Lakin N D, Boardman M, Woodland H R
Department of Biological Sciences, University of Warwick, Coventry, England.
Eur J Biochem. 1993 Jun 1;214(2):425-35. doi: 10.1111/j.1432-1033.1993.tb17938.x.
In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealis alpha 3B embryonic/larval skeletal actin gene, reside in a 156-nucleotide fragment of the promoter that spans nucleotides -197 to -42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box 3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box 1) exhibits two binding activities using bandshift analysis. One of these binding activities contains components antigenically related to a serum-response factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility-shift analysis. Although the shifted complex coelectrophoreses with the CArG box 1/serum-response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides -197 and -167. In the actin promoter, a downstream region can apparently fulfil this function.
在本研究中，我们证明了北方爪蟾α3B胚胎/幼虫骨骼肌肌动蛋白基因表达所必需且充分的所有序列，都位于启动子的一个156个核苷酸的片段中，该片段跨度为核苷酸-197至-42。启动子的这一区域包含CC(A/T)6GG（CArG）框基序的三个不完全重复，已证明这些重复在其他肌节肌动蛋白基因的表达中很重要。利用爪蟾显微注射技术作为启动子活性的瞬时检测系统，对肌动蛋白启动子进行缺失分析，结果表明最远端的CArG框（CArG框3）对于该基因的完全表达至关重要。在我们的检测条件下，最近端的CArG框（CArG框1）通过凝胶迁移分析显示出两种结合活性。其中一种结合活性包含与血清反应因子（转录因子）抗原相关的成分，而另一种则不包含。相比之下，CArG框3通过电泳迁移率变动分析仅产生一条滞后带。尽管该迁移复合物与CArG框1/血清反应因子复合物共迁移，但CArG框3产生的条带似乎与血清反应因子不同。除了CArG基序外，在肌动蛋白启动子中核苷酸-197和-167之间还鉴定出了一个进一步的上游调控元件。在肌动蛋白启动子中，一个下游区域显然可以发挥这一功能。
