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

3. Principle of Fluorescence
Cell with
fluorescent molecule

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 Emission (e.g. 490 nm) Wavelength

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

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

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

11. Light Microscope

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

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

15. *

16. Model for “Septin Ring” Formation

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

18. Project Overview Purification of Genomic DNA from yeast
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

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

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

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

24. CDC10 Gene Primers CDC10-For CDC10-Rev
5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’
CDC10-Rev
5’ – GTGGTGAAGCTTCTAGCAGCAGCAGTACCTGT – 3’

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

26. Three Cycles of PCR
Pg. 349

27. Three Cycles of PCR
Pg. 349

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

29. CDC10 Gene
Sequence

30. Ethidium Bromide

31. Lab #1 Purify genomic DNA PCR Copies of CDC10 Gene Open Reading Frame
15 million bp
~ 6500 genes
PCR
Copies of
CDC10 Gene
Open Reading Frame
Pg. 350

32. +
Wells
2000 bp
500 bp

33. +
Wells
2000 bp
500 bp

34. Restriction Endonuclease Reaction5’ 3’ 3’ 5’
HindIII (37o C.) 5’ 3’

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

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

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

38. CDC10 ORF DNA from PCR 5’ GTGGTGAAGCTTATGTCCATCGAAGAA
CACCACTTCGAATACAGGTAGCTTCTT
ACTGCTGCTGCTAGAAAGCTTCACCAC
TGACGACGACGATCTTTCGAAGTGGTG 5’ 3’

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

40. Ori
AmpR
pGFP Plasmid
HindIII

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

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

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

44. Transformation of E. Coli
plasmid

45. DNA Cloning Pg. 344 Bacterial Transformation (Lab #4) Plasmid
pCDC10-GFP
Plasmid
Purification (Lab #5)
(AmpR)
(Ampicillin sensitive)
(LB growth medium
with ampicillin)
Pg. 344

46. DNA Cloning Pg. 344 Bacterial Transformation (Lab #4) Plasmid
pCDC10-GFP
Plasmid
Purification (Lab #5)
(AmpR)
(Ampicillin sensitive)
(LB growth medium
with ampicillin)
Pg. 344

47. DNA Cloning Pg. 344 Plasmid Purification (Lab #5) (AmpR)
(LB growth medium
with ampicillin)
Pg. 344