McCarthy J J, Vyas D R, Tsika G L, Tsika R W
Department of Veterinary Biomedical Sciences, School of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA.
J Biol Chem. 1999 May 14;274(20):14270-9. doi: 10.1074/jbc.274.20.14270.
Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (betaMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct betaMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair betaMyHC promoter fused to chloramphenicol acetyltransferase (beta293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the betaMyHC negative regulatory element (-332 to -300; betaNRE) within transgene beta350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the betaNRE sense strand (dbetaNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the betaNRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated betaMyHC promoter elements transcriptionally regulate betaMyHC transgene expression in response to two diverse modes of neuromuscular activity.
我们之前的转基因分析表明，一段600个碱基对的β-肌球蛋白重链（βMyHC）启动子赋予了氯霉素乙酰转移酶报告基因对机械过载（MOV）和非负重（NWB）的反应性。在体内，MOV或NWB活动后是否相同的DNA调控元件指导βMyHC表达仍不清楚。我们现在表明，与氯霉素乙酰转移酶融合的293个碱基对的βMyHC启动子（β293）对MOV有反应，但对NWB活动无反应，这表明这两种不同的元件是分离的。在转基因β350中包含βMyHC负调控元件（-332至-300；βNRE）会抑制所有转基因品系中的表达。电泳迁移率变动分析显示，仅在NWB比目鱼肌核提取物中存在高度富集的结合活性，该活性对βNRE有义链的远端区域（dbetaNRE-S；-332至-311）具有特异性。超迁移电泳迁移率变动分析显示，βNRE有义链远端区域的结合在抗原性上不同于细胞核酸结合蛋白和Y盒结合因子1，这两种蛋白已被证明可结合该元件。二维紫外线交联和迁移Southwestern印迹分析检测到两种与该元件结合的蛋白（50和52 kDa）。这些体内结果表明，分离的βMyHC启动子元件通过转录调控βMyHC转基因表达以响应两种不同的神经肌肉活动模式。
