Cell-Free Translation of Messenger RNA for a Myeloma Light Chain Prepared from Synchronised Plasmacytoma Cells

Abstract

Crude preparations of biologically active mRNA, which code for a myeloma (MPC-11) light chain, were isolated by two successive sucrose gradient centrifugations of RNA extracted from membrane-bound ribosomes, mRNA thus obtained was separated into a poly(A)-rich and a poly(A)-poor fraction by oligo(dT)-cellulose chromatography. Both these fractions were able to direct the synthesis of light chains in reconstituted cell-free systems derived from heterologous cells (ascites tumor lysates) and homologous cells (MPC-11 cells grown in suspension culture). The identity of the products in vitro was confirmed by comparing their migration with that of light chains produced in vivo upon electrophoresis in sodium dodecylsulphate/polyacrylamide gels, and from the profiles of tryptic peptides obtained by chromatography on Aminex A-5 ion-exchange columns. Template activity of the poly(A)-rich light chain mRNA fraction showed very little variation during the cell cycle. The activity of the poly(A)-poor fraction on the other hand was maximal during the early S phase. It is concluded that maximal synthesis of immunoglobulins observed in vivo during the late G1 phase of the cell cycle is achieved by translational control mechanisms.