猕猴(Macaca mulatta)肝5S核糖核蛋白体RNA的分离提纯

应用酚-去污剂和1摩尔/升NaCl抽提低分子量RNA,通过DEAE-葡聚糖A-50离子交换层析提纯后,经含有7摩尔/升尿素的10%聚丙烯酰胺凝胶垂直板电泳制备纯化猕猴肝5S核糖核蛋白体RNA是简易而行之有效的方法。制纯的5SrRNA经聚丙烯酰胺凝胶电泳鉴定呈现单一的一条区带。与大肠杆菌5SrRNA标准品具有相同的电泳迁移率。

ISOLATION AND PURIFICATION OF 5S RIBOSOMAL RNA FROM THE RHESUS MONKEY (MACACA MULATTA) LIVER

5SrRNA is a small molecular ribonucleic acid existing in the large subunit of ribosomes of prokaryotic and eukaryotic cells. It is one of the most suitable molecule for studying molecular evolution and plays an important role in protein biosynthesis.A simple and reliable procedure of the purification of 5S ribosomal RNA from the Rhesus monkey liver is described.The liver was removed from a freshly killed Rhesus monkey and homogenized with two volumes of the "TSMK" buffer containing 0.005mol/L tris-HCl pH7.5, 0.25mol/L Sucrose. 0.005mol/L MgCl₂,0.025mol/L KC1 and 0.2% bentonite.The homogenate was centrifuged at 10000 Xg for 20 min. The supernatant (cytoplasmic fraction) was treated with water-saturated phenol containing 0.1% 8-hydroxyquinoline and 0.2% sodium dodecyl sulfate-bentonite, centrifuged at 10000 xg 40 min and then total RNAs was precipitated by cold 95% ethanol. Low-molecular weight RNAs was extracted from the precipitate with 1 mol/L sodium chloride solution. Then,the low molecular weight RNAs was purified by ion-exchange chromatography of DEAE-Sephadex A-50 and eluted with a linear concentration from 0.375mol/L to 0.525mol/L NaCl.The 5SrRNA was purified from low molecular weight RNA₂ by 10% preparative polyacrylamide gel slab electrophoresis in the presence of 7mol/L urea, using 90mmol/L tris-boric acid pH8.3, and 2.5mmol/L ethylenediamine tetraacetic acid disodium buffer system. The 5SrRNA band was excised from the preparative slab gel under 254nm ultraviolet light,eluted with 10mmol/L tris buffer (pH8.O) containing 350mmol/L KC1, lOmmol/L MgCl₂, 1mmol/L EDTA and dried in vacuo.Identification of polyacrylamide gel electrophoresis and ultraviolet absorption spectrum for pure 5SrRNA of the liver of Rhesus monkey indicated that only a single band on denatured polyacrylamide gel and a typical ultraviolet absorption peak of nucleic acid were shown. The A₂₆₀/A₂₃₀ ratio was 2.08 and A₂₆₀/A₂₈₀ ratio esd 2.07.