Gedamu L, Iatrou K, Dixon G H
Biochem J. 1978 Jun 1;171(3):589-99. doi: 10.1042/bj1710589.
Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.
通过采用温和条件的简单程序，从鲑鱼睾丸组织中制备出了毫克量的纯化聚腺苷酸[poly(A)+]（多聚腺苷酸化）鱼精蛋白mRNA。这一过程包括：用25 mM乙二胺四乙酸（EDTA）解离多核糖体以释放信使核糖核蛋白颗粒，从而分离出总核酸，再用0.5%十二烷基硫酸钠（SDS）于0.25 M氯化钠中处理线粒体后上清液，通过将其结合到二乙氨基乙基纤维素（DEAE-纤维素）柱上进行脱蛋白。在这些条件下，总RNA会被结合，缺乏18S和28S RNA的低分子量RNA可用0.5 M氯化钠洗脱，并在寡聚（dT）-纤维素柱上进行层析以选择聚腺苷酸[poly(A)+]RNA。在蔗糖密度梯度上对未结合的聚腺苷酸[poly(A)-]RNA和结合的聚腺苷酸[poly(A)+]mRNA进行进一步纯化，结果表明18S和28S核糖体RNA均不存在，它们在DEAE-纤维素层析步骤中被去除。聚腺苷酸[poly(A)-]RNA沉降在4S区域，而结合的聚腺苷酸[poly(A)+]RNA部分在6S处显示出一个主峰[聚腺苷酸[poly(A+)]鱼精蛋白mRNA]，在3 - 4S区域有一个肩峰。在第二个梯度上对主峰和肩峰进行分析表明，主峰的大部分沉降在6S，而肩峰沉降速度比4S慢。聚腺苷酸[poly(A)+]鱼精蛋白mRNA的身份通过以下标准得以确定：（1）纯化的鱼精蛋白mRNA在尿素/聚丙烯酰胺凝胶电泳上迁移为一组四条带；（2）通过淀粉凝胶电泳分析小麦胚芽提取物中合成的多肽，显示出一条放射性带，其迁移位置与载体鲑鱼睾丸鱼精蛋白标准品完全一致；（3）多肽产物在羧甲基纤维素（CM-纤维素，CM-52）上的层析显示存在三或四种放射性标记的鱼精蛋白成分，它们与作为载体添加的未标记鲑鱼睾丸鱼精蛋白成分共洗脱。现在，大量纯化的鱼精蛋白mRNA的可得性应能使其物理和化学性质得到更全面的分析。
