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Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidase-negative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading.

Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidase-negative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading.

The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa3 content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14-and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.

The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.

The 15,697-nucleotide sequence of Paracentrotus lividusmitochondrial DNA is reported. This genome codes for 2 rRNAs, 22 tRNAs, and 12 mRNAs which specify 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement differs from that of other animal species. The two ribosomal genes 16 S and 12 S are separated by a stretch of about 3.3 kilobase pairs which contains the ND1 and ND2 genes and a cluster of 15 tRNA genes. The ND4L coding sequence is not contained in the ND4 mRNA but has its own mRNA which maps between the tRNAArgand the Co II genes. The main noncoding region, located in the tRNA gene cluster, is only 132 nucleotides long, but contains sequences homologous to the mammalian displacement loop. Other short noncoding sequences are interspersed in the genome: they contain a conserved AT consensus which probably has a role in transcription or RNA processing. As regards the mitochondrial genetic code, the codons AGA and AGG specify serine and are recognized by a tRNA with a GCU anticodon, whereas AUA and AAA code for isoleucine and asparagine rather than for methionine and lysine. Except for ND4L which starts with AUC and ATPase 8 which starts with GUG, AUG is used as the initiation codon. In 11 out of 13 cases the genes terminate with the canonical stop codons UAA or UAG. These observations suggest that during invertebrate evolution each lineage developed its own mechanism of mitochondrial DNA replication and transcription and of RNA processing and translation.

A quantitative analysis of the frequency of the supercoiled mitochondrial DNA molecules containing the D-loop in rat heart and cerebral hemispheres, at different ages, is presented. Both tissues of aged animals exhibit a remarkable reduction in the content of supercoiled D-loop containing molecules compared to the adults. This alteration could be responsible for the age-dependent reduction of mitochondrial DNA transcription previously observed in rat brain and heart.

Isolated rat liver mitochondria in the presence of an appropriate incubation buffer are able to support DNA transcription and RNA processing in a way qualitatively and quantitatively similar to that used by intact cells. This system is also able to synthesize an RNA species of 155–175 nucleotides which probably corresponds to the 7S RNA, a type of RNA found so far only in growing cells. The role of this RNA in the mitochondrial replication and transcription processes, in relation to the cell metabolism, is discussed.

Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.

We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNAThr and tRNAPro genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence of unique mechanisms for the regulation of gene expression in this organism.

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.