UNIPARENTAL INHERITANCE OF CHLOROPLAST DNA

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Eukaryotes have evolved several strategies to ensure uniparental inheritance of organellar DNA. The goal of the proposed work is to elucidate the mechanism of uniparental inheritance using an experimentally tractable organism, the unicellular algae, Chlamydomonas reinhardtii. Chloroplast inheritance is controlled by the two mating type alleles in Chlamydomonas, mt+ and mt-. After haploid cells mate and undergo meiosis, the progeny retain only mt+ chloroplast DNA. The strategy that Chlamydomonas utilizes for maintaining uniparental inheritance appears to involve selective protection of mt+ chloroplast DNA from a zygotic nuclease that is active in both mt- and mt+ chloroplasts. The proposed experiments are designed to isolate the key components of this system, the nuclease and the protection factor(s). First, strains with sequence specific chloroplast tags will be constructed so that degradation of mt- chloroplast DNA can be quantitatively monitored. Second, these tagged strains will be used to develop an in vitro assay for mt- specific chloroplast DNA degradation, and mt+ specific protection. This assay will be used to purify the nuclease and protection activities. Finally, mutants that are defective for the nuclease and protection activities will be isolated and cloned. The biochemical and genetic characterization of this system will allow a dissection of the molecular mechanisms underlying uniparental inheritance. Besides gaining insight into a fundamental property of eukaryotic organellar biology, this work may also have implications for aging and cancer. Deletions and transpositions of mitochondrial DNA and alterations in mitochondrial metabolism are associated with cancer and the aging process. Understanding how a cell such as Chalmydomonas eliminates undesirable organellar genomes will provide important basic information on how cells recognize and regulate the fate of their organellar DNA.