Chapdelaine P, Potvin C, Ho-Kim M A, Larouche L, Bellemare G, Tremblay R T, Dubé J Y
Laboratoire de Biorégulation Hormonale, Le Centre Hospitalier de l'Université Laval, Ste-Foy, Que., Canada.
Mol Cell Endocrinol. 1988 Mar;56(1-2):63-70. doi: 10.1016/0303-7207(88)90009-3.
Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.
犬前列腺精氨酸酯酶互补DNA已被克隆到一种新的克隆载体pPBS27中。完整犬前列腺中雄激素调节的mRNA的相对丰度通过以下发现得以体现：cDNA文库中的高比例克隆通过噬菌斑或菌落杂交与来自完整犬前列腺的聚腺苷酸加尾（poly(A)+）RNA探针强烈杂交，但不与来自去势犬前列腺的聚腺苷酸加尾RNA探针杂交。选择了一个携带400个碱基对cDNA插入片段的克隆进行进一步研究。在无细胞系统中对杂交选择的RNA进行翻译，产生了一种31 kDa的肽，该肽可被抗精氨酸酯酶抗体免疫沉淀。通过对cDNA的部分序列分析证实了这一鉴定，该分析揭示了一种与已知激肽释放酶具有高度同源性的编码蛋白。对聚腺苷酸加尾RNA和总RNA的Northern印迹分析表明，精氨酸酯酶mRNA的大小约为1.0 kb，这对应于一种主要的雄激素调节RNA种类，在对完整犬前列腺的聚腺苷酸加尾RNA进行变性琼脂糖凝胶电泳后可以观察到。斑点印迹分析表明，在3周前被去势的犬的精氨酸酯酶mRNA水平比完整犬或用长效睾酮治疗的去势犬低100倍以上。
