62. Cloning and expression of recA<sup>+</sup>e -like gene from rice mitochondria in Escherichia coli

62. Cloning and expression of recA⁺ -like gene from rice mitochondria in Escherichia coli

Laboratory of Radiation Genetics and Chemical Mutagenesis, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan

It is well known that the recA⁺ gene in Escherichia coli plays an important role in genetic recombination with homologous pairing, DNA repair, mutagenesis and cell division (Witkin 1976).

A recombinant DNA technique was used to determine whether a gene having the recA⁺ function exists in higher plants. The DNA extracted from mitochondria of rice was digested by restriction endonuclease BamH1, and these BamH1 fragments were randomly inserted into pBR 322 plasmid vector DNA by T\4\ DNA ligase. Cells of recA^- E. coli were used as the recipient. By genetic complementation, two transformants were obtained and named pBM 10 and pBM 328. The BamH 1 fragments of the recombinant plasmid DNA were recovered from the intact DNA of pBM 10 and pBM 328, and they were electrophoresed in agarose gel (0.9%) (Fig. 1). The length of inserts from pBM 10 and pBM 328 were calculated to be aoorixunatekt 5000 and 9500 base pairs, respectively. Three fragments isolated from pBM 328 plasmid suggested that the pBM 328 contained another proximal restriction site in addition to the distal BamH1 site.

Fig. 1. Agarose gel electrophoresis of lambdaDNA digested with EcoR1(lane 1) and BamH1(lane 2), pBR322 plasmid DNA digested with BamH1(lane 3), pBM10(lane 4) and pBM328(lane 5) digested with BamH1.

Fig. 2. The survival rate after UV irradiation. Strains were grown to 2X10⁸ cells/ml and samples of various dilutions were irradiated on the surace of 1B agar. Colonies of survivors were scored after 24-36 hours incubation. O, KY 700 (recA⁺`2 lexA⁺); square symbol, KY 705 (recA); shaded square symbol, pBM 10; shaded triangle symbol, pBM 328.

After UV irradiation, the high survival rate was observed in both pBM 10 and pBM 328 compared to recA^- recipient cells (Fig. 2), suggesting that the mitochondrial DNA of rice contained a recA⁺ -like gene.

Witkin, E.M., 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40: 869-907.