Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.

ChemInform Abstract The synthesis of silyl-protected ribonucleoside 3'-O-phosphoramidites (I), (II) and the preparation of the controlled-pore glass supports needed for the solid-phase chemical synthesis of oligoribonucleotides are described. These reagents are evaluated in the synthesis of a series of oligomers consisting of the pentadecameric homopolymers of adenosine, cytidine, guanosine, uridine, and various sequences of mixed-base composition. The results of these studies are applied to the successful chemical synthesis of the 43-mer (III), corresponding to the 3'-terminus of a formylmethionine (fMet) + RNA of Escherichia coli, in which the modified bases are substituted by their unmodified parent nucleosides. The effectiveness of alkylsilyl ethers for the protection of the 2'-hydroxyl of the ribose ring, when used in conjunction with the phosphoramidite method for the formation of the phosphotriester linkages in RNA synthesis, is clearly established.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.

Detailed knowledge of gene maps or even complete nucleotide sequences for small genomes leads to the feasibility of evolutionary inference based on the macrostructure of entire genomes, rather than on the traditional comparison of homologous versions of a single gene in different organisms. The mathematical modeling of evolution at the genomic level, however, and the associated inferential apparatus are qualitatively different from the usual sequence comparison theory developed to study evolution at the level of individual gene sequences. We describe the construction of a database of 16 mitochondrial gene orders from fungi and other eukaryotes by using complete or nearly complete genomic sequences; propose a measure of gene order rearrangement based on the minimal set of chromosomal inversions, transpositions, insertions, and deletions necessary to convert the order in one genome to that of the other; report on algorithm design and the development of the DERANGE software for the calculation of this measure; and present the results of analyzing the mitochondrial data with the aid of this tool.Evolutionary inference based on DNA sequences traditionally compares homologous versions of a single gene in different organisms. These comparisons are generally reliable indicators of phylogenetic relationships, even for very divergent organisms, but are limited in being based on point mutations only. In particular, homology between related mitochondrial genes may become difficult to distinguish from noise levels due to rapid nucleotide substitution (1), and this is not the only context in which the degree of sequence homology between genes having common origin is not a useful measure. Availability ofcomplete nucleotide sequence for organellar genomes suggests the possibility of inferring phylogenetic distances from their gene orders instead offrom sequences of individual genes (2). Analyses ofevolution at the genome level necessarily differ from sequence comparisons of individual genes. Though the processes of insertion and deletion of sequence elements have direct counterparts at the genomic level, the predominant process, nucleotide substitution, does not, whereas other processes assume major importance, such as the transposition of a segment from one region of a chromosome to another or the inversion of a chromosomal segment. Here we propose a quantitative analysis of transposition, inversion, and insertion/deletion, leading to the reconstruction of a mitochondrial phylogeny.Though the inference of evolutionary history through genomic rearrangements is well-established (3-5), it has been the goal of our work to define a general edit distance that combines a variety of order-disrupting events, to devise and implement a combinatorial algorithm capable of estimating this distance, and to apply these tools in a uniform way across a wide spectrum of eukaryotic organisms to generate input suitable for phylogenetic tree construction methods. Our results generally agree with evolutionary relationships inferred from gene...