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EDUCATION:
Genetics Graduate Program: Faculty |
Genetic controls of the integrity of organelle DNA.
Although chloroplasts and mitochondria are derived from prokaryotes that at one time were self-sufficient, they evolved into an endosymbiotic existence, losing many gene functions along the way. Even the replication and repair of their DNAs is completely dependent on nuclear gene products. We are studying one such nuclear gene that contributes to the integrity of the chloroplast DNA (cpDNA). When the plastome mutator (pm) allele of Oenothera is homozygous, cpDNA mutations occur at a frequency that is 200-fold above spontaneous levels. Our characterizations of DNA lesions caused by plastome mutator have shown that duplications/deletions occur at sites defined by short direct repeats in the cpDNA, suggesting that mutations occur from an increase in slipped strand mispairing during replication or from elevated levels of recombination. Most likely, pm encodes an enzyme that may be active in both DNA replication and repair/recombination. We have tested chemical agents for their impact on the plastome mutator line, looking for a synergistic effect that would implicate a particular DNA metabolic pathway. Current efforts focus on the development of an in vitro assay for replication slippage and comparative analyses of replication complexes isolated from wild-type plants and the plastome mutator line.
Controls of chloroplast multiplication and inheritance.
In crosses of Oenothera, the evening primrose, chloroplasts are transmitted to the progeny from both parents (biparental non-Mendelian inheritance). Chloroplasts from different species of Oenothera have intrinsic differences in their "strength" of transmission. Our working hypothesis has been that the variation in heritance may be due to differences in the ability of cpDNA to replicate. Techniques of DNA spreading and electron microscopy were used to identify and map the origins of replication of the cpDNA of Oenothera, and we are currently using the plastome mutator system to obtain deletions in or near one of the replication origins. We hope to establish a correlation between the presence of particular DNA sequences and the multiplication strength of the plastids (as measured through their ability to be transmitted in crosses).
Hupfer, H.,M. Swiatek, S.Hornung, R.G. Herrmann, R.M. Maier, W.L. Chiu, and B.B. Sears. 2000. Complete nucleotide sequence of the Oenthera elata plastid chromosome, representing plastome I of the five distinguishable Euoenothera plastomes. Mol Gen Genet 263:581-585.
Stoike, L. and B. B. Sears. 1998. Plastome mutator induced alterations arise in Oenothera chloroplast DNA through template slippage. Genetics 149:347-353.
Chang T. L., L. L. Stoike, D. Zarka, G. Schewe, B. B. Sears. 1996. Characterization of primary lesions caused by the plastome mutator of Oenothera. Current Genetics 30:522-530.
Sears B. B., L. L. Stoike, W. L. Chiu. 1996. Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication. Mol. Biol. Evol. 13:850-863.
Glick, R. and B. B. Sears. 1994. Genetically-programmed chloroplast dedifferentiation as a consequence of plastome-genome incompatibility in Oenothera. Plant Physiol. 106:367-373.
Chiu, W. -L. and B. B. Sears. 1993. Plastome-genome interactions affect plastid transmission in Oenothera. Genetics. 133:989-997.
