Chloroplasts and mitochondria contain the eukaryotic cell's machinery for the photochemical or oxidative formation of ATP and synthesis or metabolism of reduced, energy-rich carbohydrates. The unclear eukaryote Chlamydomonas is an excellent model system in which to study the molecular biology and genetics of these important organelles whose biogenesis depends on both nuclear and organelle encoded gene products. Successful transformation of the chloroplast genome of Chlamydomonas now allows one to determine the consequences in vivo of chloroplast genes manipulated in vitro. The first problem to be studied involves biogenesis of chloroplast ribosomes and the chloroplast ATP synthase. Antibiotic resistance mutations in chloroplast and nuclear genes which affect the translational efficiency and fidelity of the small subunit of the chloroplast ribosome will be characterized to identify the gene products involved. Mutations in selected regions of the 16S ribosomal RNA gene and the gene encoding the S12 ribosomal protein will be made in vitro and their effects studied on ribosomes isolated from the transformants. Similar studies will examine the effects on ATP synthase function of altering specific amino acid residues in the catalytic beta subunit. An investigation of messenger RNA domains and possible trans acting factors responsible for translational regulation of chloroplast genes will also be initiated. The second problem involves understanding the molecular cytogenetics of the chloroplast genome. While recombination of chloroplast and mitochondrial genomes probably occurs generally, the process can only be studied systematically in a few organisms with good genetic and molecular markers and having organelle fusion as some part of the life cycle. Chlamydomonas is ideally suited for these studies as chloroplast gene recombination is easily studied, numerous mutants have been mapped physically in specific genes and recombination patterns can be traced using restriction fragment length polymorphisms that differentiate several commonly used strains. The role of short repeat sequences ubiquitous to intergenic regions in legitimate and illegitimate recombination events in the chloroplast genomes will be defined. While mobile genetic elements have been will characterized in yeast mitochondrial genomes, they are only now being identified in chloroplasts. Two mobile chloroplast elements containing open reading frames, which may encode proteins responsible for their movement, will be studied using molecular techniques including mutagenesis in vitro and transformation. The third problem involves understanding the mechanism by which organelle genes are selectively transmitted by one parent in sexual crosses where gametes make equal cytoplasmic contributions to the zygote. In Chlamydomonas chloroplast and mitochondrial genomes are transmitted by opposite parents under the control of the nuclear mating type locus. Mechanisms responsible for the postulated protect and destruct functions for each organelle genome will be dissected through the isolation of specific mutations and their biochemical and molecular characterization. Conceivably, this research could lead to the discovery of enzyme systems analogous to the restriction- modification systems of bacteria.
