McAndrew S J, Chen N Y, Wiehl P, DiCaprio L, Yun J, Wagner T E, Okada S, Kopchick J J
Department of Zoological and Biomedical Sciences, Ohio University, Athens 45701.
J Biol Chem. 1991 Nov 5;266(31):20965-9.
A synthetic oligonucleotide, 5'-d(CTAGT-CTAGACTAG)-3' which encodes translational termination codons in three reading frames, was inserted into either exon IV (pbGH-4A) or V (pbGH-5A) of the bovine growth hormone gene. The resultant plasmids, under the transcriptional regulation of the mouse metallothionein 1 promoter, were introduced into cultured mouse L-cells or rat GH3 cells. Compared to wild type bGH RNA, bGH-specific RNA transiently expressed from pBGH-5A or pBGH-4A DNA in mouse L-cells was similar or slightly smaller in size, respectively. Unexpectedly, bGH-4A RNA lacked exon IV sequences. Immunofluorescence and immunoprecipitation analyses revealed that wild type bGH was localized to the Golgi apparatus, while truncated hormones were confined to the cytoplasmic compartment of transfected cells. In addition, truncated hormones were shown to be secretion-defective albeit the bGH signal peptide was efficiently and precisely processed. Thus, structural alterations in the bGH gene can dramatically affect bGH precursor mRNA processing and hormone localization within cultured mouse fibroblast or rat pituitary cells.
一种合成寡核苷酸5'-d(CTAGT-CTAGACTAG)-3'，它在三个阅读框中编码翻译终止密码子，被插入到牛生长激素基因的外显子IV（pbGH-4A）或外显子V（pbGH-5A）中。在小鼠金属硫蛋白1启动子的转录调控下，将所得质粒导入培养的小鼠L细胞或大鼠GH3细胞。与野生型bGH RNA相比，在小鼠L细胞中从pBGH-5A或pBGH-4A DNA瞬时表达的bGH特异性RNA大小分别相似或略小。出乎意料的是，bGH-4A RNA缺少外显子IV序列。免疫荧光和免疫沉淀分析表明，野生型bGH定位于高尔基体，而截短的激素局限于转染细胞的细胞质区室。此外，尽管bGH信号肽被有效且精确地加工，但截短的激素显示出分泌缺陷。因此，bGH基因的结构改变可显著影响培养的小鼠成纤维细胞或大鼠垂体细胞内bGH前体mRNA的加工和激素定位。
