Different levels of human intervention in domestic rabbits: effects on genetic diversity.

The effects of human interaction on domestic rabbits were evaluated through the analysis of animals (up to 267) belonging to fancy breeds (22), a commercial breed (1), and selected strains (2). Microsatellite loci and mtDNA polymorphism revealed that the genetic pool of domestic rabbits studied only originated from that available in France. The good conservation of the original diversity was probably ensured through the multiplicity of samplings from wild populations. Selected strains, because of the breeding strategy, keep a fairly high level of diversity compared to other breeds.

Mitochondrial genes collectively suggest the paraphyly of Crustacea with respect to Insecta.

Complete sequences of seven protein coding genes from Penaeus notialis mitochondrial DNA were compared in base composition and codon usage with homologous genes from Artemia franciscana and four insects. The crustacean genes are significantly less A + T-rich than their counterpart in insects and the pattern of codon usage (ratio of G + C-rich versus A + T-rich codon) is less biased. A phylogenetic analysis using amino acid sequences of the seven corresponding polypeptides supports a sister-taxon status for mollusks-annelid and arthropods. Furthermore, a distance matrix-based tree and two most-parsimonious trees both suggest that crustaceans are paraphyletic with respect to insects. This is also supported by the inclusion of Panulirus argus COII (complete) and COI and COIII (partial) sequence data. From analysis of single and combined genes to infer phylogenies, it is observed that obtained from single genes are not well supported in most topologies cases and notably differ from that of the tree based on all seven genes.

Nonneutral evolution of tandem repeats in the mitochondrial DNA control region of lagomorphs.

The mitochondrial DNA of the European rabbit (Oryctolagus cuniculus) contains a tandem array of 153-bp repeats in the vicinity of the replication origin of the H-stand. Variation among molecules in the number of these repeats results in inter- and intraindividual length polymorphism (heteroplasmy). Generally, in an individual, one predominant molecular type is observed, the others representing a low percentage of the mtDNA content. At the tissue level, we observe a particular distribution of this polymorphism in the gonads compared with liver, kidneys, or brain, implying a relationship between the differentiation status of the cells and the types of new mtDNA molecules which appear and accumulate during lifetime. Similar tandem repeats were also found in the mtDNA noncoding region of European hares (Lepus europaeus), a cottontail (Sylvilagus floridanus), and a pika (Ochotona rufescens). The lengths and the sequences of these units evolve rapidly and in a concerted way, but the number of repeats is maintained in a narrow range, and an internal 20-bp segment is highly conserved. Constraints restrict the evolution of the primary sequence of these repeated units, the number of which is probably controlled by a stabilizing selection.

Partial sequence of the shrimp Penaeus notialis mitochondrial genome.

About half of the mitochondrial DNA of the shrimp Penaeus notialis (Crustacea: Decapoda) has been cloned (in 2 overlapping fragments of 7.9 kb and 1 kb) and partially sequenced. The gene content and arrangement are identical to that of the homologous domain in Drosophila yakuba. Intergenic nucleotides are scarce and a 982 bp non-coding sequence exhibit features similar to that of mtDNA control regions. The gene organization and the tRNA structures differentiate the Penaeus notialis mitochondrial genome from that of Artemia franciscana. Paraphyletism of crustacean mtDNA with respect to Insecta is discussed. A secondary structure of s-rRNA is proposed.

Rabbit mitochondrial DNA diversity from prehistoric to modern times.

The mitochondrial genetic variability in European rabbit (Oryctolagus cuniculus) populations present in Europe and North Africa from 11,000 years ago to the present day has been analyzed using ancient DNA techniques. DNA was extracted from 90 rabbit bones found in 22 archaeological sites dated between the Mesolithic and recent times. Nucleotide sequences present in a variable 233-bp domain of the cytochrome b gene were compared to those present in modern-day rabbits. The results show that the structure of ancient populations of wild rabbit exhibited remarkable stability over time until the Middle Ages. At this time, a novel type of mtDNA molecule abruptly appears into most wild populations studied from France. This mtDNA type corresponds to that currently present in the domestic breeds of rabbit examined so far. The relative rapidity by which this mtDNA type established and its absence in all sites examined before 1,700 years ago lend support to the hypothesis that between 2,000 and 1,000 years ago, man may have favored the development, into all regions of France, of animals carrying this particular mtDNA molecule. The origin of such animals has still to be found: animals previously living outside of France or within France but in very restricted areas? This event was concomitant with the documented establishment of warrens after the tenth century A.D. in Europe.

Genetic analysis of systematic mitochondrial heteroplasmy in rabbits.

One unusual property of rabbit mitochondrial DNA (mtDNA) is the existence of repeated 153-bp motifs in the vicinity of the replication origin of its H strand. Furthermore, every individual is heteroplasmic: it carries mtDNA molecules with a variable number of repeats. A systematic study of 8 females and their progeny has been devised to analyze mtDNA transmission through generations. The results suggest that three mechanisms are acting simultaneously. (1) Genetic drift in the germ line is revealed by the evolution of heteroplasmy when two major molecular forms are present in a female. (2) A high mutation rate (around 10(-2) per animal generation) generating molecular diversity, by deletion and addition of repeated units, is required to explain the observation of heteroplasmy in every individual. Moreover, the rates of mutation from the most frequent type to the other types are unequal. The deletion of one unit is more frequent than a deletion of two units, which is in turn more frequent than a deletion of three. (3) Selection for shorter molecules in somatic cells is probable. The frequency distribution of mtDNA types depends on the organ analyzed (kidney-spleen and liver vs. gonads).

Ancient DNA from Bronze Age bones of European rabbit (Oryctolagus cuniculus).

The European rabbit (Oryctolagus cuniculus) is now widely distributed throughout the world as a result of transportation by man. The original populations, however, were confined to southern France and Spain. In order to investigate the role of human intervention in determining the genetic diversity of rabbit populations, we are studying the origin of rabbits introduced onto a small Mediterranean island (Zembra) near Tunis over 1400 years ago, by examining ancient DNA extracted from rabbit bones found both on Zembra and on the European mainland. Ancient DNA was successfully extracted from rabbit bones found at two archaeological sites dated to at least the Early Bronze Age (more than 3500 years ago) in south-central France, and compared to that found in modern mainland and island populations using a small variable region of the cytochrome b gene. The results confirm that the Zembra Island population is descended from that present over 1400 years ago. The technical aspects of DNA extraction from bones and the implications of this type of research for determining the origin of introduced rabbit populations are discussed.

Mitochondrial DNA variability in Drosophila simulans: quasi absence of polymorphism within each of the three cytoplasmic races.

Nucleotide variability of mtDNA extracted from 144 isofemale lines collected in the whole range of D. simulans was analysed with 10-15 restriction enzymes and 73 lines were studied using one or a few enzymes. All clones were distributed into 3 mitochondrial genomes, siI, siII and siIII. These types are allopatric and can define geographic races. Mixed populations occur only in Madagascar and Réunion, where siII and siIII are found together. Among 40 sites detected with 10 enzymes, the variability of the coding region is extremely low, with one or no polymorphic restriction sites depending on the type. The control A + T-rich region is more variable in length and in restriction sites, and allows subtypes to be designated. Several lines were heteroplasmic for the length of the genome. These results are relevant to the evolutionary history of the species, its recent worldwide extension and to probable founder effects at the origin of each of the three types.

Co-linear organization of Xenopus laevis and mouse mitochondrial genomes.

Cloned fragments of Xenopus laevis mitochondrial DNA and Pleurodeles waltlii mitochondrial cDNAs have been hybridized together and with mouse mtDNA. In the three cases cross-hybridization was observed. The overall organization of the X. laevis fragments appeared to be co-linear with the mouse mtDNA, most sequences being conserved except for the D-loop and the URF6 regions. The use of mouse mtDNA has enabled us to identified several mitochondrial genes in X. laevis and P. waltlii.

Changes in D-loop frequency and superhelicity among the mitochondrial DNA molecules in relation to organelle biogenesis in oocytes of Xenopus laevis.

Changes in mitochondrial DNA (mtDNA) replication activity are known to occur during oogenesis in Xenopus laevis. Electron microscopic and electrophoretic analyses carried out on mtDNA molecules at different vitellogenic stages show that 1. The frequency of displacement loop (D-loop) forms is correlated with the intensity of mitochondrial biogenesis. 2. Most of the native molecules as well as the D-loop carrying ones are superhelical. 3. Four families of different superhelicity may be distinguished and D-loops are found only in the most superhelical ones. To account for the changes in the frequencies of the D-loop forms and of the different topological types during cell differentiation, it is suggested that the initiation of a new replication occurs only on the most superhelical molecules and that some control of superhelicity may exist in mitochondria.

Development of the mitochondrial mass and accumulation of mtDNA in previtellogenic stages of Xenopus laevis oocytes.

The development of the mitochondrial mass of the previtellogenic oocytes of Xenopus laevis has been analysed by a morphometric method and it has been correlated with the growth of the occyte itself. The study which was performed with oocytes from females of very different ages shows that the relative size of the mitochondrial mass in the smallest oocytes decreases gradually with the age of the females. Cytophotometric analysis of the Feulgen-stained mitochondrial mass enabled the mtDNA content of that structure to be quantified during its development. This development involves about 12 rounds of replication out of the total 16--17 rounds of replication during complete differentiation of large oocytes. It is possible to estimate the mean replication rate of the mtDNA during the course of the previtellogenic period. The calculated rate of replication is very high in the mitochondrial mass of all the growing oocytes of very young females(about 1--2 months old), then it decreases rapidly when the oocytes reach 120 microns in diameter. These results suggest that the mitochondrial masses found in small oocytes of old females do not correspond to sites of active mitochondrial biogenesis but rather they represent a physiologically arrested collection of organelles. Thus the aggregation of mitochondria is the cytological expression of rapid and strictly localized mitochondriogenesis only in very young females.

KINETIC ANALYSIS OF ENTIRE OOGENESIS IN XENOPUS LAEVIS.

In our breeding conditions (without artificial hormonal stimulation) two years after metamorphosis are necessary for a Xenopus laevis female to produce mature oocytes. Four periods of different growth rates can be distinguished into this first wave of oogenesis: 1) oocytes reach 120 µm in diameter (early stage I according to Dumont (5)) in a few weeks after metamorphosis; 2) growth slows down and the size of 250-300 µm (late stage I) is obtained 6-7 months later; 3) a rapid growth resumes during vitellogenesis (stages II, III and IV) and a 1,000-1,100 µm diameter is reached in 2-3 months; 4) the last period spans for a year and the oocyte grows up to 1,200 µm. This phase covers many physiological changes and it should be critical to size carefully the oocytes for biochemical investigations and comparisons. At last most of the oocytes in a young female do not proceed through this entire oogenesis, they are stopped at the end of the second growth phase (about 250 µm in diameter).

Inserted sequence in the mitochondrial 23S ribosomal RNA gene of the yeast Saccharomyces cerevisiae.

The sequence organization of the yeast mit-DNA region carrying the large ribosomal RNA gene and the polar locus omega was examined. Hybridization studies using rho- deletion mutants and electron microscopy of the heteroduplexes formed between 23S rRNA and the appropriate restriction fragments, lead to the conclusion that the 23S rRNA1 gene of the omega+ strains is split by an insertion sequence of 1,000-1,100 bp. In contrast, no detactable insertion was found in the 23S rRNA gene of the omega- strains. The size and the location of the insert found in the 23S rRNA gene of the omega+ strains appear to be identical to those of the sequence delta which had previously been found to characterize the difference (at the omega locus) between the mitDNA of the wild type strains carrying the omega+ or omega- alleles (Jacq et al., 1977).