Phi-Van L, von Kries J P, Ostertag W, Strätling W H
Institut für Physiologische Chemie, Universitäts-Krankenhaus Eppendorf, Hamburg, Federal Republic of Germany.
Mol Cell Biol. 1990 May;10(5):2302-7. doi: 10.1128/mcb.10.5.2302-2307.1990.
Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.
基质附着区域(MARs)是一种DNA元件,通过与染色体骨架结合将基因组切割成拓扑分离的结构域。本研究探讨了位于鸡溶菌酶基因5'端的MAR对异源细胞系统中异源基因表达的潜在影响。在稳定转染大鼠成纤维细胞后,检测了由单纯疱疹病毒胸苷激酶启动子(TC)控制的氯霉素乙酰转移酶(CAT)指示基因构建体,以及相同转录单元两侧带有鸡溶菌酶5'MARs的构建体(MTCM)的表达情况。MTCM转染子中每个拷贝数的CAT活性中位数相对于TC转染子提高了约10倍。标准化CAT活性的总变异系数从TC转染子中的100倍以上降至MTCM转染子中的近6倍。如S1核酸酶和连续转录分析所示,MTCM转染子中转录本的稳态水平和相对转录速率均有所提高。因此,鸡溶菌酶5'MAR可通过增加转录RNA聚合酶分子的密度,赋予不同物种细胞中异源启动子更高且位置依赖性更低的表达。MAR介导的转录增强表明,MAR不仅对DNA包装很重要,对基因表达也很重要。