Isolationand in vitro translation of leghaemoglobin mRNA from yellow lupin root nodules

Abstract

Messenger RNA for leghaemoglobin, extracted from polysomes of yellow lupin root nodules, was purified by chromatography on oligo(dT1-cellulose followed by sucrose-gradient centrifugation. Leghaemoglobin mRNA activity was assayed in a cell-free protein synthesizing system derived from wheat germ. The purified mRNA was sedimented in surcrose gradient in the 9S region which corresponds to a molecular weight of about 225,000. The poly(A) segment was estimated to be 66 nucleotides in length, based on hybridization with [3H]poly(U). Analysis of the translation product using immunoprecipitation and polyacrylamide-gel electrophoresis in denaturing conditions revealed that the product made in vitro was identical with authentic leghaemoglobin.