Erin Dougher & Lauren Hartigan
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Transcript of Erin Dougher & Lauren Hartigan
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Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization
Sophie Sanchez,1 Stéphane Hourdez,1 and François H Lallier1
1Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, UMR 7144 CNRS UPMC, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, FranceCorresponding author.
Sophie Sanchez: [email protected]; Stéphane Hourdez: [email protected]; François H Lallier: [email protected] Received June 8, 2007; Accepted September 24, 2007.
Erin Dougher &
Lauren Hartigan
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Background• Riftia pachyptila live around
hydrothermal vents in the East Pacific Rise
• 2600 meters-depth• Gut-tubeworms who form
dense communities that are dependent on chemosynthetic primary production
• Adults lack a mouth, gut and anus
• They make up a major component of biomass in the deep-sea
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Background• They have specialized tissue
called a trophosome that contains a symbiotic sulfide oxidizing bacteria
• They acquire bacteria by interacting with the environment via their skin and mouth during the larva stage
• As they mature bacteria become trapped in the trophosome (mesodermal tissue)
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Goal of the Experiment
• To determine what molecular components were present in the various tissues of the worm body through SSH and PCR techniques
• They essentially used the equation (Bacteria DNA + Worm DNA) – (Worm DNA) = Bacteria DNA to discern species specific DNA sequences in the variety of bacteria involved in the Riftia, bacteria symbiosis
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Table 6• Standard curve equation and efficiency (E) in the different tissues (calculated from one sample each time• Transcript Branchial plume Trophosome Body wall•• 18S y = -3.31x + 24.67E = 101 % y = -3.38x + 28.30 E = 98 % y = -3.29x + 24.55 E = 101 %• RpCAbr y = -3.24x + 39.13E = 103 % y = -3.37x + 51.40 E = 98 % y = -3.30x + 44.66 E = 101 %• RpCAtr y = -3.30x + 49.19 E = 101 % y = -3.21x + 38.84 E = 105 % y = -3.25x + 47.08 E = 103
%• MVP y = -3.35x + 39.63 E = 99 % nd y = -3.24x + 42.51 E = 104 %• Cathep y = -3.22x + 43.13 E = 104 % y = -3.33x + 47.41 E = 100 % y = -3.26x + 43.98 E = 102
%• ChPr y = -3.37x + 39.57 E = 98 % nd nd• 16S y = -3.31x + 29.86 E = 101 % y = -3.17x + 38.77 E = 107 % y = -3.43x + 34.80 E = 96 %• ccoxI y = -3.54x + 36.05 E = 92 % y = -3.42x + 40.74 E = 96 % y = -3.46x + 38.51 E = 94 %• ATPF1 y = -3.43x + 41.54 E = 96 % y = -3.41x + 44.95 E = 96 % y = -3.34x + 43.14 E = 99 %
• MH nd y = -3.24x + 41.08 E = 103 % nd• TCR nd y = -3.27x + 41.34 E = 102 % nd• TRbwC27 nd y = -3.36x + 39.07 E = 98 % nd
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Experimental Interest• The plume is only organ in direct
contact with sea-water• The trophosome is the only
organ in contact with the symbiotic bacteria
• The plume is strongly exposed to hydrogen sulfide and other toxic molecules (heavy metals-abundant in vent environment)
• But the trophosome is the one dealing with the symbiotic relationship not the plume
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Methods
• Tissue samples were taken from the plume, body wall, and the trophosome
• The body was used as reference tissue to find specific proteins expressed in the gills (main metabolite exchange organ) and the trophosome (organ that houses the symbiotic bacteria)
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Suppression Subtractive Hybridization (SSH)
• Takes PCR-based amplifications of complementary DNA fragments
• It removes double stranded DNA formed by hybridization between a control and test group so that only complementary DNA or genomic DNA is left in similar quantities
• Shows the variability of sequences and expressed by genes and transcripts– For example it allows us to see how many transcripts are
being copied and the genetic differences between species
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Figure 3
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Polymerase Chain Reaction (PCR)
• It used to amplify or single or a few copies of DNA
• It generates thousands or millions of copies of particular DNA sequences
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Protein Degradation and Turnover
• Electron dense organelles structures are common in tissues of sulfide adapting marine annelids
• In order for the symbiosis to work the lysosomes have to maintain mitochondrial integrity in the harsh sulfide environment
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Protein Degration and Turnover in Branchial Plume TIssue
• Cathespin transcript was expressed more so in the bronchial plume tissue than the body wall tissue and trophosome tissue because of its direct contact with the sea water
• Sulfide exposure poisons the mitochondria leading to irreversible depolarization and the presence of lysosomes helps degrade damaged mitochondria– Other similar degradation proteins were also found in
the plume tissue (Valosin-Containing Protein (VCP))
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Hydroxlamine Reductase Protein
• Plays a role in nitrogen matabolism • Catalyses reduction of hydroylamine to form
ammonia using NADH• Sequences for this protein did not match the
Riftia tissue samples, but it did match the bacteria tissue sequences
• This could be due to contamination because the bacteria live so close to the plume
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Major Vault Protein (MVP) Gene Expression
• Major protein component of vaults• These vaultes are involved with nucleocytoplasmic
transport of ribosome and/or mRNA• This showed up in branchial plume tissue and
body wall tissue but it was not detected in the trophosome tissue
• Presence of protein in branchial plume tissue may be used to temporarily stop toxic molecules from being processed
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Chitinase Gene Expression
• Branchial plume specific transcript
• Involved in tube growth and tube shape modifications
• Produced in the body wall and the vestimentum
• Potential host defense against pathogens
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Tissue-Specific Expression of Carbonic Anhydrase
• Carbonic Anhydrase converts CO2 and water to bicarbonate and protons
• There was a high abundance of RpCAbr transcript in branchial plume tissue compared to the trophosome and body wall tissue
• RpCAtr was found in high abundance in the trophosome tissue in comparison to the branchial plume tissue
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Myohemerythrin, T-Cell Receptor and Unidentified Transcripts
• Myohemerythrin is an oxygen-binding protein that participates in the storage of oxygen in muscles– It can deprive bacteria of iron essential for growth
• Cellular recognition by T-Cells helps the trophosome function with the bacteria– TCR transcripts were found that code for T-Cell
receptors
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Results• Four cDNA libraries were
produced1. Body wall subtracted
bronchial plume (BR-BW)
2. Bronchial plume subtracted body wall (BW-BR)
3. Body wall subtracted trophosome (BW-TR)
4. Trophosome subtracted body wall (TR-BW)
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Results
• PCR analysis was done that showed Major Vault Proteins in use
• Carbonic anhydrase, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and body wall tissues
• Carbonic anhydrase, myohemerythrin (putative T-Cell receptor), and one non identified transcript were highly specific of the trophosome tissue
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Conclusion
• The PCR analysis matched Table 1 library which confirmed the existence of tissue-specific transcripts
• Even with all the research some sequences could not be identified.
• Further research would have to be done for those individual sequences in order to verify expression level transcripts in the different parts of the worm
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References• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175520/• http://invertebrates.si.edu/Features/stories/vestimentifera.html• http://foundation.total.com/environment/processes-and-dynamics-of-marine-
life/ongoing-projects/world-inventory-of-marine-biodiversity-census-of-marine-life-800069.html
• http://bioweb.uwlax.edu/bio203/s2007/rossing_jaco/introduction__the_worm_under_inv.htm
• http://en.wikipedia.org/wiki/Polymerase_chain_reaction• http://www.mbari.org/staff/vrijen/dives/sspring/sspgs/riftia.htm• http://publishing.cdlib.org/ucpressebooks/view?docId=kt167nb66r&chunk.id=ch
17&toc.id=ch17&brand=eschol• http://www.marine-genomics-europe.org/index2.php?rub=b&pid=419• http://en.wikipedia.org/wiki/Carbonic_anhydrase• http://en.wikipedia.org/wiki/Complementary_DNA• http://en.wikipedia.org/wiki/gDNA• http://en.wikipedia.org/wiki/polymerase_chain_reaction