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.