The 16S ribosomal RNA of the Euglena gracilis chloroplast has been characterized in terms of its two-dimensional electrophoretic "fingerprint" (TI ribonuclease). Results show it to be a typically prokaryotic 16S rRNA. By the present criterion, different chloroplasts are shown to be related to one another and at least distantly to blue-green algae and perhaps to Bacillaceae. These results argue in favor of an endosymbiont origin of the chloroplast.Evidence suggests that various intracellular eukaryotic organelles evolved from endosymbiotic prokaryotes (1). For chloroplasts, similarities with prokaryotes are found for photosynthetic membranes (2, 3), ribosomes (4), and pathways of CO2 fixation and photoelectron flow (5, 6). Also, symbiosis between blue-green algae and a variety of eukaryotic cells occurs frequently (7). Thus, the most likely endosymbionts postulated to give rise to the chloroplast are the Cyanophytes (1, 7). Although there is much circumstantial evidence for both the general and the specific conjectures concerning endosymbiosis, a definitive molecular support for any endosymbiotic origin of eukaryotic organelles has yet to be furnished.Phylogenetic relationships among prokaryotes can be established by means of primary structural characterizations of the larger ribosomal RNAs (8, 9). Thus, if the chloroplast arose from an ancestral prokaryote, the chloroplast ribosome should carry this organelle's pedigree. A primary structural characterization of the chloroplast 16S rRNA should then test the validity of the prokaryote-endosymbiont hypothesis.To date, more than 30 prokaryotic 16S rRNAs and eukaryotic 18S rRNAs have been characterized in terms of their (T1 ribonuclease) oligomer "fingerprints" Woese et al., unpublished). These rRNAs exhibit primary structural features common to all and/or common to various subgroups, but show little, if any, resemblance to the eukaryotic 18S rRNAs. Against this molecular background, then, it is possible to make reasonably definitive conjectures about the origin of the chloroplast.We present here the results of a "fingerprint" analysis of the chloroplast 16S rRNA of the unicellular flagellate, Euglena gracilis. This organism was chosen since chloroplasts and chloroplast ribosomes can be isolated from it free of significant contamination by ribosomes from any other cellular compartment (12, 13). Thus, any confusion with the mitochondrial 16S rRNA, for example, is eliminated.
MATERIALS AND METHODSOrganism and Growth Conditions. E. gracilis, strain Z, was grown in 100-125 ml of defined medium with ethanol as carbon source (12) in the dark at 27°with limiting phosphate concentrations (15). At densities of 1 to 1.5 X 106 cells per ml, cultures were supplemented with 10-25 mCi of carrierfree 32Pi and incubated in the light for 87-90 hr at 7.2 J m-2 sec-1.Isolation of Chloroplasts. Chloroplasts were isolated as described (13). All buffers were supplemented with heparin, rat liver RNase-inhibitor, and 2-mercaptoethanol to inhibit RNase activity (12). Chloroplasts w...
