Preparation of Immunoglobulin Light‐Chain mRNA from Microsomes without the Use of Detergent

Abstract

A technique which releases ribosomes from membranes without the use of detergent described in the preceding paper is shown to allow the separation of two distinct pools of poly(A)‐containing RNA in microsomes. Thus, using preparations from mouse myeloma tissue‐culture cells, essentially 100% release of immunoglobulin mRNA occurs under conditions in which only 50–60% of the total microsomal poly(A)‐containing RNA is released. The RNA which is not released is principally mitochondrial ribosomal RNA and the method thus provides a good preparative procedure for this fraction. Messenger ribonucleoprotein particles involved in immunoglobulin biosynthesis have been prepared from the released material. The heavy‐chain mRNA‐containing particle sediments in a neutral sucrose gradient at about 22 S, and that containing the light‐chain mRNA at about 18 S. The ribosome‐release technique has been used for the rapid preparation of light‐chain mRNA in good yield and at over 60% purity after a single sucrose gradient centrifugation. Further purification has been achieved by polyacrylamide gel electrophoresis. Using [ 32 P]RNA, the contaminants can be estimated directly at the chemical level using a fingerprint assay.