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Marine Animal Health Genes and Genomics

Marine Animal Health Genes and Genomics. Paul S. Gross, Ph.D. Marine Biomedicine and Environmental Sciences Program Medical University of South Carolina Charleston, South Carolina, USA. “Genome Projects”. THE PARADIGM OF GENETICS. DNA. RNA. Proteins. Everything Else. Genomics defined:.

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Marine Animal Health Genes and Genomics

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  1. Marine Animal Health Genes and Genomics Paul S. Gross, Ph.D. Marine Biomedicine and Environmental Sciences Program Medical University of South Carolina Charleston, South Carolina, USA

  2. “Genome Projects”

  3. THE PARADIGM OF GENETICS DNA RNA Proteins Everything Else

  4. Genomics defined: • the scientific study of genomes, esp. of their organization and evolution. - OED • the comprehensive study of the genetic information of a cell or organism - NIH • the study of how an individual's genes interact with each other and with the environment to create the complexity of life. - Mayo Clinic

  5. “OMICS” (a.k.a. Genomics in the generic sense)(a.k.a. Biology with fancy machines) Genomics (strictly referring to the “genome” or DNA) Lipidomics (referring to lipid manufacture) Transcriptomics (expressed sequences or RNA a.k.a ESTs) Metabolomics (referring to metabolism cascades) Proteomics (referring to expressed proteins) Ecogenomics Toxicogenomics etc…

  6. “OMICS” Genomics (strictly referring to the “genome” or DNA) Lipidomics (referring to lipid manufacture) Transcriptomics (expressed sequences or RNA a.k.a ESTs) Metabolomics (referring to metabolism cascades) Proteomics (referring to expressed proteins) Ecogenomics Toxicogenomics etc…

  7. “OMICS” Genomics (strictly referring to the “genome” or DNA) Lipidomics (referring to lipid manufacture) Transcriptomics (expressed sequences or RNA a.k.a ESTs) Metabolomics (referring to metabolism cascades) Proteomics (referring to expressed proteins) Ecogenomics Toxicogenomics etc…

  8. “OMICS” Genomics (strictly referring to the “genome” or DNA) Transcriptomics (expressed sequences or RNA a.k.a ESTs)

  9. WHOLE GENOME SEQUENCING: Genomes available • 478 Bacterial genomes • 49 Eukaryotic genomes www.genomesonline.org/

  10. WHOLE GENOME SEQUENCING: problems, pitfalls and failures • The Human Genome Project using the ‘Celera’ Scheme of 10 X coverage • Regions not covered • Regions very densely covered • Contigs 1.0 -15 kb • # Gaps? >100,000? • Base Quality High or Low? • Mis-Assemblies? • Duplications?

  11. WHOLE GENOME SEQUENCING: problems, pitfalls and failures • Other projects: • Zebrafish: used more than one individual to “try to simultaneously capture variability with the draft genome sequence” • Ciona: selected a wild animal off a rock so not lineage to go back to for further analysis

  12. “OMICS” Genomics (strictly referring to the “genome” or DNA) Transcriptomics (expressed sequences or RNA a.k.a ESTs)

  13. TRANSCRIPTOMICS • Transcript libraries • Microarrays

  14. GENOME FUNCTIONAL GENOMICS CONCEPT STIMULUS RESPONSE • PHYSIOLOGICAL • Hormonal, • Metabolic • SYSTEMIC: • Development? • Differentiation? • IMMUNITY: • Survival? • Mortality? INFECTION • ENVIRONMENT • Physical, • Chemical, • Biological stress • STRESS: • Physiological? • Pathological? GENE EXPRESSION

  15. EXPRESSED SEQUENCE TAGS (ESTS) What are they? - Randomly selected clones from a cDNA library - Single-pass uncorrected sequences, usually from the 5’ end What is their value? - Novel Gene Discovery - Transcript profiling- distribution of metabolic energy - Source of “unigenes” for microarrays

  16. TRANSCRIPTOMICS: problems, pitfalls and failures • Whole transcriptome is not really possible only a general representation • Incomplete and short sequences • Redundancy • Gene identification <60% for most organisms (human is still 30-40% unknown)

  17. TRANSCRIPT LIBRARIES: sequences • Number of ESTs at NCBI = 61,132,599 • Number of Unigenes at NCBI > 17,960,667 • Number of bases at NCBI > 65,369,091,950 • Searching resources abound • NCBI • EMBL (Swissprot) • Etc…(over a million hits on the web)

  18. TRANSCRIPTOMICS • cDNA libraries • Microarrays

  19. Microarrays • Snap shot of the status of all gene expression for those genes (features) on the array • Comprehensive? • Massive data collection • Repeatable?

  20. Overview Test Animal Microarray Analysis Quantification of gene expression Classical Health Assessment Environmental Parameters + Predictive Models

  21. Atlantic bottlenose dolphin:Tursiops trucatus Class: Mammalia Superorder: Cetartiodactyla Order: Cetacea Suborder: Odontoceti Family: Delphinidae Genus: Tursiops Species: Tursiops truncatus • Length is from 8-12 feet (2.5-3.8 m). May weigh as much as 1,430 pounds (650 kg) (Great Britain). • Males are larger than females. • An adult bottlenose dolphin may consume 15-30 pounds (8-15 kg) of food each day (fish; squid; crustaceans). • The average lifespan is estimated to be about 25 years, although some live to their late 40's. • Bottlenose dolphins are found worldwide in temperate and tropical • waters (harbors, bays, lagoons, estuaries, and river mouths). • There appear to be two ecotypes: a coastal and an offshore form. • The bottlenose dolphin is protected in U.S. waters by the Marine Mammal Protection Act (1972).

  22. Why the dolphin? • Good sentinel organism in aquatic and coastal environments • (Bossart, 2005): • - Long life span • - Feed at a high trophic level • Have extensive fat stores deposit for anthropogenic toxin • (Reddy et al, 2001; Wells et al., 2004) • Sentinels for: • Infectious diseases such as toxoplasmosis, urogenital cancer • (Goldston et al., 1990; Jenson et al., 2006) • Anthropogenic pollutants such as organohalogen and heavy metal contaminants • (Stein et al., 2003; Houde et al., 2005) • Harmful Algal Blooms (HABs) and their toxins such as brevetoxins, saxitoxins, okadaic acid and others • (Gulland, 2000; Bossart et al., 2002; Flewelling et al., 2002; Landsberg, 2002)

  23. Dolphin Peripheral Blood Leukocyte cDNA Microarray • 3600 clones from cDNA libraries • IL-2 and LPS stimulated PBL (T and B cell biased) • and targeted PCR • 52 TARGETED STRESS & IMMUNE FUNCTION GENES Pattern and intensity of the spots TRANSCRIPTOMIC SIGNATURE

  24. Analysis of differential gene expression in wild dolphins: What can a microarray tell us? Association with location, sex, stress, health?

  25. Samples collection 0 30’-40’ pre blood sample post blood sample FB833 Samples: summary • Blood samples from 20 dolphins • 2 samples for each animal: pre and post Experimental design CAPTURE RELEASE time • 40 samples: microarray hybridization and analysis

  26. CHS IRL RNA samples from Wild Dolphins CHS: Charleston, SC IRL: Indian River Lagoon, FL

  27. CHS, SC 122 genes differentially expressed IRL, FL Cluster analysis: pre samples

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