The research described in this project is designed to elucidate the mechanism of genetic control of uniparental inheritance of the chloroplast genome in the green alga C. reinhardtii by studying mutant strains in which inheritance of the chloroplast genome is altered. Uniparental inheritance of organelle genomes is an important fundamental biological process which is poorly understood. The results obtained from analysis of genes (and ultimately gene products) involved in organelle inheritance in Chlamydomanas could help explain this phenomenon in other organisms. In C. reinhardtii, chloroplast genes are normally transmitted by the mating type plus (mt+) parent. Irradiation of mt+ gametes with ultraviolet light prior to mating markedly increases transmission of chloroplast genes from the mating type minus (mt-) parent as do expression of the nuclear mt+- linked mat-3-3 and the unlinked nuclear uvsE1 mutations. The two mutations also inhibit the degradation of mt- DNA which normally occurs during mating. As the uvsE1 mutant strain is also deficient in repair of damaged DNA and reduces recombination of nuclear genes in meiosis, the possibility exists that repair and/or recombination are involved in the maintenance of uniparental inheritance of the chloroplast genome. To determine whether deficiency in DNA repair or recombination is sufficient to alter chloroplast gene transmission, several additional mutant strains in which recombination and/or repair are altered will be tested for their effect on transmission of chloroplast DNA. Each mutant in which transmission of the chloroplast genome is affected will be tested for degradation of paternal chloroplast DNA during mating and for its effect on recombination between chloroplast genomes (by looking for both genetic recombinants and recombinant DNA molecules in meiotic progeny). Isolation of revertants and/or suppressor mutations of uvsE1 will be attempted to determine whether alteration of chloroplast gene transmission and repair deficiency can be separated. In addition, proteins from matings in which mt- is degraded will be compared with those in which it is not in an attempt to isolate and characterize those proteins associated with degradation of mt- DNA. Finally, the effects of ultraviolet irradiation of mt+ gametes on proteins produced during mating, on the degradation of mt- DNA during mating and on chloroplast gene transmission in strains in which the uvsE1 and/or mat-3-3 alleles are expressed (to test for synergistic effects) will be studied. Minority students will be involved in all of the above research projects and will learn a wide variety of techniques that will be generally applicable to research in the fields of molecular genetics, molecular biology and biochemistry. The students will also be expected to become involved in publication and presentation of their data.
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