Characterization of a toll-interacting protein (TOLLIP) gene in the black abalone ( Haliotis cracherodii ) Cullen Taplin M...
Presentation Outline <ul><li>Background to abalone and withering syndrome </li></ul><ul><li>Introduction to the Toll-like ...
Black Abalone <ul><li>Single shelled gastropods </li></ul><ul><li>Found in intertidal zone at depths from 0 – 5 m </li></u...
Abalone Distribution in Western U.S. <ul><li>Black abalone are confined to the west coast of the U.S., extending from Coos...
Black Abalone Declines <ul><li>Overharvesting </li></ul><ul><li>Environmental Contaminants </li></ul><ul><li>Climate chang...
Withering Syndrome <ul><li>Caused by a bacterium which infects the post esophagus, followed by a change in tissue morpholo...
Abalones’ Immune Response <ul><li>Abalones’ immune response is not well understood. </li></ul><ul><li>Like all invertebrat...
Identifying Immune Related Genes <ul><li>Used comparative bioinformatic techniques </li></ul><ul><li>Several immune relate...
Toll-like Receptor Pathway <ul><li>Recognizes molecules and patterns produced exclusively by bacteria and viruses. </li></...
Research Goal & Objectives <ul><li>The goal of my research was to characterize the toll-interacting protein in black abalo...
Abalone Used In Research <ul><li>Visually healthy black abalone were collected and maintained in Dr. Carolyn Friedman’s la...
Research Methods <ul><li>Tissues were isolated and RNA was extracted via tri-reagent method. </li></ul><ul><li>cDNA was ma...
Partial TOLLIP sequence and comparison to the Pacific abalone ( H. discus ) <ul><li>Using primers designed from a TOLLIP e...
TOLLIP Sequencing Results (cont.) <ul><li>Complete genomic sequencing continues. </li></ul><ul><li>Preliminary evidence in...
Results: Discovering  Other TLR Genes <ul><li>Classifies genes by their functions. </li></ul><ul><li>Can predict the funct...
Results: TOLLIP Expression in Digestive Gland Tissue Five months after initial exposure. N=8 for control groups, N=9 expos...
Results: TOLLIP Expression in Gill Tissue Eight months after initial exposure. N = 6 for both treatment groups. Means were...
Results: Correlation of TOLLIP Expression in and Bacterial Loads No significant correlations between TOLLIP expression and...
Summary of Results <ul><li>Obtained TOLLIP coding sequence & isolated genomic DNA </li></ul><ul><li>Identified other TLR m...
Future Work <ul><li>Explore other intermediates in the toll-like receptor pathway. </li></ul><ul><li>Examine expression in...
Acknowledgements <ul><li>Dr. Carolyn Friedman and her lab staff. </li></ul><ul><li>Sam White </li></ul><ul><li>Dr. Steven ...
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Taplin Capstone

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Taplin Capstone

  1. 1. Characterization of a toll-interacting protein (TOLLIP) gene in the black abalone ( Haliotis cracherodii ) Cullen Taplin May 16, 2008
  2. 2. Presentation Outline <ul><li>Background to abalone and withering syndrome </li></ul><ul><li>Introduction to the Toll-like Receptor Pathway </li></ul><ul><li>My research goal and objectives </li></ul><ul><li>Results </li></ul><ul><li>Future work and conclusions </li></ul>
  3. 3. Black Abalone <ul><li>Single shelled gastropods </li></ul><ul><li>Found in intertidal zone at depths from 0 – 5 m </li></ul><ul><li>Can reach shell sizes of 200 mm </li></ul>
  4. 4. Abalone Distribution in Western U.S. <ul><li>Black abalone are confined to the west coast of the U.S., extending from Coos Bay, Oregon to Cabo San Lucas, Baja California. </li></ul>
  5. 5. Black Abalone Declines <ul><li>Overharvesting </li></ul><ul><li>Environmental Contaminants </li></ul><ul><li>Climate change </li></ul><ul><li>Disease </li></ul><ul><ul><li>Withering syndrome </li></ul></ul>1986 1988 1999
  6. 6. Withering Syndrome <ul><li>Caused by a bacterium which infects the post esophagus, followed by a change in tissue morphology of the digestive gland. </li></ul><ul><li>The abalone is forced to rely on glycogen stores from the foot. </li></ul>Healthy black abalone (left) and one suffering from withering syndrome (right). J. Moore
  7. 7. Abalones’ Immune Response <ul><li>Abalones’ immune response is not well understood. </li></ul><ul><li>Like all invertebrates, abalones only have an innate immune system. </li></ul><ul><li>Several immune components are conserved among invertebrates, including toll-like receptors, lectins in non-self recognition, and the prophenoloxidase system. </li></ul>
  8. 8. Identifying Immune Related Genes <ul><li>Used comparative bioinformatic techniques </li></ul><ul><li>Several immune related genes were found in black abalone. </li></ul><ul><li>TOLLIP, an intermediate in the toll-like receptor pathway was chosen for study. </li></ul>
  9. 9. Toll-like Receptor Pathway <ul><li>Recognizes molecules and patterns produced exclusively by bacteria and viruses. </li></ul><ul><li>It functions to signal the presence of a pathogen and produce proinflammatory cytokines. </li></ul>Singh, Bhanu P., R. S. Chauhan, and Kokesh K. Singhal. 2003. Toll-like Receptors And Their Role In Innate Immunity . Current Science. vol. 85, no. 8, pgs. 1156 – 1164.
  10. 10. Research Goal & Objectives <ul><li>The goal of my research was to characterize the toll-interacting protein in black abalone. </li></ul><ul><li>Objectives to accomplish the goal were: </li></ul><ul><ul><li>Obtain more TOLLIP sequence information </li></ul></ul><ul><ul><li>Discover other TLR genes in black abalone. </li></ul></ul><ul><ul><li>Compare differential expression of TOLLIP </li></ul></ul><ul><ul><li>Correlate TOLLIP expression to bacterial loads </li></ul></ul>
  11. 11. Abalone Used In Research <ul><li>Visually healthy black abalone were collected and maintained in Dr. Carolyn Friedman’s lab at the University of Washington. </li></ul><ul><li>Control and bacterium exposed treatments were then constructed. </li></ul>
  12. 12. Research Methods <ul><li>Tissues were isolated and RNA was extracted via tri-reagent method. </li></ul><ul><li>cDNA was made via reverse transcription. </li></ul><ul><li>qPCR was carried out using an Opticon 2 System and SYBR Green Fluorescent Dye. </li></ul><ul><li>Data was analyzed with Real-time PCR Miner (www.miner.ewindup.info/miner/) </li></ul><ul><li>Bacterial Loads were quantified via qPCR methods. </li></ul>
  13. 13. Partial TOLLIP sequence and comparison to the Pacific abalone ( H. discus ) <ul><li>Using primers designed from a TOLLIP expressed sequence tag from the Pacific abalone (gene accession #CX726806), partial sequence information was obtained for TOLLIP in black abalone and submitted to GenBank. Comparisons show 97.5% similarity. </li></ul>Results: Sequence Information
  14. 14. TOLLIP Sequencing Results (cont.) <ul><li>Complete genomic sequencing continues. </li></ul><ul><li>Preliminary evidence indicates the gene is over 10,000 bp long. </li></ul><ul><li>Comparisons to the fully sequenced sea urchin genome indicates six introns. </li></ul>10kbp – 4kbp – 2kbp – 800bp –
  15. 15. Results: Discovering Other TLR Genes <ul><li>Classifies genes by their functions. </li></ul><ul><li>Can predict the function of genes based on relationships. </li></ul><ul><li>Searched for genes within the TLR pathway. </li></ul><ul><li>Identified several other TLR genes in black abalone, including toll-like receptor #9, NF κ B and several TRAFs. </li></ul>Protein Analysis Through Evolutionary Relationships www.pantherdb.org/
  16. 16. Results: TOLLIP Expression in Digestive Gland Tissue Five months after initial exposure. N=8 for control groups, N=9 exposed groups. Means were not significantly different (p > 0.05).
  17. 17. Results: TOLLIP Expression in Gill Tissue Eight months after initial exposure. N = 6 for both treatment groups. Means were not significantly different (p > 0.05).
  18. 18. Results: Correlation of TOLLIP Expression in and Bacterial Loads No significant correlations between TOLLIP expression and bacterial loads (p > 0.05).
  19. 19. Summary of Results <ul><li>Obtained TOLLIP coding sequence & isolated genomic DNA </li></ul><ul><li>Identified other TLR members </li></ul><ul><li>3. </li></ul><ul><li>4. </li></ul>Data suggests the TLR pathway is active in gill tissue.
  20. 20. Future Work <ul><li>Explore other intermediates in the toll-like receptor pathway. </li></ul><ul><li>Examine expression in other tissues; in particular, the post-esophagus. </li></ul><ul><li>Compare laboratory results to field results. </li></ul>
  21. 21. Acknowledgements <ul><li>Dr. Carolyn Friedman and her lab staff. </li></ul><ul><li>Sam White </li></ul><ul><li>Dr. Steven Roberts </li></ul><ul><li>NOAA </li></ul><ul><li>Sea Grant California </li></ul>
  22. 22. Questions?
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