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Mitochondrial Endosymbiosis: Phylogenetically
Reconstructing the Presence of protein HSP70
Presence of a mitochondrial-type 70-kDa heat
shock protein in Trichomonas vaginalis
suggests a very early mitochondrial
endosymbiosis in eukaryotes
Agnès Germot*†, Hervé Philippe‡, and Hervé Le Guyader‡
Hervé Philippe – Phylogenist from Université de
Montréal – “"Phylogenetic structural modeling of
molecular evolution".
Terms to Keep in Mind:
Heat-Shock Proteins (HSP70)- among the most highly conserved proteins
present in all organisms, Archaebacteria, Eubacteria, and Eukaryota
Trichomonas vaginalis - flagellated protists, anaerobic, lack mitochondria
and peroxisomes
Hydrogenosomes - double membrane-bounded organelles, lacking DNA,
which have no tricarboxylic acid cycle enzymes and cytochromes and do not
carry out oxidative phosphorylation
GDAWV sequence signature - exclusively found
in mitochondrial HSP70 and in proteobacterial dnaK.
The Symbiosis In Questions: HSP70
and T.vaginalis
Established knowledge: three
independent species of protists
have consistently have held a base
position in the eukaryotic tree,
before mitochondrial
endosymbiosis
Symbiotic Question: How to
empirically prove HSP70 is
responsible for a phylogenetically
earlier than anticipated
mitochondrial endosymbiosis?
HSP70: Function and Relevance to
T.vaginalis
• Boorstein W R
• Ziegelhoffer T
• Craig E A
(1994) J Mol Evol 38:1–17.
Methods and Materials - Isolation to Analysis
1) DNA Isolation - axenic culture
2) Cloning of T.vaginalis HSP70 - fragment amplification
and via primer selectionv+ PCR
http://rbg-web2.rbge.org.uk/bbs/learning/exsitu/exsitu.htm
3) Southern Blotting - Enzyme Digestion via EcoRV, HindIII,
Sspl to obtain fragments
http://www.molecularstation.com/images/southern-blot.jpg
4) Phylogenetic Analyses - MUST Package
& PROTML program - Genetic analysis for Maximum
Likelihood, Maximum Parsimony, and Neighbor Joining
Significant Results
Significant Results: Findings to Keep In Mind
- High G+C and low G+C Gram-positive bacteria were monophyletic.
- Position of T. vaginalis within this mitochondrial group was supported by high
bootstrap values, whatever the tree reconstruction method used: maximum
likelihood, 100%; maximum parsimony, 80%; and neighbor joining, 94%.
- Two distinct signatures - GDAWV sequence & YSPSQI sequence. Confirm
phylogenetic affinity between spirochetes and monophyly of mitochondri.
Germot et Al: Implications and Discussion
- The consistencies in phylogenetic analysis suggest that
trichomonad hydrogenosomes may have in fact involved from
mitochondria.
"Phylogenetic analyses located T. vaginalis sequence inside the
mitochondrial group. In addition, this HSP70 presented
mitochondrial signatures shared only by α-Proteobacteria or the
whole group of purple bacteria."
Potential Problems:
1) Lack of current studies peer reviewing the evidence
behind the Montreal team’s work.
- Secondary mitochondrial endosymbiosis has not
been researched past phylogenetic trees.
"our data provide evidence that T. vaginalis may have
had mitochondria during its evolution as it is the case
for other extant amitochondriate protists such as
Entamoeba histolytica"
"More generally, the absence of some characteristics
in these lineages (for example, peroxisomes) has led
others to propose scenarii of progressive construction
of the eukaryotic cell"