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A method has been developed to accurately measure steady state concentrations of mitochondrial transcripts in adult rat liver. Total mitochondrial RNA has been hybridized with an excess of labelled mitochondrial DNA fragments coding for a single gene or a piece of it. The results obtained show that each mitochondrion contains 36 molecules of 16S rRNA, 81 of 12S rRNA and about 8 molecules of mRNAs coding for identified and unidentified reading frames. Thus, the rRNA/mRNA ratio in rat liver differs from that reported in HeLa cell mitochondria. These results are discussed in the light of mitochondrial DNA transcription regulation.

Rat liver mitochondria are able to incorporate labelled nucleotides in RNA species which are retained by Millipore filters or oligo(dT)-cellulose column. The radioactivity retained was completely RNAse sensitive when [3H] UTP was used as radioactive precursor whereas by using [3H] ATP this fraction was only 30% RNAse sensitive. These results demonstrate that rat liver mitochondria are able to synthesize poly(A) containing RNA species and furthermore that the polyadenylation reaction occurs within the organelle.

The nucleotide sequences of the D-loop-containing regions of three rat mitochondrial DNAs (mtDNAs), two from the species Rattus norvegicus and one from R. rattus, were determined. Comparisons made among these sequences and with the mouse sequence showed that, on the basis of both base composition and frequency of nucleotide alterations, three domains could be defined within the D-loop-containing region: a central conserved segment, poor in L-strand adenine, flanked by two divergent, adenine-rich regions. Deletions and insertions were found to occur at an unexpectedly high frequency in these sequences and the conserved sequence block called CSB-1 was found not to be intact in the R. rattus sequence. Although in comparisons of more distantly related mtDNAs the D-loop region is the most divergent on the molecule, it does not diverge more than typical protein genes between R. norvegicus and R. rattus, and its central conserved domain appears to be one of the molecule's most conserved regions. The most variable domain borders the tRNAPhe gene and contains the L and H-strand promoters and the 5' terminus for H-strand DNA synthesis. Within this region we have found sequences in all the mtDNAs we have examined, including those of human, two artiodactyls and Xenopus, that are capable of folding into cloverleaf structures. In the other divergent domain of the same mtDNAs, we find sequences capable of assuming similar secondary structural configurations at or near the sites for the termination of D-loop DNA synthesis. The evolutionary preservation of the potential to form such structures despite the high primary-structural divergence of the regions they occur in, suggests the structures are of principal importance for some processes occurring in the D-loop-containing region.

There is still much that is obscure concerning the transport of proteins into or through the mitochondrial membrane systems. In addition, as pointed out previously, it is unlikely that the details of the process are the same for proteins destined for different compartments of the organelle. A brief summary of the process for matrix proteins might be as follows: The proteins are synthesized on free polysomes as precursors of higher molecular weight than the native forms. These precursors are liberated into the cell cytosol and subsequently translocated into the mitochondria. This timing might be different in yeast under some circumstances, synthesis being completed in association with the mitochondria. The precursors interact with a receptor in the outer mitochondrial membrane interaction being mediated by the presequences of the precursors. The presequences therefore act as addressing signals as well as possibly playing a role in one or all of (a) solubilization of precursors, (b) prevention of premature assembly into multimeric structures, or (c) maintenance of nonnative configurations required for transport. Interaction occurs with a second receptor, this time in the inner membrane of the mitochondria, interaction being with multiple sites in the polypeptide chain. Transport across the inner membrane then occurs, this transport depending on a transmembrane electrochemical gradient of which the proton component is the essential part. Transport is accompanied or followed by proteolysis of the prepiece, and formation of the native structure. While steps 1 and 2 of this sequence can be considered well established, the remaining steps are still poorly understood or purely hypothetical. Nevertheless, this sequence of events is consistent with known facts about the process and provides a framework for future investigations.

RNA synthesis in vitro by mitochondria of sea urchin has been characterized using embryos isolated at the mesenchyme blastula stage. This synthesis is supported by a specific mitochondrial RNA polymerase which possesses peculiar properties similar to that of the same enzyme found in other organisms. Synthesis of RNA in sea urchin mitochondria is inhibited by rifampicin, acriflavin and actinomycin D and unaffected by α-amanitin. Measurements of the reaction at different developmental stages demonstrate that RNA synthesis already increases significantly a few minutes after fertilization.

When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p -hydroxymercuriphenyl sulfonic acid, but not N -ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N -Ethylmaleimide does not react with this group. N -Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.

We present a further application of the stochastic model previously described (Lanave et al., 1984, 1985) for measuring the nucleotide substitution rate in the mammalian evolution of the mitochondrial DNA (mtDNA). The applicability of this method depends on the validity of "stationarity conditions" (equal nucleotide frequencies at first, second and third silent codon positions in homologous protein coding genes). In the comparison of homologous sequences satisfying the stationarity condition at the silent sites, only the four codon families (quartets) for which both transitions and transversions are silent at the third position are considered here. This has allowed us to estimate the transition and transversion rates for any pair of species. We have analyzed the third silent codon position of the triplet rat-mouse-cow, of a series of slightly divergent primates and of two Drosophila species. In terms of two external dating input we have then determined the phylogenetic trees for rat, mouse, and cow as well as for a number of primates including man. The phylogenetic tree that we have derived for the triplet rat, mouse and cow agrees with that we had previously determined by analyzing the first, second and third silent codon positions (in both duets and quartets) of mt genes (Lanave et al., 1985). For primates our method leads to the following branching order from the oldest to the most recent: Gibbon, Orangutan, Gorilla, Chimpanzee and Man. In absolute time, fixing the distance Chimpanzee-Man as 5 million years (Myr) we estimate the dating of the divergence nodes as: Gorilla 7 Myr; Orangutan 16 Myr; Gibbon 20 Myr. In all cases analyzed, the transition rate has been found to be substantially higher than the transversion rate. Moreover we have found that the transition/transversion ratio is different in the various lineages. We suggest that this fact is probably related to the nucleotide frequencies at the third silent codon position.

Publisher Summary This chapter discusses the biochemical analysis of mitochondrial transcription and translation. It is well known that mitochondria, like chloroplasts, are semiautonomous organelles. This implies that two genetic systems cooperate in their biosynthesis: an extramitochondrial system and an intramitochondrial system. The extramitochondrial system is formed by the nuclear genome which codes for the majority of mitochondrial proteins through the formation of mRNA translated mostly or completely at the level of the cytoplasmic machinery for protein synthesis. The intramitochondrial system is formed by a mitochondrial genome whose genetic continuity and expression is ensured by the existence, within the organelle itself, of enzymes responsible for DNA and RNA synthesis, and complete protein-synthesizing machinery with properties completely different from those of its cytoplasmic counterpart. Two approaches are used in the study of mitochondrial transcription: studies in vitro and studies in vivo. The chapter summarizes the main results obtained in the study of mitochondrial transcription with different organisms, those most commonly used being yeast, Neurospora crassa, Tetrahymena pyriformis, HeLa cells, Xenopus, and rat liver. The experimental approaches used to study mitochondrial translation products have been reviewed.

We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria.

This paper reports the nucleotide sequence of rat mitochondrial DNA, only the fourth mammalian mitochondrial genome to be completely sequenced. Extensive comparative studies performed with similar genomes from other organisms revealed a number of interesting features. 1) Messenger RNA genes: the codon strategy is mainly dictated by the base compositional constraints of the corresponding codogenic DNA strand. The usage of the initiation and termination codons follows well-established rules. In general the canonical initiator, ATG, and terminators, TAA and TAG (in rat, only TAA), are always present when there is gene overlapping or when the mRNAs possess untranslated nucleotides at the 5' or 3' ends. 2) Transfer RNA genes: a number of features suggest the peculiar evolutionary behavior of this class of genes and confirm their role in the duplication and rearrangement processes that took place in the evolution of the animal mitochondrial genome. 3) Ribosomal RNA genes: accurate sequence analysis revealed a number of significant examples of complementarity between ribosomal and messenger RNAs. This suggests that they might play an important role in the regulation of mitochondrial translation and transcription mechanisms. The properties revealed by our work shed new light on the organization and evolution of the vertebrate mitochondrial genome and more importantly open up the way to clearly aimed experimental studies of the regulatory mechanisms in mitochondria.

The mitochondrial DNA (mtDNA) from Paracentrotus lividus (sea urchin) eggs, a circular molecule of about 15 500 bp, has been cloned in plasmid vectors after cleavage with various restriction enzymes. By a combination of Northern blot hybridization and nucleotide sequence analysis we have characterized most of the P. lividus mitochondrial transcripts and determined the basic gene organization of the mtDNA. The nucleotide sequence of a gene for one NADH dehydrogenase (ND) subunit, ND4L, has also been determined. Our results show the existence of a novel gene order. The 12 S and 16 S rRNA genes are not contiguous but are separated from each other by ND 1 and ND2 genes. The ND4L gene is not adjacent to ND4 but is located between the tRNA Arg gene and the gene for subunit II of cytochrome oxidase (CoII). The tRNA genes are reshuffled and contrary to all vertebrate mitochondrial genomes studied so far, there are no intergenic regions between the tRNA Phe and the cytochrome b genes. These characteristics suggest a peculiar mechanism for the regulation of gene expression in this organism and provide information on the evolution of the mitochondrial genetic system in animal cells.

1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.

The most relevant and peculiar feature of mitochondrial (mt) DNA in animal cells is its compact gene organization. Genes coding for transfer (t), ribosomal (r) and messenger (m) RNAs are often contiguous, sometimes slightly overlap or are separated by only a few nucleotides. One main non-coding region is usually present in the genome. Its length, largely variable, can be reduced, as in sea urchins, to a hundred nucleotides (Attardi, 1981a, b; 1985; Cantatore and Saccone, 1987 for review). As a consequence of such economic gene organization the size of mitochondrial DNA in metazoa is extremely reduced compared with that of other eukaryotes. Among the mitochondrial genomes completely sequenced so far, whose length is shown in Table 1, the smallest size pertains to Ascaris mt DNA with 14,284 base pairs (bp), the largest to a frog, Xenopus laevis with 17,553 bp. More recently numerous cases in which the length of mt DNA exceeds the normal, rather constant average, size found in animal cells have been described in the literature (Moritz et al., 1987 for review). Such length variations can span an almost two-fold size range (14–28 kb) or even more (39 kb as in sea scallop, Snyder et al., 1987). However it seems that in such cases there is always a duplication of some genomic regions. Length variations are more frequent in invertebrates and in poikilotermic vertebrates and can be distributed within (heteroplasmy) and between individuals and thus may be generated very rapidly. In general length differences are confined to the control region but cases in which length variations appear to be dispersed and/or include structural genes have also been described. Deletion of mt genome tracts have been reported, particularly in heteroplasmic animals. Large deletions of the mt genome have been described in mice (Boursot et al., 1987).

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 Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments of Eco RI, Hind III, Bam 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 methods applied and to the lower specific radioactivity of the ribosomal RNAs used in the first series of hybridization experiments.

During the evolution of sea urchins, a transfer RNA gene lost its tRNA function and became part of a protein-coding gene. This functional loss of a tRNA with specificity for one group of leucine codons (CUN, where N is any base) was accompanied by the gain of a new tRNA with that specificity. The new tRNA gene for CUN codons appears to have evolved by duplication and divergence from a tRNA gene specific for another group of leucine codons (UUR, where R is a purine). These proposals account for (1) the strong sequence resemblance between the modern tRNA genes for CUN and UUR codons in Paracentrotus, (2) the altered location of the CUN gene in mitochondrial DNA of this urchin, and (3) the persistence of a 72-base pair sequence containing a trace of the old CUN gene at its original location. The old CUN gene now codes for an extra 24 amino acids at the amino end of subunit 5 in NADH dehydrogenase. Besides giving clues about the mechanisms by which tRNA genes move during mitochondrial DNA evolution, this finding leads us to propose a pathway relating the arrangements of other genes in mitochondrial DNAs from four animal phyla.

Rat liver mitochondria contain an endogenous factor highly specific in stimulating the homologous poly(A) polymerase. By using an in vivo labelling with [32P] orthophosphate it is possible to prepare a labelled factor and to demonstrate that it is stably incorporated in an acid insoluble molecule. This suggests that the factor probably acts as a primer in the polymerization of ATP molecules, being involved in the recognition between the mitochondrial poly(A) polymerase and the homologous RNA molecules which have to be polyadenylated.

Publisher Summary This chapter presents an up-to-date account of the organization, structure, and evolution of mammalian mitochondrial genes, with the belief that the knowledge of these features should supply valuable information to understand the interrelationships between the mitochondrion and the rest of the cell. The concerted effort of several laboratories has produced a great deal of information on the structure of mitochondrial genomes of several mammalian species. Mitochondria1 DNA ( MtDNA) sequence of man, cow, and mouse as well as a large part of rat sequence have been determined and the transcription products of these DNAs have been identified and mapped. Mammalian MtDNA is a circular closed double-stranded molecule, containing about 16,500 base pairs. This gene organization is it extremely compact with the exception of the region containing the origin of replication of the mtDNA heavy strand (D-loop region), the heavy (H)-strand is completely filled with discrete products. The mammalian transcripts are colinear with the DNA, showing a relevant difference in the gene organization as compared to lower eukaryote mtDNA such as yeast and N. crassa —that is,the lack of introns.

It is well known that mitochondria and chloroplasts possess their own DNA. Eukaryotic cell contains therefore at least two or, in the case of plants and plastid-containing micro-organisms, even three different genetic systems (1–4). The organelle genomes show a pattern of cytoplasmic and maternal inheritance. For a number of features in lower organisms this has been known already for a long time. Recently it has been shown that the same holds for the mitochondrial genome of animal cells (5–8). Although the genetic function of the organelle genomes is not yet known in all details, it is evident they are indispensable and vital for eukaryotic organisms. For the expression of their genomes, the organelles depend on the nucleus. They constitute heteronomous genetic entities, their expression and continuity being ensured by the existence within the organelles of elements coded for and synthesized by the main genetic system of the cell, the nuclear-cytoplasmic genetic system. This implies that a much higher level of genetic complexity is reached in the eukaryotic cell as compared to the prokaryotic cell. Although also in prokaryotes extrachromosomal genetic elements such as plasmids may exist, the obvious difference is that the products of transcription and translation of these elements are not obligatory for the cells to survive as is the case for the mitochondrial and chloroplast genomes.

Publisher Summary This chapter describes the experiments that suggest that a protein receptor exists in the mitochondrial membrane system for uptake of aspartate aminotransferase, and moreover that uptake of the enzyme via this receptor is inhibited by malate dehydrogenase. Uptake of aspartate aminotransferase into mitochondria can be measured by two methods, one of which involves the measurement of intramitochondrial enzyme activity and its increase after incubation of the organelles with purified isoenzyme. The other method involves radiolabeling of the enzyme by reduction of the cofactor-apoprotein linkage with [ 3 H]-sodium borohydride and then using liquid scintillation counting to determine the amount of the enzyme imported after incubation with the organelles and their recovery by centrifugation.

We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria.

Theoretical analysis and experimental approaches by gel electrophoresis in retarding conditions allowed us to identify the presence of an intrinsic bending in the D-loop containing region of the rat mitochondrial genome. The curvature was located in the right domain of the sequence analyzed, between the origin of replication of the heavy strand and its promoter. The preliminary evidence of a specific recognition of the bent DNA with mitochondrial matrix proteins suggests a probable role of this DNA conformation in the duplication and/or expression of the mammalian mitochondrial genome.

It is well known that all mitochondrial (mt) genomes contain the information for a number of proteins which are part of the inner mitochondrial membrane among which the three subunits of cytochromeoxidase (CoI, CoII, CoIII) and the apocytochrome b (Cyt b). In spite of the rather constant information content other features of mtDNA,such as the genome size and the gene structure and organization,appear to be very different in the various organisms. With regard to the evolutionary rate of mt genes we have only a limited number of data, particularly in some classes of organisms, which however already suggest that also this parameter is extremely variable. In this paper we report our quantitative analyses on the evolution of the mitochondrially coded cytochrome genes in animals and in a few plants applying a method recently developed in our laboratory for the measurement of the base substitution rate1.

The procedure used to obtain mitochondrial ribosomes from rat liver highly active in catalyzing a poly(U)-dependent polyphenylalanine synthesis is described. Two kinds of particles with sedimentation coefficients of 80-S and 55-S, present in the mitochondrial lysate, are able to catalyze protein synthesis in vitro. Sensitivity to specific inhibitors of protein synthesis demonstrates that the activity of 55-S particles is only of mitochondrial origin whereas that of 80-S particles is partly due to mitochondrial ribosomes and partly to contaminating cytoplasmic ribosomes. Characterization of the two kinds of mitochondrial particles has been carried out by studying their sedimentation pattern in different experimental conditions and by analyzing the RNA extracted by polyacrylamide gel electrophoresis. 16-S and 12-S RNA seem to be specific mitochondrial ribosomal species. The results suggest that the 55-S particle represents the basic unit for protein synthesis in rat liver mitochondria. Particles with higher sedimentation value probably represent aggregates of mitochondrial monosomes.

Many authors have recently demonstrated that mitochondria are able to carry out the synthesis of RNA. However, concerning the existence of an active RNA-polymerase (EC 2.7.7.6) in the mitochondria, the main problem is the distinction between true mitochondrial RNA-polymerase and the same nuclear enzyme which is expected to be present as a contaminant in mitochondrial preparations. In this paper we have studied the effect of atractyloside on [ 14 C]ATP incorporation into RNA of mitochondria or nuclei of rat liver. We have found that this substance inhibits the incorporation of labeled ATP only in the presence of intact mitochondria. As it is well known that atractyloside completely abolishes the utilization of external adenine nucleotides by mitochondria, we can assume that the labeled ATP must enter the mitochondrial membrane to be incorporated into RNA. This suggests the existence of an active RNA-polymerase inside the mitochondria.

1. 1. At least 85% of the RNA synthesized by isolated swollen rat-liver mitochondria specifically hybridized with rat-liver mitochondrial DNA. With RNA from intact mitochondria up to 35% hybridized with mitochondrial DNA but no saturation plateau was reached. 2. 2. Like the RNA synthesized in vivo (cf. ref. 5), the RNA synthesized by isolated mitochondria preferentially hybridized with one of the complementary strands of mitochondrial DNA. Hybridization with the other strand was low and maximally 20% of the newly synthesized RNA could be converted into a double-stranded form by annealing in the presence of an excess of cold mitochondrial RNA. These results show that RNA synthesis in isolated mitochondria has largely retained the asymmetry characteristic of RNA synthesis in vivo.

1 Incorporation of the four labeled ribonucleoside triphosphate into rat liver mitochondrial RNA has been measured. 2 Using 32P-labeled nucleotide as precursor, nearest-neighbour analysis of isolated RNA indicates that the labelling does not result from the formation of homopolymer since all four ribonucleoside monophosphates are found to be labeled. 3 Synthesis of RNA in rat liver mitochondria is inhibited by actinomycin D and by low concentrations of acriflavin indicating that mitochondrial DNA is involved in this process. 4 RNA synthesized in vitro by rat liver mitochondria, during a pulse of 10 min in the presenceof a labeled precursor, has been characterized by sucrose-density centrifugation analysis. RNA synthesized by intact organelle appears to be similar to RNA made by mitochondria with altered permeability of membrane. 5 [3H]AMP-labeled RNA after alkaline hydrolysis showed a higher percentage of radio-activity in the nucleoside fraction than RNA labeled with the other ribonucleoside triphosphates. This suggests that in rat liver mitochondria there may be present also an enzyme responsible for terminal addition of adenylate to transfer RNA molecules.

1. In rat-liver mitochondria both the dehydrogenase and transaminase routes participate in glutamate oxidation. However, the rate of ammonia production by the dehydrogenase pathway progressively decreases with the time of incubation. 2. Glutamate deamination is stimulated by blocking the transaminase pathway with arsenite or malonate. On the other hand, this process is completely suppressed by succinate, malate, pyruvate and oxaloacetate. Succinate and pyruvate inhibit, whereas malate and oxaloacetate stimulate, aspartate formation. 3. Glutamate deamination increases with increasing concentrations of 2,4-dinitrophenol from 0·05 to 0·2mm, and then becomes inhibited, together with the rate of oxygen consumption. Aspartate formation is progressively inhibited with increasing 2,4-dinitrophenol concentration from 0·05 to 0·8mm. In the presence of 0·20mm-2,4-dinitrophenol the rate of ammonia production is higher than in the presence of phosphate acceptors and decreases much slower and linearly with the time of incubation. 4. The addition of NAD+ enhances glutamate deamination without affecting oxygen uptake.

CUG, a universal leucine codon, has been reported to be read as serine in various yeast species belonging to the genus Candida. To gain a deeper insight into the origin of this deviation from the universal genetic code, we carried out a phylogenetic analysis based on the small-subunit ribosomal RNA genes from some Candida and other related Hemiascomycetes. Furthermore, we determined the phylogenetic relationships between the tRNA(Ser)CAG, responsible for the translation of CUG, from some Candida species and the other serine and leucine isoacceptor tRNAs in C. cylindracea. We demonstrate that the group of Candida showing the genetic code deviation is monophyletic and that this deviation could have originated more than 150 million years ago. We also describe how phylogenetic analysis can be used for genetic code predictions.

Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organellar genomes. Mitochondrial genomes have been extensively sequenced and analysed and the data collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc and MitoAln, two related databases containing, respectively, detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa and yeast, and the multiple alignments of the relevant homologous protein coding regions. MitoNuc and MitoAln retrieval through SRS at http://bio-www.ba.cnr.it:8000/srs6/ can easily allow the extraction of sequence data, subsequences defined by specific features and nucleotide or amino acid multiple alignments.

A 328-bp sequence from exon 1 of the gene for aquaporin-2 (AQP2) was compared in 12 mammalian species, representing as many eutherian orders. This sequence encodes the N-terminal half of this kidney-specific water channel protein. Most amino acid replacements, as well as an insertion, have occurred in extracellular loops connecting the transmembrane helices, in agreement with a lower functional importance of these loops. Phylogenetic analyses were performed with parsimony, distance, and maximum-likelihood methods. The AQP2 data set, alone as well as in combination with previously published alpha A-crystallin protein sequences, strongly supports a clade consisting of elephant, hyrax, aardvark, and elephant shrew, reaching bootstrap values of 99%. This finding fully agrees with the only other presently available sequence data sets that include these taxa, those of von Willebrand factor and interphotoreceptor retinoid-binding protein, and suggests that this extended paenungulate clade is one of the most conspicuous superordinal groupings in eutherian phylogeny. Some support was obtained for an artiodactyl/perissodactyl clade, while the grouping of pholidotes with edentates was contradicted.

In this paper we report a detailed comparative and evolutionary analysis of the sequences of constant T-cell receptor (Tcr) C gamma delta genes of artiodactyls compared to the homologous sequences of rodents and primates. Because of the frequency and physiological distribution of gamma delta T-cells in different animals, rodents and humans are defined as "gamma delta low" species and ruminants as "gamma delta high" species. Such a characteristic seems to be due to an adaptive role of gamma delta T-cell function. By analyzing the ruminant gene phylogeny of Tcr C gamma we were able to estimate the distance between cattle and sheep at 18 million years ago, a time that is in agreement with other nonmolecular estimates. For Tcr C gamma delta genes a peculiar phylogenetic relationship was found, with human and mouse clustering together and leaving artiodactyls apart. By using appropriate outgroups, the same phylogenetic pattern was obtained with other T-cell related sequences: namely, Tcr C alpha chain, CD3 gamma and delta invariant subunits. Interleukin-2. Interleukin-2 receptor alpha chain and Interleukin-1 beta with the exception of Tcr C beta chain and Interleukin-1 alpha. In contrast, the analysis of all other T-cell nonrelated genes, available in primary databases reveals a different tree, where primates and artiodactyls are sister taxa and rodents are apart in accordance with the current view of mammalian phylogeny. These data are relevant to important evolutionary issues. They show how misleading a phylogeny based on a single or on a few homologous genes may be. In addition they demonstrate that genes with correlated functions may evolve in a lineage specific manner probably in relation to environmental conditions.

The evolution of the prokaryotic glutamine synthase (GS) genes, namely the GSI and GSII isoforms, has been investigated using the second codon positions, which have previously proven to behave as a good molecular clock. Our data confirm the early divergence between prokaryotic and eukaryotic GSII before the splitting between plants and animals. The phylogenetic tree of the GSI isoforms shows Archaebacteria to be more closely related to Eubacteria than to Eukaryotes. This finding is confirmed by the phylogenetic analysis carried out on both large and small subunits of rRNA. However, differently from the rRNA analyses, Crenarchaeota and Euryarchaeota Archaebacteria, as well as high- and low-GC gram-positive bacteria, appear to be polyphyletic. We provide evidence that the observed polyphyly of Archaebacteria might be only apparent, resulting from a gene duplication event preceding the split between Archaebacteria and Eubacteria and followed by the retention of only one isoform in the extant lineages. Both gram-negative bacteria and high-GC gram-positive bacteria, which appear closely related, have GS activity regulated by an adenylylation/deadenylylation mechanism. A lateral gene transfer from Archaebacteria to low-GC eubacteria is invoked to explain the observed polyphyly of gram-positive bacteria.

Molecular data represent an important source of information for systematic and evolutionary studies; ideally this information should be evaluated together with data coming from other approaches (e.g., paleontological and morphological), since the molecular approach is limited by the availability of suitable homologous sequences, and these may be taxonomically informative only if they derive from speciation events (i.e., orthologous) and not from gene duplications (i.e., paralogous). This study focuses on the availability of sequences from the vertebrate class of Mammalia. The results show a strong bias in databases as, for some orders, little or no sequences are stored. For nuclear genes it is often difficult to discriminate between orthologous and paralogous ones. Therefore we advise the use in comparative studies of entire mitochondrial genomes which are easy to sequence and only contain orthologous genes. We report here the genetic organization of the mitochondrial DNA in mammals. In addition ...

The base composition of 25 complete mammalian mitochondrial (mt) genomes has been analyzed taking into account all three codon positions (P1230 and fourfold degenerate sites (P4FD) of H-strand genes. In the nontranscribed L strand, G is the less represented base and A is the most represented one in all cases, while C and T differ among species. H-strand protein-coding genes show an asymmetric distribution of the four bases between the two strands. The asymmetry indexes AT and GC skews on P4FD are much higher than those on P123, suggesting the existence of asymmetrical directional mutation pressure. Relationships between the compositional features and transcription of replication processes have been investigated in order to find a possible mechanism that could explain the origin of this asymmetry. AT and GC skews, the base composition in fourfold degenerate sites, and the number of variable sites for each gene are significantly correlated with the duration of single-stranded state of the H-stranded genes during replication. We tested different replication-related hypotheses, such as the existence of biased dNTP pools, gamma DNA polymerase mispairing, and the asymmetric replication itself. Most of them failed to explain the observed results, hydrolytic deaminations being the only one in agreement with our data. Thus, we hypothesize that one of the crucial processes for the origin of asymmetric and biased base composition of mammalian mitochondrial genomes is the spontaneous deamination of C and A in the H strand during replication.

In this paper we discuss and demonstrate the importance of several factors relative to the relationship between time and evolution of biosequences. In both quantitative and qualitative measurements of the genetic distances, the compositional constraints of the nucleotide sequences play a very important role. We demonstrate that when homologous sequences significantly differ in base composition we get erratic branching order and/or wrong evaluation of the evolutionary rates. We must consider that every gene may have a different evolutionary dynamic along its sequence, generally linked to its functional constraints; this too can seriously affect its clock-like behavior. We report some cases showing how these factors can affect the quantitative measurements of the genetic distances of biosequences.

The discovery that the p53 family consists of three members (p53, p63 and p73) in vertebrates and of a single homolog in invertebrates has raised the challenge of understanding the functions of the ancestor and how they have evolved and differentiated within the duplicated genes in vertebrates. Here, we report that the fatty acid synthase (FAS) gene, encoding for a key enzyme involved in the biogenesis of membrane lipids in rapidly proliferating cells, is a conserved target of the p53 family throughout the evolution. We show that CEP-1, the C. elegans p53 homolog, is able to bind the two p53 family responsive elements (REs) identified in the worm fasn-1 gene. Moreover, we demonstrate that fasn-1 expression is modulated by CEP-1 in vivo, by comparing wild-type and CEP-1 knockout worms. In human, luciferase and chromatin immunoprecipitation assays demonstrate that TAp73alpha and DeltaNp63alpha, but not p53, TAp73beta and TAp63alpha bind the two p53 REs of the human FASN gene. We show that the ectopic expression of TAp73alpha and DeltaNp63alpha leads to an increase of FASN mRNA levels, while their silencing produces a decrease of FASN expression. Furthermore, we present data showing a correlation between DeltaNp63alpha and FASN expression in cellular proliferation. Of relevant importance is that fasn-1 is the first CEP-1 direct target gene identified so far in C. elegans and our results suggest a new CEP-1 role in cellular proliferation and development, besides the one already described in apoptosis of germ cells. These data confirm the hypothesis that the ancestral functions of the single invertebrate gene may have been spread out among the three vertebrate members, each of them have acquired specific role in cell cycle regulation.

In order to assay the functionnal significance of the D-loop containing region of mitochondrial DNA, the authors characterized the transcripts from the replication origin of rat mt DNA