The study of organelle genomes and genes is essential to our understanding of eukaryotic cellular and molecular biology. The proper functioning of organelles in all eukaryotic cells involves a precise cooperation between nuclear and organelle genetic systems. This proposal addresses two interconnected problems in organelle molecular genetics. First, mechanisms and consequences of the exchange of DNA sequences between cellular genomes will be explored. Two genes that have departed the chloroplast genome will be studied in their putative new home - the nucleus - in terms of their mode of expression and the targeting of their products back to the chloroplast. We will investigate the hypothesis that two novel families of dispersed repeats, putative transposable elements, residing in the chloroplast genomes of clover and geranium have invaded these genomes from the nucleus. Several chloroplast pseudogenes that recently entered the mitochondrial genome will be characterized as to their mode of exchange and the nature of the intermediate molecules involved. Finally, studies will test the hypothesis that most of the plant mitochondrial genome is derived from the nucleus. Second, internal rearrangements (inversions and transpositions) within organelle genomes will be examined in terms of how they occur and how they affect gene function. The hypothesis that the chloroplast genomes of clover and geranium were recently destabilized by invading repeated, putatively transposable, elements will be tested by examining the role of these repeats in generating specific and very recent rearrangements. The effects of these rearrangements on proper chloroplast gene function will also be examined. The extraordinary instability of plant mitochondrial genomes will continue to be explored using the convenient-sized genomes of Brassica species as a model system. These studies will involve sequencing the endpoints of rearrangements to understand how they are generated, and following the dynamics of mitochondrial DNA rearrangement in cell culture and in plants regenerated from fused protoplasts. A major focus will be on rearrangements and disruptions in gene structure and function associated with mitochondrial dysfunction in two male-sterile Brassica cytoplasms.
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