9319595 Goodenough The research proposed would provide detailed molecular understanding of the mechanisms of uniparental inheritance of organelle DNA in Chlamydomonas reinhardtii. Uniparental inheritance of the plastid genome in C. reinhardtii involved two events: protection of the chromosome contributed to the zygote by the mating-type plus (mt+) gamete, and nuclease destruction of the unprotected mt- chromosome in the zygote. A cloned gene cluster, ezy-1 linked to both mt loci, has been shown by this lab to be involved in destruction. Experiments using native and/or recombinant ezy-1 protein are designed to learn whether ezy-1 functions as the nuclease or as an activator of nuclease activity. Genetic and biochemical experiments will determine whether protection is conferred directly on the mt+ chloroplast DNA (e.g. via methylation) or via protein(s) that associate with this DNA. And finally, insertional mutant strains defective in protection will be sought and analyzed. %%% Eukaryotic organisms are composed of cells that contain organelles. Of these, mitochondria (animals and plants) and chloroplast (plants only) contain their own complements of DNA, derived from their evolutionary origin as free-living bacteria. During sexual reproduction, organelle DNA is invariably transmitted to offspring via only one parent, usually via the maternal egg. In the unicellular alga Chlamydomonas, both parental gametes contribute equal amounts of organelle DNA to the zygote, but one set is then destroyed. The lab has cloned a gene whose protein product appears to participate in this selective degradation of chloroplast DNA. The project proposes to test this inference via genetic, molecular, and cell biology experiments, the goal being to learn how one parental set of DNA is destroyed and the other is preserved within a single cell. ***
