Continuing the analysis of rat liver mitochondria) DNA, we have determined the sequences of two segments containing several tRNA genes. One of these segments also comprises the L-strand origin of replication. The characteristics of the 15 rat liver mitochondrial tRNA structures known at present are compared with those from other mammalian mitochondria.

The sequence of a 6.24 kb DNA segment of the mitochondrial genome from rat liver has been determined. It comprises several genes coding for mitochondrial protein subunits and five tRNA genes in the following order: cytochrome oxidase subunit I - tRNA (UCN) (Ser) -tRNA(Asp) - cytochrome oxidase subunit II - tRNALys -ATPase subunit - cytochrome oxidase subunit III -tRNA(Gly) - potential open reading frame - tRNA(Arg) -two potential open reading frames. The tRNA genes were detected by a computer search programme. The assignments for the protein coding sequences were made through comparison with known sequences, mainly from the yeast mitochondrial proteins (e.g. Bonitz et al. 1980). Our data are discussed with regard to the features of gene arrangement, codon usage, and tRNA structure in mammalian mitochondria (Anderson et al. 1981).

In addition to recently characterized DraI (1), two new Type II restriction endonucleases, DraII and DraIII, with novel site-specificities were isolated and purified from Deinococcus radiophilus ATCC 27603. DraII and DraIII recognize the hepta- and nonanucleotide sequences (sequence in text) The cleavage sites within both strands are indicated by arrows. The recognition sequences were established by mapping of the cleavage sites on pBR322 (DraII) and fd109 RF DNA (DraIII). The sequence specifities were confirmed by computer-assisted restriction analyses of the generated fragment patterns of the sequenced DNA's of the bacteriophages lambda, phi X174 RF, M13mp8 RF and fd109 RF, the viruses Adeno2 and SV40, and the plasmids pBR322 and pBR328. The cleavage positions within the recognition sequences were determined by sequencing experiments.

A restriction endonuclease cleavage map of rat liver mitochondrial DNA is presented for the following enzymes: Xba I, Bgl II, Hae II, Bam HI, Hpa I, Hha I, Bcl I, Hind II, Hind III, Eco RI, Hpa II, Hae III, and Sau 3A. It was derived from complete and partial digestions with these enzymes, double digestions, and redigestions of defined fragments obtained with one enzyme with other restriction enzymes. By the use of these and further enzymes (Taq YI, Alu I) the mitochondrial DNA (ca. 15.6 Kb) can be dissected into a large number of defined fragments.

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