86] Fractionation of RNA's by countercurrent distribution

Abstract

Publisher Summary This chapter presents three procedures that are commonly used for the separation and/or purification of specific transfer RNA. Countercurrent distribution procedures have been employed successfully by several investigators for the separation and purification of transfer RNA isolated from the yeast, Escherichia coli , and the rat liver. In these procedures, it is possible to fractionate grams of transfer RNA and obtain tens of milligrams of purified specific RNA. These procedures are advantageous in that, using theoretical calculations, the extent of purity of a single homogeneous RNA can be determined. In order to recover purified transfer RNA by countercurrent distribution procedures, it is essential that no amino acid acceptor activity be lost during the process of purification. All the procedures described here are designed for a 200-tube apparatus, each tube having the capacity to hold l0 ml of each phase. It is preferable to perform initial countercurrent distribution in one solvent system, isolate the RNA fractions having the highest amino acid acceptor activity, and redistribute in another solvent system having a lower partition coefficient for the material to be redistributed. Procedure for the purification of yeast alanine RNA is described here. The method is presented in such a manner that it can be extended for the purification of any other RNA's.