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The complete sequence of the mitochondrial genome of Tetrahymena thermophila has been determined and compared with the mitochondrial genome of Tetrahymena pyriformis. The sequence similarity clearly indicates homology of the entire T.thermophila and T.pyriformis mitochondrial genomes. The T.thermophila genome is very compact, most of the intergenic regions are short (only three are longer than 63 bp) and comprise only 3.8% of the genome. The nad9 gene is tandemly duplicated in T.thermophila. Long terminal inverted repeats and the nad9 genes are undergoing concerted evolution. There are 55 putative genes: three ribosomal RNA genes, eight transfer RNA genes, 22 proteins with putatively assigned functions and 22 additional open reading frames of unknown function. In order to extend indications of homology beyond amino acid sequence similarity we have examined a number of physico-chemical properties of the mitochondrial proteins, including theoretical pI, molecular weight and particularly the predicted transmembrane spanning regions. This approach has allowed us to identify homologs to ymf58 (nad4L), ymf62 (nad6) and ymf60 (rpl6).

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Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.