Large subunit ribulosebisphosphate carboxylase messenger RNA from Euglena chloroplasts (oligo(dT)cellulose chromatography/protein synthesis in vitro/wheat germ extract/aplastidic mutants)

RNA from wild-type Euglena, aplastidic mu- tant cells, and purified chloroplasts was chromatographed on oligo(dT)-cellulose at 4?. The poly(A)-containing and poly(A- lacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abili- ties to specifically stimulate synthesis of large subunit ribu- losebisphosphate carboxylase (EC 4.1.1.39; 3-phospho-D-glyc- erate carboxy-lyase (dimerizing)). The large subunit polypep- tide (59,000 molecular weight) was identified in the in vitro reaction by two-dimensional electrophoresis involving iso- electric focusing and size filtration on polyacrylamide gels. Template activity for the large subunit was detected in wild- type cells but not in aplastidic mutant strains; it was highly enriched in the isolated chloroplasts. This messenger was present only in the poly(A)lacking RNA fraction, where it constituted the most prominent template species of chloro- plast RNA. The large subunit message was freed of consider- able non-messenger RNA contamination and localized to the 10-20S fraction by sucrose gradient centrifugation.