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Analysis of Genes Isolated from Plated Hemocytes
of the Pacific Oyster, Crassostrea gigas
Steven RobertsSchool of Aquatic and Fishery Sciences
University of Washington
Rick Goetz Great Lakes WATER Institute
University of Wisconsin
Goal
Better understand stress response
Laura Canesi, Gabriella Gallo, Miriam Gavioli, and Carla PruzzoBacteria–hemocyte Interactions and Phagocytosis in MarineBivalves. 2002. Microscopy Research And Technique 57:469–476
Immune System
Specific Objectives
• Identify genes expressed in oyster hemocytes
• Characterize response of select molecules following Vibrio challenge
Outline
•cDNA Library Description
•Select Functional Categories• Proteases and Protease Inhibitors
• Receptor and Associated Proteins
• Transcription/Translation/Cell Cycle
Pacific OysterC. gigas
Incubate 24 hours Wash in sterile seawaterExtract with Tri-Reagent
+ sterilesaltwater
Plated hemocyte cDNA library
Plated hemocyte cDNA library
Prior to washing After washing
Gene - DNA
Transcript - mRNA
Extract from tissue
Make cDNA
Clone into a “Vector”
Randomly sequence clones = “Expressed Sequence Tags”
(ESTs)
ESTs
(Complementary DNA = Copy)
= cDNA Library
C. gigas plated hemocytes
Roberts et al 2009
C. gigas plated hemocytes
modified from Roberts et al 2009
Markers &more
Markers &more
C. gigas Vibrio challenge
Roberts et al 2009
Proteases and AntiproteasesGene Function
Proteases and AntiproteasesGene Function
Proteases and AntiproteasesGene Function
Inhibitors
Proteases and AntiproteasesGene Function
Cathepsins
Proteases and AntiproteasesGene Function
Cathepsins
Proteases and AntiproteasesGene Function
C. virginica Homologs
Proteases and AntiproteasesGene
C. virginica Homologs
Receptors and other proteinsGene Function
Prostaglandin E2EP4 subtype
Prostaglandin
15-hydroxyprostaglandin dehydrogenase
PGE2 breakdown
lipoxygenases production of leukotrienes,
lipoxins,hydroperoxy fatty acids
other ESTs present
Prostaglandin
Mackenzie Gavery - Poster
Receptors and other proteinsGene Function
Ectodysplasin A2Receptor
Transcription / Translation / Cell CycleGene Function
Erythroid differentiation-related factor
Transcription / Translation / Cell CycleGene Function High-mobility
group proteins
Transcription / Translation / Cell CycleHigh-mobilitygroup proteins
Transcription / Translation / Cell CycleHigh-mobilitygroup proteins
Conclusions
• Several novel genes were identified from C. gigas hemocytes
• Hemocyte signaling plays important role in immune response
• Stress response is diverse and complex
•
Conclusions
• Several novel genes were identified from C. gigas hemocytes
• Hemocyte signaling plays important role in immune response
• Stress response is diverse and complex
•
h/t
