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The GFP revolution!

The GFP revolution!

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The GFP revolution!

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  1. The GFP revolution!

  2. Green beings….

  3. Fluorescence: A property of certain molecules that absorb light of a specific wavelength, then emit at another (longer) wavelength.

  4. Fluorescent variants of GFP….

  5. Plasmodesmata: Communication channels in plants

  6. Viral movement protein trafficking.

  7. pSUC2 expression in root apex. pSUC2::GFP.ER pSUC2::GFP

  8. pAtML1-GFP~GUS

  9. pAtML1-GFP~KN1 pAtML1-GFP~MP L1 specific expression.

  10. Developing a Strategy to Determine the Subcellular Localization of Unknown Genes in Arabidopsis Charles Kopec Amitabh Mohanty

  11. Predicted Gene Function in Arabidopsis thaliana

  12. Goal • Determine the subcellular, tissue, and developmental localization of ~4000 genes of unknown function.

  13. YFP Fusion Constructs • Internal YFP fusion • 10 aa from C-terminus • Flanked by 10x Ala and Gly linkers • Designed to: • Not disrupt protein folding • Not block C-terminal localization signals • Contain flanking intergenic regions FseI SfiI 4kb Upstream Promoter 1.5 kb Exons 10x Glycine linker YFP 10x Alanine linkers

  14. P3 P1 P4 P2 PCR: Triple Template Method • Does not require restriction enzymes to insert the YFP cassette. • No cloning before bombardment. • Design primers • P1 upstream primer • P2 points up, contains 5’ portion of YFP cassette • P3 points down, contains 3’ portion of YFP cassette • P4 downstream primer

  15. P1 / P2 YFP P3 / P4 P1 / P2 YFP P3 / P4 First round of PCR. P1 / P2 P3 / P4 YFP Fse1 only Sfi1 only Double Undigested

  16. P1 / P2 YFP P3 / P4 P1 P4 PCR: Triple Template Method • Second Amplification: Overlap

  17. At2g215240 1 kb ladder 1 kb ladder At2g222170 At2g16530 At2g20830 At2g27080 kb 12 7 6 5 4 3 2 1.5 TTPCR Amplification TTPCR for TIP Unknown genes 1OO bp Ladder P1-P2 PCR kb 10 8 6 5 4 1 Kb Ladder P2-P3 PCR kb TTPCR YFP 10 8 6 5 4 3 2 1.5 1 0.5 TTPCR of reference protein genes and some of the putative/hypothetical protein genes. TIP, tonoplast intrinsic protein.

  18. attL1 & L2 attL1 and P2 attL2 and P3 Gy2 (GSR_YFP) VLY4 (VILLIN_YFP) VLY4 (VILLIN_YFP) VLC3 (VILLIN_CFP) VY4 (VIP_YFP) VC1 (VIP_CFP) VLC3 (VILLIN_CFP) GC3 (GSR_CFP) VY4 (VIP_YFP) TC5 (TIP_CFP) VC1 (VIP_CFP) Gy2 (GSR_YFP) GC3 (GSR_CFP) TC5 (TIP_CFP) TY2 (TIP_YFP) 1 kb Ladder TY2 (TIP_YFP) pDONOR207 Gene P1-P2 size GSR ~5.5 kb VIP1 ~3.9 kb TIP ~3.7 kb VILLIN ~5.0 kb Clone confirmation by PCR for verification of entry clones, colony PCR was performed with primers from the vector (attL1 or L2) and either P2 or P3 primer from the gene specific part of the reference protein genes.

  19. CFP/Citrine YFP Att B Att B + (pDONOR 207) BP Clonase + Insertion of PCR products into Gateway entry vector. The recombination reaction is catalyzed by BP clonase TM Transfer of the fragments into binary vectors Schematic representation of the LR recombination reaction. Transfer of entry clone to destination vector is catalyzed by an invitro recombination reaction by Gateway LR clonaseTM.

  20. Bombardment • Coat 1mm gold particles with constructs. • Bombard into Arabidopsis leaves and onion scales. • Determine subcellular localization using fluorescence dissecting, compound, and confocal microscopy.

  21. VIP1 bombarded into onion.

  22. 3707 Bombarded Arabidopsis

  23. Reference Genes Gene Number Gene Name Subcellular Location Assigned to Lab Primers 1 SYP41 Trans-Golgi    NR(Chary) 2 SYP42 Trans-Golgi    NR(Chary)    Primers for SYP42 3 Mannosidase Golgi stacks    VC(Gouwei) 4 SYP21 Prevacuol    NR(Chary)    Primers for SYP21 5 Delta TIP Vacuol Membrane   DJ(Amitabh) 6 SEC12 ER    NR(Chary)    Primers for Sec12 7 SYP51 Late Secretory System   NR(Chary)    Primers for SYP51 8 SYP61 Late Secretory System   NR(Chary)    Primers for SYP61 9 MAF1 Nuclear Membrane   DJ(Amitabh) 10 VIP1 Intranuclear    DJ(Amitabh) 11 Toc75 Chloroplast    VC(Brigitte) 12 AOX2 Mitochondria    VC(Brigitte) 13 PIP2A Plasmamembrane    NR(Chary)    Primers for PIP2A 14 PME Cell Wall    VC(Gouwei) 15 PRP2 Cell Wall    NR(Chary)    Primers for PRP2 16 TMV MP Plasmodesmata    VC(Brigitte) 17 TVCV MP Plasmodrsmata    DJ(Amitabh) 18 VLN Microfilament arrays   DJ(Amitabh) 19 Fim1 Microfilament arrays   VC(Gouwei) 20 TUA6 Microtubule arrays   VC(Brigitte) 21 MFP2 Peroxisome    VC(Guowei) 22 PEX5 Peroxisome    VC(Guowei) 23 GSR1 Cytosol    NR(Chary) 24 GSK B Cytosol    VC(Brigitte) 25 AtELP Prevacuol    NR(Chary)    Primers for AtELP ------------------------------------------------------------------------general comments or questions to: curator@arabidopsis.org comments or questions about seed stocks to: seedstock@arabidopsis.org comments or questions about DNA stocks to: dnastock@arabidopsis.org

  24. Collaborators: Andy Maule Rick Nelson Wes Bruce Mike Muszynski Hajime Sakai Ed Buckler Sarah Hake Bob Schmidt Volker Brendel Toby Kellogg Rob Martienssen Vitaly Citovsky Natasha Raikhel Sue Rhee David Erhardt Jim Haseloff Kathy Barton Alain Joliot Dave Jackson Jae Yean Kim Michelle Cilia Namiko Satoh Jing Wang Wim Deleu Zhuang Yuan Anna Giulini Fumio Shiobara Funding: NSF, USDA, CSHL