Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts.
Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts.
复制标题
GH3 垂体无细胞提取物对大鼠催乳素基因的选择性转录和 DNase I 保护。
DOI：
10.1073/pnas.84.15.5211
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发表时间：
1987
影响因子：
11.1
通讯作者：
Loukin,S
中科院分区：
文献类型：
--
作者：
Gutierrez-Hartmann,A;Siddiqui,S;Loukin,S
催乳素（PRL）是生长激素基因家族的成员，在垂体前叶的催乳细胞中特异性表达。从培养的GH 3大鼠垂体瘤细胞中制备全细胞提取物，以在体外转录测定中研究大鼠PRL（rPRL）基因的催乳素特异性表达。人α 1-珠蛋白和劳斯肉瘤病毒启动子在HeLa和GH 3细胞提取物中有效地启动转录，而含有425个碱基对的5'侧翼DNA的rPRL启动子仅在GH 3垂体无细胞提取物中有活性。通过添加GH 3细胞提取物在HeLa细胞提取物中重建rPRL基因的转录。rPRL启动子的DNA酶I消化揭示了位于位置-55（I）和-160（III）的两个保护区，其是GH 3细胞特异性的和rPRL启动子选择性的。这些“足迹”覆盖高度保守的8碱基对基序，CCTGATAATA。相比之下，在位置-125的足迹II是共同的HeLa和GH 3细胞提取物和覆盖在生长激素基因家族的所有成员中发现的15个碱基对的序列。因此，GH 3垂体无细胞提取物选择性地转录rPRL基因，并含有直接与rPRL启动子相互作用的细胞特异性因子。这些研究提供了有用的分析，以进一步识别，纯化和表征垂体特异性转录因子，并解决涉及rPRL基因表达的生化机制。
Prolactin (PRL) is a member of the growth hormone gene family that is specifically expressed in the lactotroph cells of the anterior pituitary. Whole cell extracts have been prepared from cultured GH3 rat pituitary tumor cells to study lactotroph-specific expression of the rat PRL (rPRL) gene in an in vitro transcription assay. The human alpha 1-globin and Rous sarcoma virus promoters efficiently initiate transcription in both HeLa and GH3 cell extracts, whereas the rPRL promoter containing 425 base pairs of 5' flanking DNA is active only in GH3 pituitary cell-free extracts. Transcription of the rPRL gene was reconstituted in HeLa cell extracts by the addition of GH3 cell extracts. DNase I digestion of the rPRL promoter reveals two protected regions centered at positions -55 (I) and -160 (III) that are GH3 cell-specific and rPRL promoter-selective. These "footprints" overlie a highly conserved 8-base-pair motif, CCTGATAATA. By contrast, footprint II at position -125 is common to both HeLa and GH3 cell extracts and overlies a 15-base-pair sequence found in all members of the growth hormone gene family. Thus, GH3 pituitary cell-free extracts selectively transcribe the rPRL gene and contain cell-specific factors that directly interact with the rPRL promoter. These studies provide useful assays to further identify, purify, and characterize pituitary-specific transcription factors and to address the biochemical mechanisms involved in rPRL gene expression.