THE CHLOROPLAST-LOCATED HOMOLOG OF BACTERIAL-DNA RECOMBINASE

The cDNA for the chloroplast-located homolog of bacterial RecA protein , designated recA-AT, was placed in a plasmid appropriate for in vitro transcription and translation, Translation with S-35-labeled Met perm itted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size, Preliminary evidence f or the first amino acid was estimated from results using both S-35-Met and H-3-Leu for in vitro transcription and translation, followed by u ptake into chloroplasts and processing, The labeled protein was subjec t to sequential amino acid hydrolyses, and radioactivity was measured in each round, Induction of gene transcription in leaves infiltrated w ith the DNA-damaging agent, methyl methanesulfonate was shown by North ern blot analysis, Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production o f some soluble protein. Extracts from transformed bacteria were shown to have RecA activity using the ''POM'' assay [Bertrand et al, (1993) Nucl. Acids Res, 21: 3653] for DNA strand transfer. The protein was pu rified to close to homogeneity using methods developed for E. coil Rec A isolation.