Evolution of Mitochondrial Genome Architecture in Nematodes

Sita PingDepartment of ZoologyMentor: Dr. Dee Denver

HHMI Summer 2010

Background: Mitochondriaorganelle in eukaryotic cells that is

responsible for energy productionElectron 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 electronspeculated to be a part of the

aging process, and causes genetic mutations

Background: MtDNAHas genome separate from the nucleus

Small, circular chromosomeMany 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 relationshipsThought to be very slow evolving

Used in evaluating arthropod evolution

Human vs. Nematode mitochondria

(Ss) Strongyloides stercoralis human parasite 600 million infections

estimated worldwide1(Rs) Rhabditophanes sp KR3021

Close relative to Strongyloides spp.Non-parasiticFound in Oregon

(Pr) Panagrolaimus rigidusAble to grow in labFound 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

Ce

Pr

Alloionema appendiculatum

Rs

Parastrongyloides trichosuri

Ss

Conventional mt gene order; single chromosome

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 stercoralisPr = Panagrolaimus rigidus

Rb = Rhabditophanes sp KR3021Ce = Caenorhabditis elegans

Parastrongyloides tricorhosuri Australian possum parasite Has both free-living and parasitic lifecyclespossible model mammalian parasite?

Ce

Pr

Rs

Ss

Aa

Pt

Alloionema appendiculatumSlug parasitePossible bio-control agent?

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

Amplicon 1 Amplicon 2

~2800bp~3100bp

~2500bp

~11000bp~10000bp ~5000bp

~2200bp

~500bp

12

3

4

1 2

3

4

1: C. elegans = ~13,800 bp2: P. trichosuri = ~13,100 bp 3: A. appendiculum = ~4,700 bp4: Rhabditophanes spp. = ~5,500 bp

Gene order results

A. appendiculatum - Amplicon 1, Reverse

rrnScox3

rrnS728

EWND4L-proteinND6-protein1092

VPLKATPase6~2305

P. trichosuri - Amplicon 1, Reverse

C. elegans MtDNA

~1000

Project Reflection

Ce

Pr

Rs

Ss

Pt

Aa

Conventional mt gene order; single chromosome

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

Future DirectionsIllumina sequence

Sequence the other A. appendiculatum chromosome(s)

Evaluate mtDNA of more Strongyloides species

Acknowledgements

Howard Hughes Medical Institute (HHMI) OSU Computational Genome Bio Initiative

Dr. Dee Denver and Denver LabDana HoweLarry WilhelmKatie ClarkMichael RaboinDanika KusumaKristin Gafner

Dr. Kevin AhernDr. Sparky Lok

Irma DeLeyOSU CGRB

References1. 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.