The chloroplast [of Arabidopsis thaliana]-located homolog of bacterial DNA recombinase

• Published in: Plant and cell physiology Vol. 38; no. 12; pp. 1319 - 1325
• Main Authors: Cao, J. (Cornell Univ., New York (USA)), Combs, C, Jagendorf, A.T
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.12.1997
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN RECOMBINADO; ADN RECOMBINE; Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; ARABIDOPSIS THALIANA; BACTERIA; Biological and medical sciences; Biological Transport; chemistry; Chloroplast; CHLOROPLASTE; CHLOROPLASTS; Chloroplasts - enzymology; Cloning, Molecular; CLOROPLASTO; complementary DNA; DNA damage; DNA Nucleotidyltransferases; DNA Nucleotidyltransferases - chemistry; DNA Nucleotidyltransferases - genetics; DNA Nucleotidyltransferases - isolation & purification; DNA Nucleotidyltransferases - metabolism; DNA recombinase; DNA repair; DNA, Single-Stranded; DNA, Single-Stranded - metabolism; enzyme activity; Enzymes; ENZYMIC ACTIVITY; enzymology; Escherichia coli; Fundamental and applied biological sciences. Psychology; gene expression; Genes, Plant; genetic regulation; genetics; Genic rearrangement. Recombination. Transposable element; Homologous recombination; Integrases; isolation & purification; Metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Peas; peptidases; Pisum sativum - enzymology; Plant physiology and development; Plant Proteins; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Protein Processing, Post-Translational; protein transport; Rec A Recombinases; Rec A Recombinases - chemistry; RecA; RECOMBINANT DNA; Recombinases; RNA, Messenger; RNA, Messenger - metabolism; RNA, Plant; RNA, Plant - metabolism; Substrate Specificity; transcription (genetics); translation; Molecular processing; Escherichia coli; Homologous recombination; Molecular targeting; Arabidopsis thaliana; Characterization; Enzymatic activity; Cruciferae; Dicotyledones; Angiospermae; N terminal-Sequence; Bacteria; Chloroplast; Enterobacteriaceae; Leader peptide; Intracellular transport; Heterologous system; Purification; Genetic transformation; Enzyme; Gene expression; Leguminosae; Complementary DNA; Pisum sativum; Spermatophyta; Experimental plant
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/oxfordjournals.pcp.a029124

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 35S-labeled 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 estimated from results using both 35S-Met and 3H-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 infiltrated with the DNA-damaging agent, methyl methanesulfonate 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 RecA activity using the "POM" assay for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli RecA isolation