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Evolution of Mitochondrial Genome Architecture in Nematodes. Sita Ping Department of Zoology Mentor: Dr. Dee Denver HHMI Summer 2010. Background: Mitochondria. organelle in eukaryotic cells that is responsible for energy production.
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Evolution of Mitochondrial Genome Architecture in Nematodes Sita Ping Department of Zoology Mentor: Dr. Dee Denver HHMI Summer 2010
Background: Mitochondria • organelle in eukaryotic cells that is responsible for energy production • Electron transport chain, located in the inner membrane of Mitochondria produces usable energy and in doing so gives off reactive oxygen species (ROS) • ROS is a chemical body that has an unpaired electron • speculated to be a part of the aging process, and causes genetic mutations
Background: MtDNA • Has genome separate from the nucleus • Small, circular chromosome • Many chromosomes per organelle • Mitochondrial (mt) DNA may be related to longevity, cognition, and neurodegenerative and cancer diseases2 • Mitochondrial gene order used in analyzing deep evolutionary relationships • Thought to be very slow evolving • Used in evaluating arthropod evolution
(Ss) Strongyloides stercoralis • human parasite • 600 million infections estimated worldwide1 • (Rs) Rhabditophanes sp KR3021 • Close relative to Strongyloides spp. • Non-parasitic • Found in Oregon • (Pr) Panagrolaimus rigidus • Able to grow in lab • Found in Antarctica • (Ce) Caenorhabditis elegans • first animal to have its genome sequenced • important model organism
Comparison of gene order in S. stercoralis [Ss], Rhabditophanes sp KR3021 [Rs], Panagrolaimus rigidus [Pr], and Caenorhabditis elegans [Ce], created by Dr. Dee Denver and Dana Howe of the Denver lab
Hypothesis of mt gene order rearrangement: Denver Lab Recombination base fission and fusion model Courtesy of Dee Denver and Dana Howe
Analyze mtDNA of evolutionary intermediates to evaluate both hypotheses Ss Highly scrambled mt gene order; single chromosome Parastrongyloides trichosuri Rs Semi-conserved gene order; two chromosome mtDNA Alloionema appendiculatum Pr Conventional mt gene order; single chromosome Ce Conventional mt gene order; single chromosome Ss = Strongyloides stercoralis Pr = Panagrolaimus rigidus Rb = Rhabditophanes sp KR3021 Ce = Caenorhabditis elegans
Ss • Parastrongyloides tricorhosuri • Australian possum parasite • Has both free-living and parasitic lifecycles • possible model mammalian parasite? Pt Rs Aa • Alloionema appendiculatum • Slug parasite • Possible bio-control agent? Pr Ce
If hypothesis A is true: • presence of a large super chromosome mtDNA molecule as an evolutionary intermediate is expected
If hypothesis B is true: • presence of multi-chromosome mtDNA as the evolutionary transition is expected
Method • Worm lysis • A. appendiculatum received from Irma DeLey at UC Riverside; P. trichosuri from Dr. Sparky Lok at University of Pennsylvania • Long PCR amplification in overlapping amplicons • Initial primers created by Dana Howe of the Denver Lab • Run a gel-electrophoresis to estimate amplicon size • purify PCR product with invitrogen beading • Sequence reaction • PCR product directly sequenced using the primer walk strategy • Ethanol precipitation • Sent to CGRB for sequence results
1: C. elegans = ~13,800 bp 2: P. trichosuri = ~13,100 bp 3: A. appendiculum = ~4,700 bp 4: Rhabditophanes spp. = ~5,500 bp Amplicon 1 Amplicon 2 ~11000bp ~5000bp ~10000bp 1 2 ~3100bp 4 ~2500bp ~2200bp ~2800bp 2 1 3 3 ~500bp 4
Gene order results C. elegans MtDNA A. appendiculatum - Amplicon 1, Reverse ~2305 728 1092 ATPase6 K L P V ND6-protein ND4L-protein W E rrnS P. trichosuri - Amplicon 1, Reverse ~1000 cox3 rrnS
Project Reflection Ss Highly scrambled mt gene order; single chromosome Pt unknown gene order; single chromosome Rs Semi-conserved gene order; two chromosome mtDNA Aa Semi-conserved gene order; two chromosome mtDNA Pr Conventional mt gene order; single chromosome Ce Conventional mt gene order; single chromosome
Future Directions • Illumina sequence • Sequence the other A. appendiculatum chromosome(s) • Evaluate mtDNA of more Strongyloides species
Acknowledgements • Dr. Dee Denver and Denver Lab • Dana Howe • Larry Wilhelm • Katie Clark • Michael Raboin • Danika Kusuma • Kristin Gafner • Howard Hughes Medical Institute (HHMI) • OSU Computational Genome Bio Initiative • Irma DeLey • OSU CGRB • Dr. Kevin Ahern • Dr. Sparky Lok
References • Dorris, M., Viney, M.E., Blaxter, M.L., 2002. Molecular phylogenetic analysis of the genus Strongyloides and related nematodes. • Montiel, R., Lucena, M.A., Medeiros, J., Simoes, N., 2005. The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carposcapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny.