Catterall J F, Leary S L
Biochemistry. 1983 Dec 20;22(26):6049-53. doi: 10.1021/bi00295a001.
beta-Glucuronidase mRNA was purified from androgen-induced mouse kidney by immunoadsorption of polysomes to protein A-Sepharose. Cell-free translation of mRNA isolated from the protein A bound RNA followed by immunoprecipitation revealed that beta-glucuronidase mRNA represented approximately 2% of the purified mRNA fraction. This mRNA preparation was used to produce complementary DNA clones by recombination with pBR322. Clones containing sequences that were enriched during the purification procedure were selected by differential colony hybridization. These were further screened for homology with beta-glucuronidase mRNA by hybrid-selected translation. A beta-glucuronidase cDNA clone, designated pGUS7, was identified by these criteria. With this plasmid, the abundance of beta-glucuronidase mRNA in total poly(A) mRNA from androgen-induced mouse kidney was estimated to be less than 0.04%. The beta-glucuronidase cDNA plasmid hybridized to a mRNA of 2.6 kb in length, which was induced in an androgen receptor dependent fashion over a time course of 21 days. Treatment of female mice with a single dose of testosterone (10 mg) revealed that beta-glucuronidase mRNA concentration begins to increase between 12 and 24 h after hormone administration.
通过将多核糖体免疫吸附到蛋白A-琼脂糖珠上，从雄激素诱导的小鼠肾脏中纯化出β-葡萄糖醛酸酶mRNA。对从与蛋白A结合的RNA中分离出的mRNA进行无细胞翻译，然后进行免疫沉淀，结果显示β-葡萄糖醛酸酶mRNA约占纯化mRNA组分的2%。该mRNA制剂用于通过与pBR322重组来产生互补DNA克隆。通过差异菌落杂交筛选出在纯化过程中序列富集的克隆。通过杂交选择翻译进一步筛选这些克隆与β-葡萄糖醛酸酶mRNA的同源性。根据这些标准鉴定出一个β-葡萄糖醛酸酶cDNA克隆，命名为pGUS7。利用该质粒，估计雄激素诱导的小鼠肾脏总poly(A) mRNA中β-葡萄糖醛酸酶mRNA的丰度小于0.04%。β-葡萄糖醛酸酶cDNA质粒与一个长度为2.6 kb的mRNA杂交，该mRNA在21天的时间进程中以雄激素受体依赖的方式被诱导。用单剂量睾酮（10 mg）处理雌性小鼠，结果显示激素给药后12至24小时之间，β-葡萄糖醛酸酶mRNA浓度开始增加。
