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Southwestern histochemical localization of regulatory element binding proteins for rat anterior pituitary hormones.

大鼠垂体前叶激素调节元件结合蛋白的西南组织化学定位。

基本信息

批准号：
06454143
负责人：
NAKANE Paul K.
金额：
$ 4.61万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

For the activation or suppression of a gene, the formation of stable molecule complexes between regulatory elements (RE) of the gene and binding proteins for the regulatory elements is often a prerequisite (REBP). Pit-1/GHF-1 form complex with RE of growth hormone (GH) (GH-1) and activates rat GH gene and it also binds to RE of prolactin (PRL) (P1) and activates PRL gene. At the same time, the binding of GH gene promoter proximal repressor element binding protein (PREB) to GH gene promoter proximal repressor element (PRE) suppress GH gene. In GH3 cells in vitro and in sections of rat anterior pituitary, Pit-1/GHF-1 and PREB were localized by utilizing a newly introduced Southwestern histochemical method (SWH). Pit-1/GHF-1 protein, GH and PRL were localized by immunohistochemistry (IHC). Some tissue sections were used for the double staining by SWH and IHC.For the localization of REBP,oligodeoxynucleotides (oligo-DNAs) correspond to rat GH-1 (-89 and -70) and rat PRE (-169 and -152) were used. SWH revealed that Pit-1/GHF-1 was mainly found in nuclei of GH or PRL cells as well as in nuclei of some other cells except gonadotrophs, whereas nuclear localization of the PREB was mainly in PRL cells and some other cells except GH cells and gonadotrophs in the rat pituitary tissue sections. Cell nuclei positive for Pit-1/GHF-1 by SWH were also positive for Pit-1/GHF-1 proteins by IHC.In GH3 cells in vitro, the results similar to that seen in sections of anterior pituitary were found.Our findings support a notion that the formation of complex between GH-1 and Pit-1/GHF-1 is needed for the activation of GH gene and that the formation of complex between P1 and Pit-1/GHF-1 is needed for the activation of PRL gene. If appears that the formation of complex between PRE and PREB suppressed GH gene in PRL producing cells. A mechanism that suppresses PRL gene in GH producing cells remains to be investigated.

对于基因的激活或抑制，在基因的调控元件（RE）和调控元件的结合蛋白之间形成稳定的分子复合物通常是先决条件（REBP）。Pit-1/GHF-1与生长激素（GH）的RE（GH-1）形成复合物，激活大鼠GH基因; Pit-1/GHF-1与催乳素（PRL）的RE（P1）结合，激活PRL基因。同时，GH基因启动子近端阻遏元件结合蛋白（PREB）与GH基因启动子近端阻遏元件（PRE）的结合抑制了GH基因的表达。在体外培养的GH 3细胞和大鼠垂体前叶的切片中，Pit-1/GHF-1和PREB的定位利用新引入的西南组织化学方法（SWH）。免疫组化法检测Pit-1/GHF-1蛋白、GH和PRL的表达。部分组织切片进行SWH和IHC双重染色，用大鼠GH-1（-89和-70）和大鼠PRE（-169和-152）对应的寡脱氧核苷酸（oligodeoxynucleotides，oligo-DNAs）对REBP进行定位。SWH显示Pit-1/GHF-1主要定位于GH或PRL细胞核以及除促性腺激素细胞外的其他细胞核，而PREB主要定位于PRL细胞核以及除GH细胞和促性腺激素细胞外的其他细胞核。通过SWH对Pit-1/GHF-1呈阳性的细胞核也通过IHC对Pit-1/GHF-1蛋白呈阳性。结果表明，GH-1与Pit-1/GHF-1复合物的形成是GH基因激活的必要条件，P1与Pit-1/GHF-1复合物的形成是GH基因激活的重要条件。1是激活PRL基因所必需的。PRE与PREB复合物的形成抑制了PRL分泌细胞中GH基因的表达。在GH产生细胞中抑制PRL基因的机制仍有待研究。