Holland M J, Hager G L, Rutter W J
Biochemistry. 1977 Jan 11;16(1):8-16. doi: 10.1021/bi00620a002.
Yeast poly(adenylic acid)-containing messenger RNA was isolated from total cellular RNA by affinity chromatography on poly(uridylic acid)-cellulose. The relative complexity of the isolated yeast mRNA was assessed by hybridization analysis with complementary DNA synthesized from the isolated messenger RNA (mRNA) with viral reverse transcriptase. Approximately 25% of the mRNA hybridized at an apparent Crt1/2 of 5 X 10(-3) mol sl.(-1), while the remainder hybridized at an average Crt1/2 of 10(-1) mol sl.-1. Poly(adenylic acid)-containing yeast mRNA was translated in vitro in a wheat germ cell-free extract, and the major polypeptides synthesized have the same molecular weight as the major proteins present in the cell. Four of these proteins were identified by coelectrophoresis and immune precipitation to be pyruvate kinase, enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase. These data demonstrate in agreement with the hybridization results that yeast contains major mRNA species and that some of the glycolytic enzyme mRNAs make up part of the major fraction. A procedure is outlined for the preparation of yeast mRNA which is essentially free of ribosomal RNA contamination and is further enriched in the major mRNAs present in the cell.
通过在聚（尿苷酸）-纤维素上进行亲和层析，从总细胞RNA中分离出含酵母聚（腺苷酸）的信使RNA。用病毒逆转录酶从分离的信使RNA（mRNA）合成的互补DNA进行杂交分析，评估分离的酵母mRNA的相对复杂性。约25%的mRNA在表观Crt1/2为5×10⁻³mol·s⁻¹时杂交，而其余部分在平均Crt1/2为10⁻¹mol·s⁻¹时杂交。含聚（腺苷酸）的酵母mRNA在小麦胚芽无细胞提取物中进行体外翻译，合成的主要多肽与细胞中存在的主要蛋白质具有相同的分子量。通过共电泳和免疫沉淀鉴定出其中四种蛋白质为丙酮酸激酶、烯醇化酶、醛缩酶和甘油醛-3-磷酸脱氢酶。这些数据与杂交结果一致，表明酵母含有主要的mRNA种类，并且一些糖酵解酶mRNA构成了主要部分的一部分。概述了一种制备酵母mRNA的方法，该方法基本上不含核糖体RNA污染，并且进一步富集了细胞中存在的主要mRNA。
