Structure-Function Analysis of Drosophila Notch Using Genomic Rescue Transgenes

  • Jessica Leonardi
  • Hamed Jafar-NejadEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)


One of the evolutionarily conserved posttranslational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a specific consensus sequence by the protein O-glucosyltransferase Rumi (POGLUT1 in human). Loss of rumi in flies results in a temperature-sensitive loss of Notch signaling. To demonstrate that the Notch receptor itself is the biologically relevant target of Rumi in flies, and to determine the role of the 18 Rumi target sites on Notch in regulating Notch signaling, we have performed an in vivo structure-function analysis of Drosophila Notch. In this chapter, we provide a detailed protocol for this analysis. To avoid the potential artifacts associated with overexpression of Notch and random insertion of transgenes, we have used recombineering and site-specific integration technologies, which have been adapted for usage in Drosophila in recent years. Using gene synthesis and site-directed mutagenesis, we generated a series of Notch genomic transgenes which harbor mutations in all or specific subsets of Notch O-glucose sites. Gene dosage and rescue experiments in animals raised at various temperatures allowed us to dissect the contribution of O-glucosylation sites to the regulation of the Notch signaling strength. The reagents and methods presented here can be used to address similar questions about other posttranslational modifications of Notch or other Drosophila proteins.

Key words

Notch Drosophila Recombineering Site-specific integration Glycosylation Genomic transgene 



This work was supported by the NIH grant R01GM084135. The gap-repair mutagenesis method was developed by Dr. Rodrigo Fernandez-Valdivia, a former postdoctoral fellow in our group. We thank Dr. Graeme Mardon and Dr. Barbara Jusiak for generously providing the CAT/SacB-pCR-Blunt II-TOPO-Km R construct. We thank Dr. Karen Schulze for critical reading of this chapter.


  1. 1.
    Fortini ME (2009) Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 16:633–647PubMedCrossRefGoogle Scholar
  2. 2.
    Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689PubMedCrossRefGoogle Scholar
  4. 4.
    Rubin GM, Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353PubMedCrossRefGoogle Scholar
  5. 5.
    Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415PubMedGoogle Scholar
  6. 6.
    Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72:202–230PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Lei L, Xu A, Panin VM et al (2003) An O-fucose site in the ligand binding domain inhibits Notch activation. Development 130:6411–6421PubMedGoogle Scholar
  8. 8.
    Mohr OL (1919) Character changes caused by mutation of an entire region of a chromosome in Drosophila. Genetics 4:275–282PubMedCentralPubMedGoogle Scholar
  9. 9.
    Lyman D, Young MW (1993) Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc Natl Acad Sci U S A 90:10395–10399PubMedCentralPubMedGoogle Scholar
  10. 10.
    Ramos RG, Grimwade BG, Wharton KA et al (1989) Physical and functional definition of the Drosophila Notch locus by P element transformation. Genetics 123:337–348PubMedCentralPubMedGoogle Scholar
  11. 11.
    Venken KJ, He Y, Hoskins RA et al (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751PubMedGoogle Scholar
  12. 12.
    Bischof J, Maeda RK, Hediger M et al (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104:3312–3317PubMedCentralPubMedGoogle Scholar
  13. 13.
    Groth AC, Fish M, Nusse R et al (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166:1775–1782PubMedCentralPubMedGoogle Scholar
  14. 14.
    Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769–779PubMedGoogle Scholar
  15. 15.
    Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95:5505–5510PubMedCentralPubMedGoogle Scholar
  16. 16.
    Venken KJ, Popodi E, Holtzman SL et al (2010) A molecularly defined duplication set for the X chromosome of Drosophila melanogaster. Genetics 186:1111–1125PubMedCentralPubMedGoogle Scholar
  17. 17.
    Hase S, Kawabata S, Nishimura H et al (1988) A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX. J Biochem 104:867–868PubMedGoogle Scholar
  18. 18.
    Moloney DJ, Shair LH, Lu FM et al (2000) Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275:9604–9611PubMedGoogle Scholar
  19. 19.
    Acar M, Jafar-Nejad H, Takeuchi H et al (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132:247–258PubMedCentralPubMedGoogle Scholar
  20. 20.
    Leonardi J, Fernandez-Valdivia R, Li YD et al (2011) Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling. Development 138:3569–3578PubMedCentralPubMedGoogle Scholar
  21. 21.
    Lee TV, Sethi MK, Leonardi J et al (2013) Negative regulation of Notch signaling by xylose. PLoS Genet 9:e1003547PubMedCentralPubMedGoogle Scholar
  22. 22.
    Harris RJ, Spellman MW (1993) O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3:219–224PubMedGoogle Scholar
  23. 23.
    Rana NA, Nita-Lazar A, Takeuchi H et al (2011) O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1. J Biol Chem 286:31623–31637PubMedCentralPubMedGoogle Scholar
  24. 24.
    Shao L, Luo Y, Moloney DJ et al (2002) O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase. Glycobiology 12:763–770PubMedGoogle Scholar
  25. 25.
    Sethi MK, Buettner FF, Ashikov A et al (2012) Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch. J Biol Chem 287:2739–2748PubMedCentralPubMedGoogle Scholar
  26. 26.
    Sethi MK, Buettner FF, Krylov VB et al (2010) Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285:1582–1586PubMedCentralPubMedGoogle Scholar
  27. 27.
    Warming S, Costantino N, Court DL et al (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36PubMedCentralPubMedGoogle Scholar
  28. 28.
    Hoskins RA, Nelson CR, Berman BP et al (2000) A BAC-based physical map of the major autosomes of Drosophila melanogaster. Science 287:2271–2274PubMedGoogle Scholar
  29. 29.
    Lee EC, Yu D, Martinez De Velasco J et al (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56–65PubMedGoogle Scholar
  30. 30.
    Thomason L, Court DL, Bubunenko M, et al. (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol Chapter 1, Unit 1.16Google Scholar
  31. 31.
    Kelley RL, Meller VH, Gordadze PR et al (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98:513–522PubMedGoogle Scholar
  32. 32.
    Fostier M, Evans DA, Artavanis-Tsakonas S et al (1998) Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling. Genetics 150:1477–1485PubMedCentralPubMedGoogle Scholar
  33. 33.
    De Celis JF, Barrio R, Del Arco A et al (1993) Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila. Proc Natl Acad Sci U S A 90:4037–4041PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Molecular and Human GeneticsBaylor College of MedicineHoustonUSA

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