60. Cloning of mitochondrial DNA genome in rice

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

Mitochondrial DNA (mtDNA) in higher plants is generally thought to be composed of heterogenous molecules. Recently, Yamaguchi and Kakiuchi (1983) reported that there are two plasmid-like DNAs, B-1 and B-2 DNA, in the mtDNA genome of a rice male-sterile cytoplasm. To analyze the mtDNA genome, a large quantity of homogenous mtDNA molecules need to be obtained. So we planned to clone the mtDNA genome of a rice variety (Akihikari) in Escherichia coli, using the plasmid vector YIp5.

From ten-day-old etiolated seedlings, 52 micrograms of mtDNA were extracted by the ordinary method. It was digested with restriction endonuclease Eco RI, and the band pattern produced by 0.7% agarose gel electrophoresis was analyzed. Twenty-six fragments were observed and there were differential intensities among them, suggesting that each fragment was present in different copy numbers related to repeated sequences. Table 1 shows the molecular weight of each fragment and from them, the total molecular weight of the mtDNA genome was calculated to be about 150 kil-base-pairs (kbp). But if the difference in copy number as suggested above was considered, the determined molecular weight seemed to be the minimum of the mtDNA genome.

Cloning of EcoR1-digested mtDNA was carried out by the shotgun method. After the EcoR1-digested mtDNA was inserted into the EcoR1 site of a plasmid vector XIp5, ampicilin-resistant transformants were selected. Out of 152 transformants, fourteen were recombinants. Molecular weights of inserted sequences were determined as shown in Table 2. We called these recombinant plasmids YORp. YORp 5,6 and 7 had similar molecular weights, and it remains unknown whether they are an identical sequence or not.