INACTIVATION OF THE MOLONEY MURINE LEUKEMIA-VIRUS LONG TERMINAL REPEAT IN MURINE FIBROBLAST CELL-LINES IS ASSOCIATED WITH METHYLATION AND DEPENDENT ON ITS CHROMOSOMAL POSITION
INACTIVATION OF THE MOLONEY MURINE LEUKEMIA-VIRUS LONG TERMINAL REPEAT IN MURINE FIBROBLAST CELL-LINES IS ASSOCIATED WITH METHYLATION AND DEPENDENT ON ITS CHROMOSOMAL POSITION
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DOI：
10.1128/jvi.65.2.904-912.1991
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发表时间：
1991-02-01
影响因子：
5.4
通讯作者：
VANDEREB, AJ
中科院分区：
文献类型：
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作者：
HOEBEN, RC;MIGCHIELSEN, AAJ;VANDEREB, AJ
用大肠杆菌衍生的β-半乳糖苷酶（β-gal）基因作为报告基因，监测整合到鼠成纤维细胞系基因组中后具有莫洛尼鼠白血病病毒（Mo-MuLV）长末端重复序列（LTR）启动子的逆转录病毒载体的表达。 逆转录病毒感染后衍生的单克隆细胞系在其报告基因的表达中表现出显着的异质性。 我们研究了两个单克隆细胞系与一个单一的未经重排的拷贝的载体前病毒整合到他们的基因组。 第一种是BB10，仅在其细胞群的8%中表达标记酶，而在第二种BB 16中，可以在超过98%的细胞中检测到β-gal表达。 用DNA去甲基化剂5-氮杂胞苷处理BB10使β-gal阳性细胞的数量增加到60%以上。 转染实验表明，Mo-MuLV LTR启动子-增强子在BB10和BB 16细胞系中可能是完全功能性的。 将来自BB10细胞的失活前病毒克隆并随后用于产生逆转录病毒原液。 用这些BB10衍生的病毒感染后，其LTR的启动子-增强子活性显示出与原始病毒原液相似的变化。 我们的数据表明：（1）Mo-MuLV LTR的失活是鼠成纤维细胞系中的常见事件，（2）失活与胞苷残基的从头甲基化有关，（3）前病毒的失活频率必须由其染色体位置决定，（4）LTR内序列的甲基化过程不一定与转录相同-在未分化的胚胎癌细胞中起作用的抑制机制。
The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter. Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene. We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome. The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells. Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60%. Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines. The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks. The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks. Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells.