The Chloroplast-Located Homolog of Bacterial DNA Recombinase

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The cDNA for the chloroplast-Iocated homolog of bacterial RecA protein, designated recA-A T, was placed .in a plasmid appropriate for in vitro transcription and translation. Translation with <35>^s-Iabeled Met permitted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size. Preliminary evidence for the first amino acid was estitnated from results using both <35>^S-Met and 3^H-Leu for in vitro transcription and translation, followed by uptake into chloroplasts and processing. The labeled protein was subject to sequential amino acid hydrolyses, and radioactivity was measured in each round. Induction of gene transcription in leaves in-filtrated with the DNA-damaging agent, methyl methane-sulfonate was shown by Northern blot analysis. Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production of some soluble protein. Extracts from transformed bacteria were shown to have Recta activity using the "PROM" assay [Bertrand et al. (1993) Null. Acids Ryes. 21: 3653] for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli Recta isolation.
日本植物生理学会の論文