Nicoll C S, Steiny S S, King D S, Nishioka R S, Mayer G L, Eberhardt N L, Baxter J D, Yamanaka M K, Miller J A, Seilhamer J J
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Gen Comp Endocrinol. 1987 Dec;68(3):387-99. doi: 10.1016/0016-6480(87)90077-3.
Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3'-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.
从银大麻哈鱼生长激素（sGH）细胞区域提取总RNA，并用于合成双链cDNA，该双链cDNA被插入质粒载体并用于转化大肠杆菌HB101。总RNA也通过琼脂糖凝胶电泳按大小分离，分离出在其他物种生长激素大小范围内指导无细胞蛋白质合成的组分，并用于筛选大肠杆菌的转化菌落。鉴定出一个含有假定sGH cDNA的克隆，并确定其核苷酸序列。为了验证该cDNA是sGH的cDNA，将银大麻哈鱼垂体的GH细胞区域进行器官培养。通过高效液相色谱法纯化分泌的GH，并确定其42个氨基末端氨基酸的序列。将该序列与从cDNA推导的氨基酸序列进行比较，表明它编码sGH。含有假定sGH作为唯一突出蛋白质的培养基在涉及标记牛GH和妊娠小鼠肝膜的GH放射受体测定中具有活性：sGH的活性约为bGH标准品的10%。RNA印迹分析表明，sGH是鲑鱼垂体GH细胞区域产生的主要RNA种类。sGH的mRNA与人类、大鼠和牛GH的mRNA不同，其3'非翻译区异常大（约500个核苷酸），但编码区与哺乳动物GH具有显著同源性，并且在密码子的第三位对G和C具有强烈（78%）的偏好。sGH的氨基酸序列与哺乳动物GH和催乳素分别显示32 - 34%和19 - 22%的相同同源性。还揭示了sGH与哺乳动物GH和PRL分子之间的几个保守区域，这可能表明结构和/或功能重要结构域的保守性。亲水性分析表明，尽管sGH与代表性哺乳动物（猪）的GH在某些区域具有相似的图谱，但sGH总体上比猪（p）GH更疏水。在sGH和pGH的预测二级结构中也注意到了相似性和差异。
