Mably J D, Sole M J, Liew C C
Toronto Hospital, Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
J Biol Chem. 1993 Jan 5;268(1):476-82.
A positive element between positions -924 and -851 and a negative element between -851 and -762 of the 5'-upstream region of the alpha-myosin heavy chain gene were identified through transient transfection assays in primary cultures of neonatal rat heart cells. Subsequent DNase I protection analysis revealed almost identical footprints at two positions (GAAAAATCT at -904 to -896 and GAAAATCT at -823 to -816). We have designated this sequence the GArC motif (for G,AT-rich,C). Gel mobility shift assays demonstrated the formation of specific complexes with GArC oligomers when either rat heart, rat liver, or HeLa cell nuclear extracts were used. Competition studies with unlabeled GArC oligomers resulted in a loss of binding. Oligomers were also made to the Xenopus cytoskeletal actin serum response element and to a segment of the alpha-MyHC gene (AT-core), each with a similar AT-rich core sequence. No detectable loss of binding resulted from the addition of an excess of either of these unlabeled oligomers. Southwestern blot analysis identified several proteins which interacted with the GArC element, suggesting the presence of a group of related trans-acting factors. Analysis of a sequence in the beta-MyHC gene with the same AT-rich core was negative, suggesting a role for the bases surrounding the protected area in binding. We propose that the GArC motif, together with its associated trans-acting factor(s), provides a novel mechanism of transcriptional control in addition to those previously reported for the cardiac myosin heavy chain genes.
通过新生大鼠心脏细胞原代培养中的瞬时转染实验，在α-肌球蛋白重链基因5'-上游区域的-924至-851位之间鉴定出一个正向元件，在-851至-762位之间鉴定出一个负向元件。随后的DNA酶I保护分析显示，在两个位置（-904至-896位的GAAAAATCT和-823至-816位的GAAAATCT）有几乎相同的足迹。我们将该序列命名为GArC基序（富含G、AT和C）。凝胶迁移率变动分析表明，当使用大鼠心脏、大鼠肝脏或HeLa细胞核提取物时，会与GArC寡聚物形成特异性复合物。用未标记的GArC寡聚物进行竞争研究导致结合丧失。还制备了非洲爪蟾细胞骨架肌动蛋白血清反应元件和α-MyHC基因片段（AT核心）的寡聚物，每个都有相似的富含AT的核心序列。添加过量的这些未标记寡聚物中的任何一种都未导致可检测到的结合丧失。蛋白质印迹分析鉴定出几种与GArC元件相互作用的蛋白质，表明存在一组相关的反式作用因子。对β-MyHC基因中具有相同富含AT核心序列的一段序列的分析为阴性，表明保护区周围的碱基在结合中起作用。我们提出，GArC基序及其相关的反式作用因子提供了一种新的转录控制机制，这是除了先前报道的心肌肌球蛋白重链基因的转录控制机制之外的。
