Detillieux K A, Meyers A F, Meij J T, Cattini P A
Gene Technology Group, University of Manitoba, Winnipeg, Canada.
Mol Cell Biochem. 1998 Nov;188(1-2):169-76.
We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5'-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides-793 and-720 that was greater than 97% A/G-rich. A repeat of the sequence 5'-AGGGAGGG-3' separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated approximately 3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased approximately 11 fold in both rat and human glial tumor cells. Four specific complexes (Cl-4) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 > C4) while Cl and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of Cl and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5'-gGGGAGGG-3' sequence previously implicated in the response of the atrial natriuretic factor promoter to the alpha-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (Cl-4) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5'-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.
我们克隆了大鼠成纤维细胞生长因子2（FGF - 2）启动子区域，包括1058个碱基对（bp）的5'侧翼DNA。对该启动子区域进行完全测序后发现，在核苷酸-793至-720之间有一个74 bp的结构域，其A/G含量大于97%。序列5'-AGGGAGGG-3'相隔11 bp的重复序列位于该结构域的核心。合成了一个包含这些AGGG重复序列的37 bp富含A/G的寡核苷酸，并在瞬时转染的新生大鼠心肌细胞中，对其在与萤火虫荧光素酶基因融合的最小单纯疱疹病毒胸苷激酶（TK）启动子（TKp.luc）上的功能进行了测试。在存在AGGG重复序列的情况下，启动子活性被刺激了约3倍。这种效应既不是组织特异性的也不是物种特异性的，因为在大鼠和人类胶质肿瘤细胞中，TK启动子活性都增加了约11倍。通过凝胶迁移率变动分析，在新生大鼠心脏核蛋白与37 bp富含A/G的寡核苷酸之间检测到四种特异性复合物（C1 - 4）。与过量未标记的37 bp富含A/G的寡核苷酸竞争表明，两种复合物代表了非常高亲和力/特异性的相互作用（C2 > C4），而C1和C3的亲和力较低。因此，与高达25倍摩尔过量的37 bp富含A/G的寡核苷酸竞争导致C2和C4的消失，以及C1和C3水平相应的短暂增加，随着更多竞争寡核苷酸的加入，它们自身水平也会降低。AGGG重复序列类似于先前与心房钠尿肽启动子对α - 肾上腺素能激动剂去氧肾上腺素的反应相关的5'-gGGGAGGG-3'序列。尽管在转染的心肌细胞用去氧肾上腺素处理时，在存在37 bp富含A/G的寡核苷酸的情况下检测到TK启动子活性额外增加了1.5倍，但这种效应在统计学上并不显著。此外，用37 bp富含A/G的寡核苷酸与从在有无去甲肾上腺素存在下生长的新生大鼠心肌细胞中分离的核蛋白获得的凝胶迁移率变动（C1 - 4）模式没有差异。这些数据表明，大鼠FGF - 2基因5'侧翼DNA中的富含A/G序列，包括AGGG重复序列，能够以组织和物种独立的方式赋予启动子刺激活性，但单独并不能在新生大鼠心肌细胞中诱导显著的去氧肾上腺素反应。
