猪垂体总mRNA的提取、鉴定及其cDNA的合成

 用改进的LiCl沉淀法和寡聚(dT)-纤维素亲和层析法由猪垂体制得总mRNA。在兔网织红细胞无细胞翻译体系中进行体外翻译的结果表明,制得的总mRNA具有一定的翻译活力。翻译产物与兔抗猪生长激素抗血清发生免疫沉淀,沉淀物占总翻译产物的10％左右。SDS聚丙烯酰胺凝胶电泳的结果表明翻译产物有一条很深的带,分子量约为24,000道尔顿,与猪前生长激素的分子量相近。以制备的mRNA为模板反转录合成了双链cDNA。第一链的合成产率为10—35％,第二链的合成产率为84—115％。cDNA的平均分子长度为825bp。

ISOLATION, CHARACTERIZATION OF TOTAL mRNA FROM PORCINE PITUITARY GLAND AND SYNTHESIS OF ITS cDNA

Total RNA was prepared from pocine pituitary gland by the methods of LiCl precipitation, as described by Roskam et al, and Rhoads, with modification.mRNA was purified by oligo (dT)-cellulose affinity chromatography, accordind to Mania-tis et al. Pre-growth hormone specific mRNA was characterized by translation in vitro in a rabbit reticulocyte cell-free system and of -the translation products by immunoprecipitation and electrophoresis. In the electrophotogram, a main band with a molecular weight of about 24,000 dalton was observed,which is the same as that of porcine pre-growth hormone.About 10% of the translation products could be precipitated by adding porcine pre-growth hormone specific antiserum.These results suggest that the porcine pre-growth hormone specific mRNA is a main component in our total mRNA preparation and that it has sufficient translation efficiency. The DNA complementary to the total mRNA was synthesized in vitro. The yields of the first and second strands of cDNA were 10-35% and 84-115% respectively.The average molecular size of cDNA was 825 bp.