Part of the replication origin-containing A+T-rich region of the Drosophila yakuba mtDNA molecule and segments on either side of this region have been sequenced, and the genes within them identified. The data confirm that the small and large rRNA genes lie in tandem adjacent to that side of the A+T-rich region which is replicated first, and establish that a tRNAval gene lies between the two rRNA genes and that URF1 follows the large rRNA gene. The data further establish that the genes for tRNAile, tRNAgln, tRNAf-met and URF2 lie in the order given, on the opposite side of the A+T-rich region to the rRNA genes and, except for tRNAgln, are contained in the opposite strand to the rRNA, tRNAval and URF1 genes. This is in contrast to mammalian mtDNAs where all of these genes are located on the side of the replication origin which is replicated last, within the order tRNAphe, small (12S) rRNA, tRNAval, large (16S) rRNA, tRNAleu, URF1, tRNAile, tRNAgln, tRNAf-met and URF2, and, except tRNAgln, are all contained in the same (H) strand. In D. yakuba URF1 and URF2, the triplet AGA appears to specify an amino acid, which is again different from the situation found in mammalian mtDNAs, where AGA is used only as a rare termination codon.

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From a consideration of the various structural forms of partially replicated mitochondrial DNA (mtDNA) molecules from Drosophila melanogaster embryos observed in the electron microscope, it appears that the majority of molecules are replicated by a highly asymmetrical mode in which synthesis on one strand is up to 99% complete before synthesis on the second strand is initiated. Replication of the minority of molecules involves a more nearly symmetrical synthesis of the two complementary strands. The D. melanogaster mtDNA molecules have physical features with respect to which the origin and direction of replication could be mapped. These features are (i) a single region accounting for approximately 25% of the circular contour length and rich in adenine + thymine, and (ih) four EcoRI sites, all of which lie outside of this region.Molecules of this mtDNA were subjected to partial denaturation, EcoRI digestion, or partial denaturation after EcoRI digestion and the products were examined in the electron microscope. Complex forms interpretable as originating-from replicative intermediates were observed. The size and structure of the components of these complex forms were wholly consistent with the interpretation that, in all of these mtDNA molecules, replication originates at, or close to, the center of the adenine + thymine-rich region and proceeds unidirectionally around the molecule toward the EcoRI site lying closest to-the adenine + thymine-rich region.Circular mitochondrial DNA (mtDNA) molecules (molecular weight, 12.35 X 106) of -Drosophila melanogaster are distinguished by a region, accounting for 25% of the circular contour (genome) length, that denatures at a lower temperature than does the rest of the molecule due to a high content of adenine + thymine (A+T) (1-8). By electron microscopy we have studied replicative intermediates present in D. melanogaster mtDNA and, using the relative location of the A+T-rich region and sites sensitive to cleavage by the restriction enzyme EcoRI (5) we have located the origin and determined the direction of replication in these molecules. MATERIALS AND METHODSThe D. melanogaster strain used in these experiments was Oregon R-Utah (Oak Ridge, TN) (5). Growth of flies, collection of eggs, and preparation of mtDNA from embryonated eggs by use of preparative cesium chloride centrifugation were as described (5).mtDNA was centrifuged to equilibrium in cesium chloride/ethidium bromide (9). The regions of the gradient containing two distinct fluorescent bands and a third, more diffuse, intermediately located band were collected separately and the ethidium bromide was removed as described (10). The relative concentrations of the DNA in the three regions were determined by measuring absorbance at 260 nm.Digestion of mtDNA molecules with restriction endonuclease EcoRI was accomplished as described (5), except that incubations were carried out at 250 in order to minimize branch migration (11). Partial denaturation of circular mtDNA molecules and EcoRI-produced fragments of mtDN...

Survey of twelve mouse tissues revealed the presence of appreciable phenylalanine hydroxylase activity in the pancreas and kidney as well as the liver but in no other of the tissues tested. Single cell suspensions of mouse liver were prepared by use of tetraphenylboron. The enzyme activity of such suspensions was much more stable than that of liver extracts, and permitted determination of the Michaelis-Menten constant, the pseudo-first order reaction velocity constant on a cell-number basis, and the temperature coefficient and apparent activation energy of the enzyme activity. Possible applications of these methods to problems in cellular biology have been indicated.

The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.