A novel gene order in the Paracentrotus lividus mitochondrial genome.

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 12S and 16S rRNA genes are not contiguous but are separated from each other by ND1 and ND2 genes. The ND4L gene is not adjacent to ND4 but is located between the tRNAArg 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 tRNAPhe 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.

Cloning of mitochondrial DNA from Paracentrotus lividus.

In order to study the gene organization and sequence of the mt DNA from sea urchin Paracentrotus lividus, we have cloned the two Bam HI fragments of 12 Kb and 4 Kb in the plasmid vectors pAT 153 and pBR 322 respectively. The cloning has been tested by hybridizing the DNA extracted from the recombinant plasmids with sea urchin mt DNA and by comparing the restriction patterns of the cloned fragments with those shown by fragment isolated from sea urchin mt DNA. This has allowed the determination of the size of sea urchin mt DNA that results to be about 15.000 base pairs, similar to that already reported for other animal mt DNA.