Maguire K A, Jacob S T
Biochemistry. 1986 Apr 8;25(7):1515-9. doi: 10.1021/bi00355a007.
Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.
本实验室先前的研究表明，在成年肝脏中，肿瘤型聚腺苷酸聚合酶基因要么转录水平很低，要么该酶的信使核糖核酸（mRNA）在很大程度上未表达。为了验证这些可能性，采用硫氰酸胍法从大鼠肝脏和莫里斯肝癌3924A中分离出总核糖核酸（RNA）；通过寡聚（dT）-纤维素柱层析分离出聚腺苷酸（poly(A+)）RNA和非聚腺苷酸（poly(A-)）RNA，并用于兔网织红细胞裂解液系统中的翻译。体外翻译后，用纯化的抗肿瘤聚腺苷酸聚合酶抗体或对照免疫球蛋白对产物进行免疫沉淀。当用免疫血清沉淀从肝癌poly(A+)或poly(A-)RNA翻译出的多肽时，观察到一种独特的经[35S]甲硫氨酸标记的35千道尔顿（kDa）蛋白。当使用对照血清进行免疫沉淀时，这条带不明显。在免疫沉淀前，将产物与高度纯化的肿瘤核聚腺苷酸聚合酶一起孵育时，放射性标记的35-kDa多肽不明显。将翻译产物与牛血清白蛋白而非聚腺苷酸聚合酶预先孵育，对免疫沉淀没有影响。当用肝脏poly(A+)RNA指导翻译时，这种35-kDa蛋白不明显。这些数据表明：（a）肿瘤酶不是以前体形式合成的；（b）肿瘤mRNA而非正常肝脏mRNA含有可检测到的编码肿瘤型聚腺苷酸聚合酶的序列；（c）聚腺苷酸聚合酶mRNA也以poly(A-)群体的形式存在。
