Jandreski M, Liew C C
Can J Biochem Cell Biol. 1984 Apr;62(4):185-90. doi: 10.1139/o84-026.
A procedure for the extraction of intact RNA from whole heart tissue using guanidine hydrochloride has been accomplished. Subsequent chromatography on oligo(dT)-cellulose of the total RNA and translation of the poly(A)+ RNA in a cell-free reticulocyte translation system has shown that the myosin heavy chain mRNA from rat heart tissue was present. The myosin heavy chain mRNA was identified by several criteria: (i) sizing by sucrose density gradient centrifugation, (ii) migration of a polypeptide translation product coincided with a myosin heavy chain marker in one-dimensional sodium dodecyl sulfate-- polyacrylamide gel electrophoresis, and (iii) immunoprecipitation of the mRNA translation product with a myosin heavy chain specific antibody. A myosin heavy chain mRNA enriched fraction was used to direct cDNA synthesis in an in vitro system. An optimal condition has been elucidated in which large molecular weight cDNA strands are produced. Evidence is presented here for the production of cDNA strands in excess of 4000 nucleotides which includes the heavy chain myosin in a high potassium--low sodium transcription assay, as revealed by dot blot hybridization with a cDNA clone of the myosin heavy chain.
已完成一项使用盐酸胍从全心脏组织中提取完整RNA的程序。随后，对总RNA进行寡聚（dT）-纤维素柱层析，并在无细胞网织红细胞翻译系统中对多聚（A）+ RNA进行翻译，结果表明大鼠心脏组织中存在肌球蛋白重链mRNA。肌球蛋白重链mRNA通过以下几个标准进行鉴定：（i）通过蔗糖密度梯度离心法测定大小；（ii）在一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，多肽翻译产物的迁移与肌球蛋白重链标记物一致；（iii）用肌球蛋白重链特异性抗体对mRNA翻译产物进行免疫沉淀。一个富含肌球蛋白重链mRNA的组分被用于在体外系统中指导cDNA合成。已阐明了产生大分子cDNA链的最佳条件。通过与肌球蛋白重链cDNA克隆的斑点杂交显示，在高钾-低钠转录试验中，这里有证据表明产生了超过4000个核苷酸的cDNA链，其中包括重链肌球蛋白。
