EXPRESSION OF LUTEINIZING HORMONE-BETA SUBUNIT CHLORAMPHENICOL ACETYLTRANSFERASE (LH-BETA-CAT) FUSION GENE IN RAT PITUITARY-CELLS - INDUCTION BY CYCLIC 3'-ADENOSINE MONOPHOSPHATE (CAMP)
EXPRESSION OF LUTEINIZING HORMONE-BETA SUBUNIT CHLORAMPHENICOL ACETYLTRANSFERASE (LH-BETA-CAT) FUSION GENE IN RAT PITUITARY-CELLS - INDUCTION BY CYCLIC 3'-ADENOSINE MONOPHOSPHATE (CAMP)
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DOI：
10.1016/0303-7207(91)90156-m
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发表时间：
1991-09-01
影响因子：
4.1
通讯作者：
ROBERTS, JL
中科院分区：
文献类型：
--
作者：
CLAYTON, RN;LALLOZ, MRA;ROBERTS, JL
在这项研究中，我们确定了大鼠促黄体生成素β基因启动子在异源大鼠垂体细胞系（GH 3细胞）的活动。 1.7将5 kb的LH-β 5'侧翼序列和5'非翻译区的前5 bp连接到氯霉素乙酰转移酶（CAT）受体基因（LH-β-CAT）上，并通过磷酸钙沉淀瞬时转染到GH 3细胞的亚汇合培养物中。 仅在GH 3细胞中检测到基础低水平CAT活性，在两种非垂体细胞系（BeWo和HeLa）中不存在。RNA酶分析显示，来自转染的GH 3细胞的mRNA保护了用于从LH-β-CAP位点转录起始的正确大小的标记的反义探针片段，证实启动子活性反映了正确起始的LH-β-CAT融合基因转录物。 CAT活性一致诱导的平均3-5倍，从全长1.7 kb的启动子，在剂量和时间依赖性的方式，由毛喉素，二丁酰cAMP，和8-溴cAMP，这意味着在LH-β-启动子区域的cAMP响应的顺式作用结构域的存在。 缺失突变体DELTA-615-CAT、DELTA-385-CAT和DELTA-250-CAT的转染均使毛喉素诱导作用降低至1.7倍，但未完全消除诱导作用，表明-1.7和-0.6kb之间的结构域含有cAMP反应元件（CRE）。 将LH-β 5'侧翼序列进一步缺失到DELTA-85-CAT使毛喉素诱导恢复到野生型水平（3-5倍），表明在-600和-85 kb之间存在弱抑制元件，并且在近端启动子区域存在cAMP响应结构域。 LH-β启动子不包含完美的串联重复回文CRE DNA序列，尽管有几个八核苷酸序列与AP-2结合位点、共有CRE和血管肠肽基因CRE仅相差1 bp。 尽管这些数据表明LH-β基因是cAMP响应性的，但这可能是由与近端和远端LH-β启动子增强子中的多个DNA序列的几种复杂蛋白质相互作用介导的。
In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta-CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta-promoter region. Transfection of deletion mutants DELTA-615-CAT, DELTA-385-CAT and DELTA-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to DELTA-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.