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By: Jessica Flesher Mentors: Dr. Virginia Weis Dr. Camille Paxton Zoology Department

Change in the Genetic Expression of Apoptotic and Autophagic Proteins after Thermal Stress in Symbiotic Aiptasia pallida. By: Jessica Flesher Mentors: Dr. Virginia Weis Dr. Camille Paxton Zoology Department HHMI Summer Program 2011. Aiptasia pallida. Corals. Reef-building corals

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By: Jessica Flesher Mentors: Dr. Virginia Weis Dr. Camille Paxton Zoology Department

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  1. Change in the Genetic Expression of Apoptotic and Autophagic Proteins after Thermal Stress in Symbiotic Aiptasiapallida By: Jessica Flesher Mentors: Dr. Virginia Weis Dr. Camille Paxton Zoology Department HHMI Summer Program 2011 Aiptasiapallida

  2. Corals • Reef-building corals • Biogenic habitat • Trap nutrients and provide shelter • Coral polyps contain symbiotic dinoflagellate algae, zooxanthellae Coral Reef

  3. The Symbiosis • Corals • Waste removal and photosynthetic products • Zooxanthellae • Acquire shelter and nutrients for photosynthesis Zooxanthellae in coral polyp

  4. The Symbiosis NOAA Ocean Service Education 2008

  5. Coral Bleaching • Loss of zooxanthellae from the host • Increase with ocean temperature • Worldwide in 2008 • 19% lost • 15% critical • 20% threatened • Interest in the cellular and molecular pathways Partially bleached coral head, Montastreacavernosa

  6. A Model System • Corals are hard to grow • Aiptasiapallida-Symbiodiniumsp. • Same symbiosis • Replicate pedal lacerations Aiptasiapallida Symbiodinium in tentacles of a coral polyp

  7. Mechanisms for Coral Bleaching Weis, Journal of Experimental Biology, 2008

  8. Apoptosis • Programmed cell death • Caspases • Partially characterized mechanism • Apoptosis proteins • acasp • Caspase 8 Vertebrate Apoptosis Model Cheung, Clinical Cancer Research, 2006

  9. Autophagy • Autophagy – the degradation of the symbiont by the host cell • Highly conserved pathway – yeast to mammals • Unknown mechanism • Autophagy related (ATG) proteins • ATG 7 • ATG 8 • ATG12 Mizushima, Genes and Development, 2007

  10. Hypothesis • Under stress caused by increased temperature, the expression will change of the identified caspase and ATG sequences Bleached coral, Acroporasp.

  11. Prediction • If there is an increase in thermal stress, than there will be an increase in the genetic expression of the caspase and ATG sequences Bleached coral, Acroporasp.

  12. Sequences • Acasp previously identified (Dunn, et al. 2006) • Caspase 8, ATG7, ATG8, ATG12 unknown • No full genomic sequence for Aiptasia • Initial primers using Transcriptome (Pringle Lab) • Partial genomic database • With many sequence errors • Primers used to isolate, clone, and sequence genes • Accurate sequences used to develop qPCR primers Electrophoresis Gel Aiptasiapallida

  13. Experimental Setup • Four temperatures: • 25°C (control) , 27°C, 30°C, 33°C • Length of incubation (hours): • 12, 24, 48, 96, 168 • Six-well plates • Anemones were starved • Artificial seawater, changed every other day • 12 hour light/dark cycle

  14. After Incubation • Freeze anemones • Extract RNA • Purify RNA • DNase RNA • Convert RNA to cDNA • cDNA library for use in analysis • PCR as initial test • 24 and 168 hour time points Symbiotic and bleached Aiptasiapallida

  15. PCR actin acasp caspase 8 ATG 7 ATG 8 ATG 12 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 24 hr 200bp 75bp 200bp 75bp 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33° 168 hr 200bp 75bp 200bp 75bp

  16. Further Applications • Continue RNA extractions and cDNA synthesis • Perform quantitative PCR • Determine time points for ATG8 western blot analysis and localization assays

  17. Summary • Coral bleaching occurs when zooxanthellae are lost from the host • My focus is on apoptosis and autophagy as mechanisms for coral bleaching • The sequences of Caspase 8, ATG7, ATG8, and ATG12 were identified • There are visual differences in the expression of the genes at different time points and temperatures • Future work will begin with qPCR Aiptasiapallida

  18. Acknowledgements • Dr. Virginia Weis • Dr. Camille Paxton • The rest of the Weis Lab • Angela Poole, Sheila Kitchen, Jamie Jo McGraw, and Ben Haslam • Bayne, Chappell, Pringle and Taylor Lab • CGRB • HHMI Program • Dr. Kevin Ahern Aiptasiapallida

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