Question:

2. Question: How do we know where a particular protein is located in the cell?

3. Cell with fluorescent molecule Principle of Fluorescence

4. Experimental Approaches for Protein Localization 1. Small Molecule Dyes (e.g. DAPI) 2. Immunostaining (dye-conjugated antibodies) 3. Green Fluorescent Protein (GFP) “Tagging”

5. Aequorea victoria

6. Green Fluorescent Protein (GFP)

7. GFP Excitation Wavelength (e.g. 490 nm) Emission Wavelength (e.g. 510 nm)

8. Gene Expression DNA (Gene X) Transcription mRNA Translation Protein X

9. Transcription Translation GFP Tagging Approach DNA (Gene X -GFP “Fusion”) mRNA Protein X-GFP “Fusion”

10. GFP Tagging Experiments Nuclei Mitotic Spindle Tubulin-GFP Histone-GFP

11. Question: Where is the Cdc10 protein located in a yeast cell?

12. Septin Protein Family *

13. Transcription Translation GFP Tagging Approach DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”

14. Project Overview Isolation of CDC10 gene Open Reading Frame Purification of Genomic DNA from yeast Polymerase Chain Reaction (PCR) Construction of CDC10-GFP “fusion” gene Restriction endonuclease/Ligase Cloning DNA in E. coli Introduction of CDC10-GFP “fusion” gene into yeast cells Observe Cdc10 protein localization in living cells with fluorescence microscopy

15. Transcription Translation GFP Tagging of Cdc10 DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”

16. Saccharomyces cerevisiae (Yeast) Eukaryotic cell 15 million bp DNA ~ 6000 genes Complete genome sequence known!

17. Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350

18. Taq DNA Polymerase

19. Primer DNA Synthesis Pg. 202

20. CDC10 Gene Primers CDC10-Forward 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Reverse 5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

21. CDC10 Gene Sequence (non-template strand sequence)

22. First Cycle of PCR 5’ 3’ Rev CDC10 3’ 5’ 5’ 3’ (52o C.) (94o C.) (72o C.) For 5’ 3’ Pg. 349

23. Three Cycles of PCR Pg. 349

25. Agarose Gelidium comeum (kelp)

26. Ethidium Bromide

27. +

28. +

29. 3’ 3’ 5’ 5’ 5’ 3’ 5’ 3’ Restriction Endonuclease Reaction 3’ 5’ 3’ 5’ HindIII (37o C.)

30. DNA Ligase + ATP (15o C.) 3’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ HindIII recognition site is reconstituted Ligation Reaction “Compatible” ends 1. Annealing 2. Phosphodiester bond formation

31. Construction of a Recombinant DNA Plasmid (insert) Pg. 344

32. CDC10 Gene Primers CDC10-For 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Rev 5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

33. GTGGTGAAGCTTATGTCCATCGAAGAA CACCACTTCGAATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAAAGCTTCACCAC TGACGACGACGATCTTTCGAAGTGGTG 3’ 3’ 5’ AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ CDC10 ORF DNA from PCR HindIII

34. Ori AmpR pGFP Plasmid HindIII

35. Ori AmpR pGFP Plasmid AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ HindIII CDC10 orf

36. ACT1p CDC10 orf GFP orf HindIII HindIII - Thr - Ala - Ala - Ala - Arg - Lys - Leu - Met - Ser - Lys - Gly - Cdc10 GFP pCDC10-GFP Plasmid HindIII Site ACT GCT GCT GCT AGA AAG CTTATG TCT AAA GGT 5’ 3’

37. Transformation of E. Coli plasmid

38. DNA Cloning Bacterial Transformation (Lab #5) pCDC10-GFP Plasmid Purification (Lab #6) (AmpR) (Ampicillin sensitive) (LB growth medium with ampicillin) Pg. 344

39. Ori AmpR pGFP Plasmid HindIII

40. Ampicillin Inhibits cell wall synthesis

41. DNA Cloning pCDC10-GFP (LB-amp) (AmpR) (Ampicillin sensitive) (LB-amp Plate) Pg. 344

42. Transformation of E. Coli Log Phase Growth Cold (4oC) CaCl2 plasmid