1. Evolution of Mitochondrial Genome Architecture in Nematodes Sita Ping Department of Zoology Mentor: Dr. Dee Denver HHMI Summer 2010

2. 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

3. 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 diseases 2  Mitochondrial gene order used in analyzing deep evolutionary relationships  Thought to be very slow evolving  Used in evaluating arthropod evolution

4. Human vs. Nematode mitochondria

5.  ( Ss) Strongyloides stercoralis  human parasite  600 million infections estimated worldwide 1  (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

6. 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

7. Hypothesis of mt gene order rearrangement: Denver Lab Recombination base fission and fusion model Courtesy of Dee Denver and Dana Howe

8. Ce Pr Alloionema appendiculatum Rs Parastrongyloides trichosuri Ss Conventional mt gene order; single chromosome Highly scrambled mt gene order; single chromosome Semi-conserved gene order; two chromosome mtDNA  Analyze mtDNA of evolutionary intermediates to evaluate both hypotheses Ss = Strongyloides stercoralis Pr = Panagrolaimus rigidus Rb = Rhabditophanes sp KR3021 Ce = Caenorhabditis elegans

9.  Parastrongyloides tricorhosuri  Australian possum parasite  Has both free-living and parasitic lifecycles  possible model mammalian parasite? Ce Pr Rs Ss Aa Pt  Alloionema appendiculatum  Slug parasite  Possible bio-control agent?

10.  If hypothesis A is true:  presence of a large super chromosome mtDNA molecule as an evolutionary intermediate is expected

11.  If hypothesis B is true:  presence of multi-chromosome mtDNA as the evolutionary transition is expected

12. 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

13. Amplicon 1Amplicon 2 ~2800bp ~3100bp ~2500bp ~11000bp ~10000bp ~5000bp ~2200bp ~500bp 1 2 3 4 12 3 4 1: C. elegans = ~13,800 bp 2: P. trichosuri = ~13,100 bp 3: A. appendiculum = ~4,700 bp 4: Rhabditophanes spp. = ~5,500 bp

14. Gene order results A. appendiculatum - Amplicon 1, Reverse rrnScox3 rrnS 728 EWND4L-proteinND6-protein 1092 VPLKATPase6 ~2305 P. trichosuri - Amplicon 1, Reverse C. elegans MtDNA ~1000

15. Project Reflection Ce Pr Rs Ss Pt Aa Conventional mt gene order; single chromosome Highly scrambled mt gene order; single chromosome Semi-conserved gene order; two chromosome mtDNA unknown gene order; single chromosome Semi-conserved gene order; two chromosome mtDNA

16. Future Directions  Illumina sequence  Sequence the other A. appendiculatum chromosome(s)  Evaluate mtDNA of more Strongyloides species

17. Acknowledgements  Howard Hughes Medical Institute (HHMI)  OSU Computational Genome Bio Initiative  Dr. Dee Denver and Denver Lab  Dana Howe  Larry Wilhelm  Katie Clark  Michael Raboin  Danika Kusuma  Kristin Gafner  Dr. Kevin Ahern  Dr. Sparky Lok  Irma DeLey  OSU CGRB

18. References 1. Dorris, M., Viney, M.E., Blaxter, M.L., 2002. Molecular phylogenetic analysis of the genus Strongyloides and related nematodes. 2. 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.