Maurer R A
J Biol Chem. 1980 Feb 10;255(3):854-9.
Prolactin mRNA has been isolated using immunochemical techniques. Initial experiments demonstrated that 125I-labeled prolactin antibody was able to bind to pituitary polysomes but not to liver polysomes, suggesting that the binding is specific. Prolactin-synthesizing polysomes were immunoprecipitated by reaction with antiprolactin followed by anti-antibody. Immunoprecipitated polysomal RNA was chromatographed on oligo(dT)-cellulose, and the poly(A) RNA was sedimented through a sucrose gradient. This procedure resulted in a 320-fold purification of prolactin mRNA as determined by translation in a mRNA-dependent reticulocyte lysate assay. Translation analysis also suggested that the isolated prolactin mRNA is greater than 95% pure. The molecular weight of prolactin mRNA determined by electrophoresis on agarose gels containing 10 mM mercury hydroxide was 350,000. Purified prolactin mRNA was used to synthesize full-length cDNA by means of avian myeloblastosis reverse transcriptase. Use of this cDNA as a hybridization probe demonstrated that estrogen is able to increase the concentration of prolactin mRNA sequences in the pituitary.
已使用免疫化学技术分离出催乳素信使核糖核酸（Prolactin mRNA）。初步实验表明，125I标记的催乳素抗体能够与垂体多核糖体结合，但不能与肝脏多核糖体结合，这表明这种结合具有特异性。通过与抗催乳素反应，随后与抗抗体反应，对合成催乳素的多核糖体进行免疫沉淀。将免疫沉淀的多核糖体RNA在寡聚（dT）纤维素上进行层析，然后通过蔗糖梯度对聚腺苷酸（poly(A)）RNA进行沉降。通过在依赖信使核糖核酸的网织红细胞裂解物测定中进行翻译确定，该程序使催乳素信使核糖核酸得到了320倍的纯化。翻译分析还表明，分离出的催乳素信使核糖核酸纯度超过95%。在含有10 mM氢氧化汞的琼脂糖凝胶上进行电泳测定，催乳素信使核糖核酸的分子量为350,000。利用禽成髓细胞瘤逆转录酶，使用纯化的催乳素信使核糖核酸合成全长互补脱氧核糖核酸（cDNA）。将该cDNA用作杂交探针表明，雌激素能够增加垂体中催乳素信使核糖核酸序列的浓度。
