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1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Barn HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments of Eco RI, Hind III, Barn HI and Hap II is constructed on the basis of: (a) the analysis of partially restricted fragments; (b) analysis of the double digests of total mtDNA; (c) the digestion of isolated restriction fragments with other restriction endonucleases; (d) the identification of fragments of complete single and double digestions and of partially digested fragments containing the base sequences complementary to the 12-S and 16~S RNAs of rat-liver mitochondrial ribosomes. 3. The genes for the ribosomal RNAs are shown to be closely linked. This result differs from data previously reported (Saccone, C., Pepe, G., Cantatore, P., Terpstra, P. and Kroon, A.M. (1976) in The Genetic Function of Mitochondrial DNA, pp. 27-36, Elsevier/North-Holland Biomedical Press, Amsterdam). 4. The origin of replication (D-loop) is localized in the vicinity of the small ribosomal RNA gene and the direction of replication is distant from this gene. 5. The mitochondrial tRNA genes are scattered over the genome as in other animal mtDNAs. The approximate minimal number of tRNA genes is 16-20. 6. We concluded previously that the Eco RI restriction fragments A and D are not adjacent and failed to show the overlap of the 16 S rRNA gene for the Eco RI fragment D and Hind III fragment A. This misinterpretation was due to the fact that the two smallest Eco RI fragments could not be detected with the Postal addresses: a Bloemsingel 10, Groningen; b Zernikelaan,

To obtain more information about the arrangement of Hind III restriction fragments in the tRNA-rRNA region of the Neurospora crassa mitochondrial (mt) DNA we have cleaved the mtDNA with Hpa I and Hind II. We could construct additional cleavage maps for these enzymes. Hybridization of rRNAs to Hind II fragments confirmed the existence of an intervening region of about 2,300 basepairs in the 24S rRNA (Hahn et al., Cell, in press). About seven tRNA genes, among which the genes for tRNA1Ser and tRNAMetM, are located in a segment of about 5,000 bp separating the 24S and 17S rRNA genes. Another cluster of 14 tRNA genes is found adjacent to the other end of the 24S gene. The genes for tRNALeu1 and tRNAMetF are located in this cluster.

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