Evolution of Ciliate Mitochondrial Genomes
The evolution of the mitochondrial genome has some very interesting features. Plant mitochonndrial genomes are among the largest genomes extending up to several hundred thousand base pairs (kp) in length. Most animal mitochondrial genomes are circular and only several thousand bp in lengths. Protists have a wide spectrum of mitochondrial genomes with many having circular DNA and some have linear DNA., The protist Reclinomonas americana has 97 genes while many animal mitochondrial genomes have only a dozen or so genes. The vast majority of mitochondrial genes are ribosomal proteins, proteins involved in electron transport or oxidative phosporylation, or are ribosomal RNA (rRNA) genes or transfer RNA (tRNA) genes.
The complete genomes of there ciliates, Tetrahymena thermophila, Tetrahymena pyriformis and Parmecium caudatum Each of these genomes are linear, about 50 kb and contain about 50 genes, with about 44 genes coding for proteins. In spite of the limited types of proteins found in mitochondrial genomes, only about half of the mitochondrial proteins can be identified with regard to their function. The sequence of theT. thermophila mitochondrial geneome was determined and analyzed in our laboratory. We are currently determining the mitochondrial genome sequences of additional ciliates for comparison and analysis of their evolutionary patterns. Of particular interest is the identification of function for mitochondrial genes currently unidentified. The general evolutionary processes that have lead to the dramatic divergence of ciliate mitochondrial proteins are of great interest and comparison of numerous ciliate mitochondrial genomes should shed light on these processes.
In comparing the Tetrahymena mitochondrial genomes we find that the terminal regions are virtually identical within the genome of each species. When these regions are compared between species they are diverging as would be expected. In T. thermophila the nad9 gene is tandemly repeated. These tandem copies are virtually identical and when compared with the tandem nad9 genes in the closely related species Tetrahymena malaccensis we see that the sequences are diverging between species. These duplicated regions are virtually identical within the mitochondrial genome of a species, but diverge between species. This is a clear example concerted evolution. The mechanisms underlying evolutionary process are of great interest. A comparison of additional ciliate mitochondrial genomes should shed light on these processes.
Organization of Tetrahymena thermophila Genome
The availability of complete genome sequences dramatically changes the types of biological questions that can be posed. Instead of focusing on a single gene or a small cluster of genes, the interactions of all of the genes in the genome can be studied. If all of the genes in the genome are available, questions of integrated activities can be approached. The data provided by genomic sequences transforms the study of biological systems from a data poor endeavor to a data rich enterprise with all of the advantages that entails. We are engaged in provides preliminary data sufficient to initiate a complete genome sequencing of the T. thermophila genome. The macronuclear genome of T. thermophila is naturally fragmented into relatively small automonously replicating pieces (ARP) ranging in size from 75 kb to about 1,500 kb. These T. thermophila ARPs can be separated by zero integrated field gel electrophoresis. This allows us to isolated individual ARPs and determine their DNA sequences. This allows us to examine a tiny portion of the T. thermophila genome in great detail. This provides a data set for examining the genomic organization of the T. thermophila genome.
Bacterial Identification Research
Monitoring the bacterial flora in coastal marine waters by conventional techniques has been difficult as most of the bacteria do not readily grow on culture plates and their morphologies are virtually identical in the microscope. Molecular techniques, particularly characterizing bacteria using polymerase chain reaction (PCR) amplification of their small subunit ribosomal RNA (SSU rRNA) genes, has dramatically improved the ability to identify bacteria from environmental samples. Identification of bacteria by PCR amplification is specific and very sensitive. However, it is exactly these properties of PCR amplification which make it difficult to determine the amount of each individual bacterial species in the population.
We have developed a protocol that will allow the determination of the amount of a specific bacterial species in a sample. This protocol is based on the co-amplification of a modified internal standard sequence along with SSU rRNA sequences in the sample. The internal standard sequences in the PCR product are identified by gel electrophoresis following restriction endonuclease digestion. The amount of a specific SSU rRNA sequence in the sample is calculated by knowing the ratio of SSU rRNA to internal standard sequence in the PCR product and the amount of internal standard sequence that was added to the original DNA sample. The protocol is very rapid and has the specificity and sensitivity of PCR amplification.
This protocol has been successfully applied in the laboratory and adapted for field application to be used as an alternative to the conventional culture plate assays for marine bacteria. A major advantage of this approach is a dramatic decrease in the time required to assay the marine bacteria from days to hours. This assay also significantly increases the spectrum of bacterial species that can be detected, virtually any bacteria can be monitored by this protocol. Thus, the assay can be used to determine the source of bacterial contamination by matching unique bacterial sequences from various potential pollution sources with the sequences present in the sample.
Brunk, C.F., Li, J. and Avaniss-Aghajani. E., "Molecular Approaches to Evaluation of Environmental Microbial Composition", In: In Recent Research Developments in Microbiology, Kerala, India 7 : 147-159 (2003) .
Brunk, C.F., Lee, L.C., Tran, A.B. and Li, J., "Complete Sequence of hte Mitochondrial Genome of Tetrahymena therlnophila and Comparative Methods for Identifying Highly Divergent Genes", Nuc. Acids Res, 31 : 1673-1682 (2003) .
Brunk, C.F., Li, J. and Avaniss-Aghajani, E., "Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chanin Recation", Current Issues in Molecular Biology, 4 : 13-18 (2002) .
Fogel, G.B., Collins, C.R., Li, J. and Brunk, C.F.,, "Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population", Microbial Ecology, 38 : 93-113 (1999) .
Brunk, C.F.,, "Cilate Display Promise for Foreign Gene Expression", Nature Biotechnology, 17 : 424-425 (1999) .
Brunk, C.F. and Eis, N., "Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota", Applied and Environmental Microbiology, 64 : 5064-5066 (1998) .
Fogel, G.B. and Brunk, C.F, "Temperature Gradient Chamber for Relative Growth Rate Analysis of Yeast", Analytical Biochemistry, 260 : 80-84 (1998) .
Brunk, C., "Evolution of Hydrothermal Ecosystems onn Earth (and Mars?)", Palaeogeography, Palaeoclimatology, Palaeoecology, 138 : 325-328 (1998) .
Boian, M., Avaniss-Aghajani, E., Walker, R., Aronson, T., Tran, T., Glover, N., Berlin, O.G., Woods, L., Brunk, C., Li, J-L., Froman, S. and Holtzman, A., "Identification of Mycobacteriurn genavense in intestinal tissue from a parakeet using two poloymrase chain reaction methods: are pets a reservoir of infection in AIDS patients?", AIDS (London), 11 : 255-256 (1997) .
Brunk, C.F., Avaniss-Aghajani, E. and Brunk, C.A., "A computer Analysis of Primer and Probe Hybridization Potential with Bacterial Small-Subunit rRNA Sequences", sApplied and Environmental Microbiology, 62 : 872-879 (1996) .