genetic variation of renibacterium salmoninarum genes in infected salmonids
DESCRIPTION
Genetic Variation of Renibacterium salmoninarum genes in infected salmonids. Jeffrey Burnett HHMI Summer Investigator Dr. Dan Rockey Laboratory Biomedical Sciences. Renibacterium salmoninarum. Causes bacterial kidney disease (BKD) Wild and farmed salmonid species. Introduction - PowerPoint PPT PresentationTRANSCRIPT
Genetic Variation of Renibacterium
salmoninarum genes in infected salmonids
Jeffrey BurnettJeffrey BurnettHHMI Summer InvestigatorHHMI Summer InvestigatorDr. Dan Rockey LaboratoryDr. Dan Rockey Laboratory
Biomedical SciencesBiomedical Sciences
13 Oct 2007 Jeffrey Burnett - HHMI 2
Renibacterium salmoninarum
• Causes bacterial kidney disease (BKD)• Wild and farmed salmonid species
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Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Relevance
• Why is this a problem?• We eat salmonids• We depend on salmonids to
keep an ecosystemic balance in our local rivers and streams
• R. salmoninarum devastates whole populations; endangered fish stocks
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Relevance
• All salmonids are susceptible to BKD• coho salmon (Oncorhynchus kisutch)• brook trout (Salvelinus fontinalis)• brown trout (Salmo trutta)• chinook salmon (Oncorhynchus
tshawytscha)• rainbow trout (Oncorhynchus mykiss)
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Renibacterium salmoninarum
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
http://oregonstate.edu/dept/salmon/projects/images/4BKD.jpg
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Relevance
• Prevalence• Found in majority of countries• Economic impact felt worldwide
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Relevance
• Economics close to home• Local: Oregon Hatcheries• 2004 - $143,000
• Largest Global Impact: • Chile,S.A. and Europe
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Big Picture
• Drug / Vaccine to eliminate bacteria• Difficult to treat• Current treatments ineffective
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Goal of My Project
• Genome analysis• American Tissue Culture
Collection (ATCC) 33209• Accurate representation • ERGO by Integrated Genomics
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Specific Goal
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
gene affected by frameshift # frameshifted total of type percentagetransport and binding proteins 158 346 45.66central intermediary metabolism 53 170 31.18Fatty acid and phospholipid metabolism 31 100 31.00energy metabolism 91 304 29.93regulatory functions 59 240 24.58Biosynthesis of cofactors, prosthetic groups, and carriers 26 109 23.85cellular processes 21 90 23.33DNA metabolism 23 108 21.30protein fate 29 152 19.08amino acid biosynthesis 21 111 18.92unknown/hypothetical/unclassified/not called 215 1182 18.19signal transduction 1 6 16.67cell envelope 31 215 14.42Purines, pyrimidines, nucleosides, and nucleotides 6 61 9.84protein synthesis 9 128 7.03Mobile and extrachromosomal element functions 10 151 6.62transcription 2 34 5.88total 786 3507
Renibacterium salmoninarum vs. Arthrobacter sp.
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Hypothesis
• Due to extended laboratory culture, the genome of strain ATCC33209 has extensive mutations not representative of what is found in nature
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Genetic analysis
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
• Dipeptide Permease Protein
• Citrate Synthase Protein • Tetracycline Resistance Protein P
• Fibronectin Binding Protein
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Genomic DNA
• wt - 2 fish kidneys (A,B)• Mt239• ATCC33209• 684
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Primer Design
• Flank apparent frameshifts identified by ERGOIntroduction
Hypothesis
Methods
Results
Discussion
Conclusion
• Tetracycline Resistance Protein P
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Experiment
• PCR products inserted into expression vectors
• Plasmids transformed into Escherichia coli
• Plasmids purified from bacteria• Sent for sequencing
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Results
• Center for Genome Research and Biocomputing (CGRB) - OSU
• 20 sequences in both
directions
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Results
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
ATCC mt239 684 A BFibronectin Binding Protein Y Y N N Y
dppD/F Y Y N Y N
Citrate Synthase Y Y Y Y Y
Tetracycline Resistance P Y Y Y N Y
Y = yes, the sequence is identical to the ATCC sequenceN = no, the sequence received is different from the ATCC sequence
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Results
• Verification of first round of results
• Reconstruct plasmids from different samples of DNA strains
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Results
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
ATCC mt239 684 A BFibronectin Binding Protein Y Y N N Y
dppD/F Y Y N Y N
Citrate Synthase Y Y Y Y Y
Tetracycline Resistance P Y Y Y N Y
•All of the samples marked “N” ran in duplicate, returned the same results
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Discussion
• Findings are contrary to what we had originally hypothesized
• Genes are actually more mutated in the other strain isolate DNA that we tested
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
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Conclusion
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
• My research suggests that the ATCC sequence is representative of what is found in nature
• The bacteria is acquiring more mutations in its genome than the original ATCC strain
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Acknowledgements• Howard Hughes Medical Institute• Dr. Kevin Ahern
• Dr. Dan Rockey Laboratory• Sara Weeks• Gina Capri
• Integrated Genomics