Subramaniam A, Gulick J, Robbins J
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Ohio 45267-0575.
J Biol Chem. 1990 Aug 15;265(23):13986-94.
The organization of the cis-acting regulatory elements of a chick myosin heavy chain gene has been investigated. The data show that a gene which is transcribed in vivo in the fast white embryonic musculature is also the major transcript expressed during myotube differentiation of primary myoblasts derived from 12-day embryonic chick leg muscles. The upstream region of this gene consists of 7500 base pairs, and we have tested the ability of these sequences to drive expression of the chloramphenicol acetyltransferase gene in developing primary muscle cultures. Deletion analyses of the upstream region show that negative regulatory elements are present within 2000 base pairs of the basal promoter elements, the CCAAT and TAATA boxes. Removal of these elements reveals the presence of a strong positive element located near the start site of transcription. Sequence analysis showed that the region also contains a sequence characteristic of an enhancer found in the immunoglobulin heavy chain gene, ATGCAAAT, the "octa" element. Gel band-shift assays show that this octa sequence binds a transacting factor present in muscle nuclear extracts, although footprint analysis indicates a limited interaction. Transient assays carried out with a fragment in which the octa sequence has been mutated, with the subsequent abolition of protein binding, shows that the particular interaction probably plays a role in negatively modulating the action of the strong positive promoter element.
对鸡肌球蛋白重链基因顺式作用调控元件的组织进行了研究。数据表明，一个在快速白色胚胎肌肉组织中体内转录的基因，也是源自12日龄鸡胚腿部肌肉的原代成肌细胞肌管分化过程中表达的主要转录本。该基因的上游区域由7500个碱基对组成，我们测试了这些序列在发育中的原代肌肉培养物中驱动氯霉素乙酰转移酶基因表达的能力。上游区域的缺失分析表明，在基础启动子元件（CCAAT和TAATA框）的2000个碱基对内存在负调控元件。去除这些元件后发现，在转录起始位点附近存在一个强正调控元件。序列分析表明，该区域还包含免疫球蛋白重链基因中发现的增强子特征序列ATGCAAAT，即“八聚体”元件。凝胶迁移率变动分析表明，该八聚体序列与肌肉核提取物中存在的反式作用因子结合，尽管足迹分析表明相互作用有限。用八聚体序列已发生突变且随后蛋白质结合被消除的片段进行的瞬时分析表明，这种特定的相互作用可能在负向调节强正调控启动子元件的作用中发挥作用。
