The presence of tandem repeats and the initiation of replication in rabbit mitochondrial DNA.

The non-coding region of rabbit mitochondrial DNA (mtDNA) exhibits two sets of tandem repeats between conserved sequence block 1 (CSB1) and the tRNA Phe gene. Both repeated sequences, short repeated (SR) and long repeats (LR), which contain 20 and 153 nucleotides, respectively, are involved in the generation of a high degree of mitochondrial heteroplasmy. Due to the location of these sequences in the regulatory region and their properties in terms of variable conformations, they could affect the initiation of replication of the heavy-strand DNA (H-strand DNA) and subsequently would influence the efficiency of mtDNA replication. The extremities of the displacement loop (D-loop) DNA strand and the 5' ends of RNA primers initiating the H-strand DNA synthesis were characterized in individual rabbits. Mapping at the nucleotide level of the 5' and 3' ends of the D-loop DNA strands indicates that both extremities are heterogeneous. The H-strand replication origin OH is located close to the conserved sequence block CSB1 as in other mammals. In all of the individuals studied so far, DNA molecules with a 5' end 1-2 nucleotides downstream of CSB1 were always present. As H-strand DNA replication is believed to be primed by RNA transcribed from the light-strand promoter (LSP), RNA mapping was carried out to identify the 5' end of H-strand RNA. Neighbouring initiation sites were identified at the nucleotide level in an (A+T)-rich region at nucleotide 1841 and in a stretch of cytosine residues at nucleotides 1849-1852, which are located at the beginning of the first long repeat. A detailed RNA analysis indicates that H-strand RNA molecules are initiated in each long repeat. The amplification of the regulatory region has produced multiple initiation transcription sites and a family of RNA primers of various lengths. These variations in length and the ensuing secondary structures are not critical for their potential function as H-strand DNA replication primers.

The branching order of mammals: phylogenetic trees inferred from nuclear and mitochondrial molecular data.

In order to clarify some controversial phylogenies such as those regarding the triplet of human, rodent, and cow and the evolutionary position of Lagomorpha with respect to other mammals, we have analyzed both nuclear and mitochondrial genes using the stationary Markov model developed in our laboratory. We found that the two sets of genes give different results. In particular the mitochondrial tree showed rabbit linked first to rodents and the rabbit-rodents branch linked to artiodactyls with human as the outgroup. The most favorite nuclear tree showed human linked first to artiodactyls and the human-artiodactyls branch linked to rabbit with rodents as the outgroup. The obvious questions, (1) which tree is the correct one, or (2) both trees can be incorrect, and (3) how can we explain such an evolutionary pattern, are discussed on the basis of our limited knowledge of factors that influence the clocklike behavior of biological macromolecules.

Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture.

The effect of the switch to aerobic growth conditions was examined in rabbit articular chondrocytes transferred to culture. Spectroscopic analysis of the cytochromes of the respiratory chain shows that only cytochrome b is present in chondrocytes from cartilage, cytochromes c, c1, and a.a3 being undetectable as compared with the typical spectrum found in a primary cell culture on day 4. Steady state levels of RNA transcripts of nuclear (cytochrome c) and mitochondrial genes (cytochrome b and cytochrome oxidase subunits II and III) involved in the oxidative metabolism were determined relative to the RNA transcripts of the nuclear gene for glyceraldehyde phosphate dehydrogenase involved in the glycolytic pathway and to mitochondrial ribosomal RNAs. Chondrocytes transferred to culture showed a general increase in the levels of all transcripts, but the effect on mitochondrial transcripts was much greater (x 20) than the effect on nuclear transcripts (x 3-4). These results show the absence of a coordinate regulation of the expression of mitochondrial and nuclear genes coding for components of the respiratory chain. The increase in mitochondrial DNA triggered by culture conditions does not appear to be sufficient to account for the enhanced transcription. Concomitant with these mitochondrial changes, the level of transcripts for the collagen II gene involved in the differentiation function decreases dramatically (3% of the control on day 3).

Direct repeats in the non-coding region of rabbit mitochondrial DNA. Involvement in the generation of intra- and inter-individual heterogeneity.

In rabbit we observed heteroplasmy at an exceptionally high level, the heterogeneity occurring within the non-coding region of the DNA. Mitochondrial DNA (mt DNA) was cloned in pBR322 and the nucleotide sequence analysis of an EcoRI-Hind III fragment encompassing the non-coding region revealed that although there are common features with other mammalian mtDNAs (termed large central-conserved-sequence block, conserved-sequence blocks 1, 2 and 3 and termination-associated elements) the non-coding region shows an unusual organization; two stretches of tandem repeats of 20 bp and 153 bp are present in a part containing the origin of H-strand replication (OH) and probably the promoters for transcription as judged from other vertebrates. The long repeats are located between tRNA(Phe) and conserved sequence block 3 and the short repeats are located between conserved sequence blocks 1 and 2. When cloned in Escherichia coli (recA or recBC sbcb) DNA fragments containing the short repeats show length differences corresponding to various copy numbers of repeats. Electrophoretic analysis of the appropriate restriction fragments of rabbit mtDNA reveals extended intra- and inter-individual length heterogeneity. Both sets of repeats are involved in the generation of heterogeneity and are present in variable copy numbers from one mtDNA molecule to another. Moreover, rearrangement of the motives of the short repeat are observed to different extents in the mtDNA from one animal to another. The occurrence, maintenance and possible involvement of these repeated sequences, capable of forming stable secondary structures, are discussed in relation to their location in the region of control signals.

Rhodamine 123 uptake and mitochondrial DNA content in rabbit articular chondrocytes evolve differently upon transfer from cartilage to culture conditions.

Mitochondrial DNA (mtDNA) represents 0.15% of the total cell DNA (at least an order of magnitude less than in liver or heart) of rabbit articular chondrocytes. Besides the already well-documented low respiratory activity, chondrocyte differentiation thus involves a specific control of mitochondrial biogenesis. When transferred to in vitro conditions, chondrocytes increase their stock of mtDNA at the same time they resume growth, even more efficiently (8 times) than they do for cell volume (4.4 times). On the contrary, overall mitochondrial activity, estimated as the uptake of rhodamine 123, does not follow the same trend (2.5 times increase). Chondrocytes apparently keep these functional characteristics for some generations in culture.

Segregation of mitochondria in the cytoplasm of Xenopus vitellogenic oocytes.

In actively growing vitellogenic oocytes of Xenopus laevis mitochondria segregate into 2 populations. One stays around the nucleus, actively replicates mitochondrial DNA (mtDNA), and builds up most of the stock of the mitochondria in the full-grown oocyte. The other moves toward the vegetal pole and stops replicating mtDNA early in vitellogenesis. Organelles of this population are components of the germ plasm of the cell.

Heterogeneous distribution and replication activity of mitochondria in Xenopus laevis oocytes.

In early diplotene oocytes of Xenopus laevis mitochondria are not dispersed all over the cytoplasm but gathered in a well described mitochondrial mass [18]. When tracing these organelles during active vitellogenesis we observe that some of them are involved in the elaboration of a cortical layer at the vegetative hemisphere of the cell while others stay around the nucleus. The latter contribute to the transient formation of a mitochondrial crown throughout active mitochondriogenesis. Autoradiographic studies of thymidine incorporation into mtDNA suggest a differential participation of each organelle to the final population of a full-grown oocyte according to its position in the cytoplasm.