Makarova O, Gorneva G, Wu F, Farutin V, Villeponteau B, Poliani L, Fink D, Levine M
Department of Human Genetics, University of Michigan, Ann Arbor 48109-0618, USA.
Gene Ther. 1996 Sep;3(9):829-33.
The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.
核基质在DNA复制、基因转录和RNA加工过程中起着关键作用。转录活跃基因通常通过DNA序列、基质附着区（MARs）与核基质相连，这些区域将环状DNA拴系在基质上。在稳定转染和转基因小鼠中，置于基因构建体侧翼的MAR元件可增强表达并抵御位置效应变异性，这表明转录的独立单元是在与相邻基因的调控控制相隔离的情况下建立的。单纯疱疹病毒1型（HSV-1）在受感染宿主中建立终身潜伏状态。病毒基因的潜伏抑制作用延伸至整合到病毒基因组中的外源基因。我们在此报告一项假说的验证，即在HSV-1基因组中外源基因侧翼的MAR元件可使其免受潜伏抑制，实现长期表达。构建了一种重组病毒，其在HSV-1基因组的Us3位点插入了一个表达构建体。该表达构建体一侧为A MAR元件，另一侧为驱动lacZ基因的HIV-LTR和B MAR元件。A MAR元件是鸡溶菌酶基因5'端的一个3 kb对片段，B MAR元件是人类β-珠蛋白基因座控制区5'端的一个2.6 kb对片段。LTR来源于从一名艾滋病患者大脑中分离出的人类免疫缺陷病毒。将病毒立体定向注射到大鼠脑的海马体、嗅球和纹状体中。注射后2天，在每个注射区域的细胞中发现了强烈的蓝色反应产物，表明存在β-半乳糖苷酶活性。注射后14天，在任何注射部位均未再检测到β-半乳糖苷酶活性。我们得出结论，MAR元件构建体未能逃脱潜伏抑制。
