THE MITOCHONDRIAL-DNA OF THE AMEBOID PROTOZOAN, ACANTHAMOEBA-CASTELLANII - COMPLETE SEQUENCE, GENE CONTENT AND GENOME ORGANIZATION

In phylogenetic trees based on comparison of nuclear small subunit rRN A sequences, Acanthamoeba castellanii (an amoeboid protozoon) is posit ioned near the base of the radiation leading to the animals, fungi and plants. However, the specific affiliation of this protist with the ma jor multicellular lineages of eukaryotes is currently uncertain. To fu rther explore the evolutionary position of A. castellanii, we have det ermined the complete primary sequence of its mitochondrial genome. We find that the circular mtDNA (41,591 bp; 70.6% A + T) encodes two rRNA s (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subu nits of the respiratory chain and 16 ribosomal proteins). As well, thi s genome contains eight open reading frames (ORFs) larger than 60 codo ns and of undefined function. Two of these ORFs (orf124 and orf142) ha ve homologs in other mtDNAs (''orf25'' and ''orfB'', respectively), th ree are unique to A. castellanii mtDNA (orf83, orf115 and orf349), and three are intronic ORFs. Among notable features of A. castellanii mtD NA are the following: (1) Genes and ORFs are all encoded on the same s trand and are tightly packed, with only 6.8% of the total sequence not having an evident coding function and intergenic spacer sequences ran ging from only 1 to 616 bp (average 64 bp). Ten pairs of protein-codin g genes overlap by up to 38 bp and two subunits of cytochrome oxidase (COX1 and COX2) are specified by a single continuous ORF. (2) Only thr ee introns, all group I and each containing a free-standing ORF, are p resent; these are localized in the 3'-half of the large subunit rRNA g ene. (3) The genome encodes fewer than the minimal number of tRNA spec ies required to support mitochondrial protein synthesis, suggesting th at additional tRNAs are imported from the cytosol into A. castellanii mitochondria. Of the 16 tRNAs specified by A. castellanii mtDNA (one w ith an 8-nucleotide anticodon loop), 13 have been shown or are predict ed to undergo a novel form of RNA editing within the acceptor stem. (4 ) A modified genetic code is used in which UGA specifies tryptophan. ( 5) Repeated sequences and obvious small sequence motifs that might rep resent regulatory elements are absent. In overall size, gene content a nd organizational pattern, A. castellanii mtDNA most closely resembles the mtDNA of the chlorophycean alga Prototheca wickerhamii (55,326 bp ; 74.2% A + T), but is quite different in these respects from the mtDN A of Chlamydomonas reinhardtii (15,758 bp; 54.8% A + T), another chlor ophycean alga, as well from characterized animal and fungal mitochondr ial genomes. The mtDNAs of A. castellanii and P. wickerhamii share wit h those of land plants a virtually identical set of respiratory and ri bosomal protein genes, with a number of these being arranged in the sa me way In contrast, C, reinhardtii mtDNA lacks several of the standard respiratory genes and does not encode any ribosomal proteins. In addi tion, the mitochondrial genomes of A. castellanii and P. wickerhamii ( but not that of C. reinhardtii) contain homologs of two genes (orf25 a nd orfB) that have previously been found only in land plant mtDNA. The se new genomic data provide additional phylogenetic markers linking th e mitochondrial genomes of land plants, P. wickerhamii and A. castella nii, to the exclusion of C, reinhardtii, fungi and animals. We suggest that land plant, I! wickerhamii and A, castellanii mtDNAs are relativ ely conservative, slowly evolving genomes that retain a number of ance stral features that were present in a common mitochondrial progenitor. These features have largely been lost in mtDNAs such as that of C. re inhardtii, which appear to have evolved relatively rapidly and radical ly away from the ancestral pattern.