Cell‐Free Transcription and Translation of Total Spinach Chloroplast DNA

Abstract

A highly efficient cell‐free Escherichia coli system for the coupled transcription/translation of chloroplast DNA is described. Evidence for the synthesis of more than 20 discrete size classes of [35S]‐methionine‐labelled polypeptides has been obtained by dodecylsulphate/polyacrylamide gel electrophoresis of the products formed in a reaction programmed with spinach chloroplast DNA. One of the major radioactive products co‐electrophoresed with the large subunit (Mr 55000) of ribulosebisphosphate carboxylase (fraction 1 protein). Its identity as the large subunit was established by comparing its partial proteolytic digestion products with those of purified large subunit protein. Because the level of radioactivity in many of the other polypeptides synthesized in vitro is so high, their identification likewise should be possible by comparing them with known chloroplast proteins.When spinach chloroplast DNA that had been digested with certain restriction endonucleases was used to programme the transcription/translation system, some of the polypeptides, which were observed using undigested DNA, were not detectable among the products. These results are discussed with regard to the distribution of restriction enzyme sites in the operons for some of the polypeptides, and with regard to the information they can provide on the location of genes on a restriction enzyme cleavage map of chloroplast DNA.