Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ

• Published in: Analytical biochemistry Vol. 138; no. 2; pp. 465 - 471
• Main Authors: Li, Shi-Jiang, Chang, Lee-Hong, Chen, Shiow-meei, Marshall, Alan G.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.05.1984; Elsevier
• Subjects: 5 S RNA; Analytical, structural and metabolic biochemistry; Bacillus subtilis - genetics; Bacillus subtilis, Neurospora, eucaryotic, procaryotic; Biological and medical sciences; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; isolation; Methods; Neurospora - genetics; Neurospora crassa - genetics; Nucleic acids; purification; ribosomal RNA; RNA, Bacterial - isolation & purification; RNA, Fungal - isolation & purification; Rna, ribonucleoproteins; RNA, Ribosomal - isolation & purification; RNA, Transfer - isolation & purification; Triticum - genetics; wheat germ; isolation; 5 S RNA; wheat germ; ribosomal RNA; Bacillus subtilis, Neurospora, eucaryotic, procaryotic; purification
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(84)90841-8

Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity. Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5–10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells. Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite. High-molecular-weight contaminants are suppressed by either 65°C heat treatment or lowered sodium dodecyl sulfate concentration. For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65°C heat treatment until after the phenol extraction step.