ECA2细胞和F9细胞中分别使CAT活性激活超过60倍和约8倍。（iii）胚胎LTR结合蛋白ELP的抑制作用在稳定测定中比在瞬时测定中更明显。这些数据表明，与NIH 3T3细胞相比，在该测定中LTR在胚胎癌细胞中无活性的主要因素是启动子效率低下。在胚胎癌细胞分化过程中分析了LTR的转录活性。就ECA2细胞而言，增强子激活的幅度在分化过程中没有变化。启动子活性增加约10倍，诱导分化4天后ELP的抑制作用变得微不足道。F9细胞分化后，增强子活性增加超过300倍，但启动子仍然无活性。LTR结合蛋白的模式在胚胎癌细胞分化过程中也有所不同。我们目前的数据表明，通过稳定测定法测定的LTR元件活性与通过瞬时测定法测定的活性不同。这也表明这些元件的活性在胚胎癌细胞分化过程中表现出细胞类型特异性变化。
