The green alga Chlamydomonas reinhardtii is being used as a model system to investigate three distinct problems in the genetics and biogenesis of organelles. First, we propose to continue our genetic and molecular characterization of chloroplast ribosomes with primary emphasis being placed on ribosomal proteins, a third of which are synthesized and presumably encoded in the chloroplast. We now have preliminary immunological and electrophoretic evidence that the chloroplast r-proteins encoded in the organelle may be highly conserved relative to their counterparts in E. coli, whereas those encoded in the nucleus appear to have evolved considerably. For example, 6 of the 43 chloroplast r-proteins made in the cytoplasm studied thus far are made as higher molecular weight precursors, their mRNAs have acquired poly-A tails, and they have little immunological similarity to r-proteins from E. coli. The chloroplast ribosome is ideal for the study of how a eukaryotic cell coordinates the activities of organelle genes, present in many copies, with those of nuclear genes, present in one or a few copies, to synthesize and assemble a macromolecular complex in which the component proteins are present in a 1:1 stoichiometry. Second, we propose to continue our genetic and molecular characterization of the photosynthetic CFO/CF1 ATPase complex, some of whose polypeptide subunits are coded in the chloroplast and others in the nucleus. Priority will be given to a detailed genetic and physical study of the atpB gene which is located in the chloroplast genome and codes for the beta subunit of this complex that is presumed to contain the catalytic site. A group of 11 deletion and point mutations mapping in this gene will be characterized in terms of their atpB transcripts and a subset of these will be examined for alterations in the sequence of the atpB gene. Third, we will continue experiments designed to understand the relationship of the physical organization of the chloroplast genome, with its two inverted repeats, and its ability to recombine and to be altered by mutation. In the process we should be able to correlate the genetic and physical maps of the chloroplast genome. We will also attempt to identify chloroplast genes with functions indispensable for cell survival.
