Characterization of cytoplasmic and chloroplast polysomes in plants: evidence for three classes of ribosomal RNA in nature.

Recent work, reviewed by Gibor and Granickl established that chloroplasts are endowed with their own DNA complement and thus suggested that these organelles with highly specialized functions and an intriguing life cycle are semiautonomous systems, capable of self-replication, and useful as models for the study of differentiation. In addition to their own DNA, chloroplasts were also reported to contain a unique class of 70S ribosomes that are distinct from the 80S particles of the surrounding cytoplasm.2-4 This rather unusual situation invites closer examination, since it suggests a separate origin for these organelles and hence might throw light on the evolution of the photosynthetic apparatus. The coexistence of two ribosome classes within the same cell suggests the presence of two proteinsynthesizing systems, each capable of forming polysomes and subject to different mechanisms of control. In addition, it poses the question of how the two classes of ribosomes are related, both structurally and functionally, to the ribosomes of the main phyla of the living order. Available evidence indicates that ribosomes from all sources studied so far fall into one of two classes: 70S ribosomes occurring in bacteria and 80S ribosomes characteristic of nucleated cells of animals and plants.5 Although the evidence is somewhat less clear-cut, it is generally believed that the two kinds of ribosomes have their correspondence in two types of ribosomal RNA. Thus, rRNA from 70S ribosomes of bacteria has been found to consist of a 23S and 16S component, whereas the corresponding rRNA components of the 80S particles of nucleated cells have been characterized by S-values ranging from 16 to 18S for the small, and 25 to 30S for the larger subunit.6 In this report we shall show by the use of high-resolution zone-velocity sedimentation analysis that plant cells contain cytoplasmic polysomes made up of 80S monomers and chloroplast polysomes consisting of 70S ribosomes. In addition, evidence will be presented that, contrary to general belief, there are not two, but at least three classes of ribosomes: (1) 80S ribosomes of animal origin characterized by 29/18S rRNA; (2) 80S ribosomes of plant cells containing 25/16S rRNA; and (3) 70S bacterial ribosomes made up of 23/16S rRNA. Finally, we have found rRNA from 70S chloroplast ribosomes to be indistinguishable from bacterial rRNA when compared under rigorously standardized conditions. Methods.-Isolation of chloroplast polysomes: The method described is a modification of several published procedures.'-4 Primary leaves of 5to 6-day-old pinto beans (300-500 gm fresh weight) were washed in cold distilled water and homogenized in a Waring Blendor for 30 see with 3 ml of cold buffer I (0.7 M sucrose, 0.1 M tris-HCl, pH 7.5, 0.005 M MgCl1, 0.05 M KCl, 0.005 M 2-mercaptoethanol) per gram leaf weight. All operations were performed at +40C. The homogenate was filtered through several layers of gauze, and the filtrate centrifuged for 2 min at 600 X g to remove nuclei and cell debris. Centrifugation at 1100 X g for 12 min yielded pellets of crude chloroplasts and a supernatant containing the cytoplasmic ribosomes. The crude chloro-