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
Human vs. Nematode mitochondria
 (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
Ce
Conventional mt gene order; single chromosome
Conventional mt gene order; single chromosome
Ss = Strongyloides stercoralis
Pr = Panagrolaimus rigidus
Rb = Rhabditophanes sp KR3021
Ce = Caenorhabditis elegans
Ss
Pt
Rs
Aa
Pr
Ce
Parastrongyloides tricorhosuri
Australian possum parasite
Has both free-living and parasitic lifecycles
possible model mammalian parasite?
Alloionema appendiculatum
Slug parasite
Possible 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
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
~10000bp
1
~3100bp
~2500bp
~2800bp
1
2
~5000bp
2
~2200bp
3
3
~500bp
4
4
Gene order results
C. elegans MtDNA
~2305
ATPase6
728
1092
K
L
P
V ND6-protein ND4L-protein W
E
A. appendiculatum - Amplicon 1, Reverse
P. trichosuri - Amplicon 1, Reverse
~1000
cox3
rrnS
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
Pr
Ce
Semi-conserved gene order; two chromosome mtDNA
Conventional mt gene order; single chromosome
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
Dr. Kevin Ahern
Dr. Sparky Lok
Irma DeLey
OSU CGRB
References
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.
1.