Submitted to: Plant Cell Reports
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/20/2000
Publication Date: N/A
Citation: Interpretive Summary: The plant cell carries DNA in three places, the nucleus, the chloroplast, and the mitochondrium. The chloroplast DNA is considered to be highly conserved among distantly related plants. We used a new technique, Fiber-FISH, to visualize the DNA from chloroplasts. This technique all owed us to examine large numbers of native DNA molecules at relatively low magnification. We observed that the chloroplast DNA is more variable than previously revealed by more traditional techniques. For example, incomplete DNA molecules missing large sections were frequently observed. This technique will be a quick, cheap, and useful approach for identification and evaluation of chloroplast DNAs carrying introduced foreign genes. This research provides a technique to visually look at small DNA molecules under the microscope. It should be useful to assess whether foreign genes have been inserted into the DNA of the chloroplast. This is desirable because foreign genes inserted into the chloroplast DNA will not be transferred by pollen to other plants, reducing transgene spread.
Technical Abstract: Cytogenomics is the union of cytological methods with genomic techniques to analyze the structure and organization of whole genomes at a high resolution. We used cytogenomics to analyze both the structure and organization of chloroplast DNA from Arabidopsis, tobacco and pea. We determined that 25-45 percent of the chloroplast DNA within young developing leaf tissue are circular molecules. We observed a highly significant linear relationship between kilobase size and the physical distance under the microscope. Both linear and circular DNA fibers with one to four copies of the chloroplast genome were present with monomers being the predominant structure. Arabidopsis and tobacco chloroplasts contained previously unidentified multimers (>900 kb) consisting of six to eight genome equivalents. We further discovered novel chloroplast DNA molecules of incomplete genome equivalents, confirmed by both differential hybridization and size estimations. Putative replication intermediates consistent with both the rolling circle and D-loop replication mechanisms were revealed in all three species. The unique chloroplast DNA organization and novel structures revealed in our cytogenomic study demonstrates that the higher plant chloroplast DNA is more structurally plastic than previous sequence and electrophoretic analyses would suggest. Additionally, this research demonstrates how cytogenomics allows for powerful analysis of very rare events that cannot be detected by traditional techniques such as Southern hybridizations or PCR. Applications of cytogenomics are discussed.