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Notch interaction with Delta and Serrate

Notch interaction with Delta and Serrate. By Andrea Luber 27 July 2005. Outline. Background of Notch and Delta/Serrate Biological significance Long term goals of project What we have accomplished so far What is yet to come. Notch/Delta interaction diagram. Delta EBD ~20.5 kDa.

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Notch interaction with Delta and Serrate

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  1. Notch interaction with Delta and Serrate By Andrea Luber 27 July 2005

  2. Outline • Background of Notch and Delta/Serrate • Biological significance • Long term goals of project • What we have accomplished so far • What is yet to come

  3. Notch/Delta interaction diagram Delta EBD ~20.5 kDa Serrate EBD ~20.5 kDa Notch ELR 10-13 ~15 kDa Eric C. Lai, Development 131 (5) 2004

  4. Biological Significance • Drosophila • Wing structure • Neurogenesis • Hematopoiesis • Organ development • Muscles • Heart • Intestine • Humans • Neurogenesis • Vasculature development and homeostasis • Organ development • Liver • Kidney • Heart

  5. Wild type Notch Loss of Notch Overexpression of Delta Notch and Delta expression in Drosophila Eric C. Lai Development 131(5) 165-173

  6. CADASIL : Human disease associated with Notch mutation Control CADASIL Anne Joutel Journal of Clinical Investigation 2000 105(5) 597-605

  7. Long term goal • To obtain biophysical data to characterize this interaction • Kinetics and Energetics • SPR • Structure • NMR

  8. Representation of Notch Receptor R.S. Haltiwanger, P. Stanley, Biochimica et Biophysica Acta 1573 (2002) 328–335

  9. Used PCR to amplify Notch EGF segments 10-13

  10. 700 bp Expected length of 463 bp 500 bp 300 bp DNA gel to verify presence of product

  11. Notch construct progress Notch ELR 10 thru 13 ü ü Inserted into vector ü Transformed into bacteria Isolated plasmid ü ü Sequenced and confirmed  Ready for protein expression

  12. 1000 bp 700 bp 500 bp Delta EBD Delta EBD + RE sites Delta EBD 615bp

  13. Delta construct progress Delta EBD from plasmid ü ü Delta EBD from cDNA ü Submitted for sequencing Ready for protein expression 

  14. Notch and Delta: Next step Ready for expression in bacteria Protein isolation and purification NMR and SPR

  15. Summary • Notch EGF segments 10-13 • Amplified • Sequenced • Inserted into vector • Delta EBD segment • Amplified • Sequenced • Extended to include restriction enzyme sites on 5’ and 3’ ends

  16. Aknowledgements • Dr. Tim Logan • Ms. Jacquie Kitchen • Dr. Scott Olenych • REU staff and students • National Science Foundation

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