The mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum is the smallest (14.2 kb) metazoa mtDNA molecule recorded. From analysis of nucleotide sequences of segments of this molecule we have found that gene order is greatly different to that in vertebrate and Drosophila mtDNAs, the sense strands of protein genes have an unusual base composition (50% T, 23% G, 19% A, 8% C), 89% of all codons end in T or G, and both AGA and AGG are used as sense codons. Further, although genes for tRNAs with structures similar to previously described tRNAs are not found between protein genes, inter-protein gene sequences can be folded into putative tRNA genes in which the T C arm and variable loop are replaced with a six to twelve nucleotide loop, and in which there is a remarkable conservation of some nucleotides. We plan to complete the sequence of the Ascaris mtDNA molecule to determine whether the small size results from differences in individual gene size and/or gene content and/or gene overlapping, relative to vertebrate and Drosophila mtDNAs. We propose to isolate, fractionate, sequence and attempt to charge tRNA molecules from Ascaris mitochondria to determine whether these tRNAs have the unique structure suggested from our DNA sequence analyses, or some other novel structure. Experiments are proposed to better define the translation initiation codon of some protein genes, to characterize 5' leader sequences of these genes and to determine whether mt-RNAs are polyadenylated and whether protein genes might be individually transcribed. Ribosomal RNA genes will be identified and the folding potential of these genes examined. Studies involving electron microscopy will be made to gain information on the mode of replication of Ascaris mtDNA. A segment of the mtDNA of Caenorhabditis elegans, a free-living nematode, will be sequenced to determine the extent to which the peculiar properties of Ascaris mtDNA might be a function of its parasitic mode of life. Comparisons of corresponding protein and tRNA genes in two nematode mtDNAs which have diverged by less than 10% of their nucleotides will be made to gain information on substitution frequencies in different protein genes, tRNA genes and intergenic regions, and in different positions in codons of four and two-codon families, and whether substitution frequencies are consistent with the view that AGA and AGG specifie serine in the nematode mitochondrial genetic code.
