Purification of low-abundance mRNAs by polysome immunoprecipitation and its use for cDNA cloning

低丰度mRNA的cDNA克隆是重组DNA研究中最关键的步骤之一。多体免疫沉淀是纯化低丰度mrna的最有效手段之一，纯化后的mrna应有利于cDNA克隆。我们建立了一种简单快速的从大鼠肝脏中分离多体的方法，并将该方法应用于cDNA的克隆。建立了从大鼠肝脏中制备游离多体和膜结合多体的方法。该程序包括通过肝脏匀浆的差速离心和两类多体的镁沉淀分离两类多体。这些多体基本上未被降解，在无细胞合成中具有很高的活性。用免疫沉淀法从游离多聚体中纯化了约50倍的鸟氨酸转氨基甲酰基酶多聚体类。从富集的mRNA中分离出一个含有1600个碱基插入的cDNA克隆，并确定了转甲氨基酰基酶前体的整个初级结构。3-氧酰基-辅酶a硫硫酶和3-羟基酰基-辅酶a脱氢酶mrna纯化25-40倍，并分离出这两种酶的cDNA克隆。最长的克隆分别含有1400和1200碱基插入物，预计携带超过80%的硫酶和脱氢酶mrna。磷酸氨甲酰合成酶mRNA（亚单位分子量为160,000）不能通过多体免疫沉淀法有效富集。因此，本方法可能不适合纯化非常大的mrna。

cDNA cloning of low abundance mRNA species is one of the most critical steps in recombinant DNA studies. Polysome immunoprecipitation is one of the most powerful means to purify low-abundance mRNAs and the purified mRNAs should facilitate cDNA cloning. We developed a simple and rapid procedure for isolation of polysomes from rat liver and applied the procedure for cDNA cloning.1. A procedure was developed for preparation of free and membrane-bound polysomes from rat liver. The procedure involves separation of the two classes of polysomes by differential centrifugation of liver homogenate and magnesium precipitation of both classes of polysomes. The polysomes were essentially undegraded and highly active in cell-free synthesis.2. A polysome class synthesizing ornithine transcarbamylase was purified about 50-fold from the free polysomes by immunoprecipitation. A cDNA clone with a 1600base insert was isolated from the enriched mRNA and the entire primary structure of the transcarbamylase precursor was determined.3. 3-Oxoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase mRNAs were purified 25-40-fold and cDNA clones for the two enzymes were isolated. The longest clones containing 1400- and 1200-base insert, respectively, are expected to carry more than 80% of the thiolase and dehydrogenase mRNAs.4. The mRNA for carbamyl phosphate synthetase (subunit molecular weight, 160,000) could not be effectively enriched by the polysome immunoprecipitation procedure. Thus, the present procedure may not be suitable for purification of very large mRNAs.