1. Lecture 17 Chapter 9 Marker genes Neal Stewart

2. Discussion questions 1. Why use marker genes? 2. What are some differences between selectable markers and scorable markers? 3. Discuss the relative merits of GUS and GFP as reporters. Does the profile of experimentation using these reporter genes overlap directly or partially? 4. What are the advantages, if any, for the use of the manA gene over the nptII gene as a selectable marker for food and feed crops, and would the use of the manA gene overcome public concern over the use of the nptII gene? Conversely, what are the disadvantages?

3. Using marker genes helps answers • Are my plants transgenic? • Is the gene expressed? • How is my promoter working? Negative selectable Positive selectable

4. Typically used to recover transgenic plant cells from a sea of non-transgenic cells Antibiotic resistance markers and herbicide resistance markers are most common Can help visualize transient expression Can help visualize if tissue is stably transgenic Useful for cellular and ecological studies Selectable markers Scorable markers (reporter genes)

5. Figure 9.2

6. Figure 9.3 Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed

7. Figure 9.7 Barnase kills tapetum cells (and pollen)—negative non-conditional selection useful to engineer male-sterility

8. Common reporter genes • Beta glururonidase (GUS) uidA protein from Escherichia coli– needs the substrate X-gluc for blue color • Luciferase proteins from bacteria and firefly yields light when substrate luciferin is present. • Green fluorescent protein (GFP) from jellyfish is an example of an autofluorescent protein that changes color when excited by certain wavelengths of light.

9. Figure 9.4 GUS positive plants and cells

10. Figure 9.8

11. Figure 9.9 Firefly luciferase produced in tobacco Brought to you by biotechnologist of the day David Ow—was on the cover of Science

12. 35S:GFP canola White light UV light in a darkened room

14. Pollen-tagged GFP—segregating 1:1

15. GFP-tagged pollen on a bee leg. Hudson et al 2001 Mol Ecol Notes 1:321

16. Green (and other color) fluorescent proteins • FP properties • Detection and measurement • Anthozoan FPs • Why red is better than green • Why orange is best of all!

17. http://www.youtube.com/watch?v=90wpvSp4l_0&feature=related

18. What is fluorescence? Emission 507 nm Excitation 475 nm Stokes shift* x = Brightness Quantum yield % light fluoresced Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852

19. Horseweed transformation with GFP Blue Light with GFP Filter White Light

20. Blue Light with GFP Filter White Light

21. Transgenic flower cross section Transgenic versus wild-type flowers

23. In planta fluorescence ex = 395 nm Relative fluorescence Wavelength (nm)

24. LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence

25. Journal of Fluorescence 15: 697-705

27. A brief FP history Patterson Nature Biotechnol. (2004) 22: 1524

28. Anthozoan FPs in transgenics Wenck et al Plant Cell Rep 2003 22: 244 Soybean ZsGreen Cotton AmCyan Wheat leaf DsRed Cotton ZsGreen Rice callus ZsGreen Cotton callus AsRed Corn callus AmCyan DsRed tobacco

29. Fluorescence Emission 507 nm Excitation 475 nm Stokes shift* x = Brightness Quantum yield % fluoresced Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852

30. Species and FP name Ex max nm (Ext Coef) Em max nm Reference Zoanthus sp. ZsGreen Aequorea victoria GFP 395 (27) 497 (36) 506 (63) 504 (79) Tsien 1998 Matz et al. 1999 Zoanthus sp. ZsYellow A. victoria GFP S65T 528 (20) 489 (55) 510 (64) 538 (20) Matz et al. 1999 Tsien 1998 A. victoria EGFP Anemonia majano AmCyan 488 (56) 458 (40) 508 (60) 486 (24) Tsien 1998 Matz et al. 1999 Heteractis crispa t-HcRed1 A. victoria GFP “Emerald” 590 (160) 487 (58) 509 (68) 637 (4) Tsien 1998 Fradkov et al. 2002 A. victoria GFPYFP “Topaz” Discosoma sp. DsRed 558 (75) 514 (94) 527 (60) 583 (79) Tsien 1998 Matz et al. 1999 Discosoma sp. mRFP1 A. victoria GFPYFP “Venus” 584 (50) 515 (92) 607 (25) 528 (57) Campbell et al. 2002, Shaner et al. 2004 Nagai et al. 2002 Discosoma sp. dimer2 552 (69) 579 (29) Campbell et al. 2002, Shaner et al. 2004 Discosoma sp. mOrange 548 (71) 562 (69) Shaner et al. 2004 Discosoma sp. dTomato 554 (69) 581 (69) Shaner et al. 2004 Discosoma sp. tdTomato 554 (138) 581 (69) Shaner et al. 2004 Discosoma sp. mStrawberry 574 (90) 596 (29) Discosoma sp. mCherry 587 (72) 610 (22) Shaner et al. 2004 (103 M-1 cm-1)(Quantum yield %)

31. Excitation scan:Nontransgenic leaf fluorescence—why red fluorescence is better than green

32. With GFP Why RFP is better– less fluorescence “noise” in the red

33. More colors in fluorescent proteins discovered (mostly from corals…then improved) http://www.photobiology.info/Zimmer_files/Fig6.png

34. Orange Fluorescent Protein GFP Jennifer Hinds

35. Orange Fluorescent Protein (OFP)

36. An old trick: ER targeting Signal transit peptide 5’ 3’ GFP HDEL Signal peptide directs GFP to endoplasmic reticulum for secretion But HDEL tag sequesters assembled GFP in ER—protected environment allows more accumulation. Haseloff et al 1997 PNAS 94: 2122.

37. ER retention dramatically improves OFP brightness (monomers) Mann et al. submitted 160th paper? 3x brighter!

38. Big Orange Fluorescent Proteins Mann et al. submitted.

39. Red foliage as output Arabidopsis MYB transcription factor PAP1 regulates the expression of anthocyanin biosynthesis genes: overexpression of PAP1 results in a red-plant phenotype