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

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

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

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 mRNA DNA (CDC10 -GFP “Fusion”) Cdc10-GFP “Fusion” Transcription Translation

18. 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

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

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

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

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

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

26. Three Cycles of PCR Pg. 349

27. Three Cycles of PCR Pg. 349

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

29. CDC10 Gene Sequence

30. Ethidium Bromide

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

32. 2000 bp 500 bp Wells +

33. 2000 bp 500 bp Wells +

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

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

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

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

38. GTGGTG AAGCTT ATGTCCATCGAAGAA CACCAC TTCGAA TACAGGTAGCTTCTT ACTGCTGCTGCTAGA AAGCTT CACCAC TGACGACGACGATCT TTCGAA GTGGTG 5’ 3’ 5’ 3’ CDC10 ORF DNA from PCR

39. GTGGTG AAGCTT ATGTCCATCGAAGAA CACCAC TTCGAA TACAGGTAGCTTCTT ACTGCTGCTGCTAGA AAGCTT CACCAC TGACGACGACGATCT TTCGAA GTGGTG 5’ 3’ 5’ 3’ AGCTT ATGTCCATCGAAGAA A TACAGGTAGCTTCTT ACTGCTGCTGCTAGA A TGACGACGACGATCT TTCGA 5’ 3’ 5’ 3’ CDC10 ORF DNA from PCR HindIII

40. Ori Amp R pGFP Plasmid HindIII

41. ACT GCT GCT GCT AGA AAG CTT ATG TCT AAA GGT HindIII Site - Thr - Ala - Ala - Ala - Arg - Lys - Leu - Met - Ser - Lys - Gly - Cdc10 GFP 5’3’ pCDC10-GFP Plasmid CDC10 orfGFP orfACT1p HindIII

42. Ori Amp R pGFP Plasmid HindIII AGCTT ATGTCCATCGAAGAA A TACAGGTAGCTTCTT ACTGCTGCTGCTAGA A TGACGACGACGATCT TTCGA 5’ 3’ 5’ 3’ CDC10 orf

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

44. Transformation of E. Coli plasmid

45. Transformation of E. Coli plasmid Cold CaCl 2

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

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

48. Pg. 344 (Amp R ) (LB growth medium with ampicillin) DNA Cloning Plasmid Purification (Lab #5)

49. Pg. 344 (Ampicillin sensitive) (Amp R ) DNA Cloning pCDC10-GFP (LB-amp Plate) (LB-amp)

50. E. coli Cell Wall Cell Membrane Cytoplasm (chromosome, plasmids)

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

52. ACGGGGAATTCACGCGGAGAATTCAATGGGAATCGTGGA TGCCCCTTAAGTGCGCCTCTTAAGTTACCCTTAGCACCT ACGGGG TGCCCCTTAA AATTCACGCGGAG GTGCGCCTCTTAA AATTCAATGGGAATCGTGGA GTTACCCTTAGCACCT 5’ 3’

53. Ligation Reaction “Compatible” ends DNA Ligase + ATP (15 o C.) HindIII recognition site is reconstituted 5’ 3’ 5’ 3’ 5’

54. Pg. 344 (Amp R ) (LB growth medium with ampicillin) DNA Cloning Plasmid Purification (Lab #5)

55. Pg. 344 (Amp R ) (LB growth medium with ampicillin) DNA Cloning Plasmid Purification (Lab #5) pCDCGFP or pGFP?

56. Ori Amp R pCDCGFP Plasmid CDC10GFP HindIII

57. Biol 273 Morning Section Lab #7 Results

58. Biol 273 Afternoon Section Lab #7 Results

59. pCDCGFP Yeast Cells Observe Cdc10-GFP Localization (Lab #7) (Lab #8)

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

61. pCDCGFP Yeast Cells Observe Cdc10-GFP Localization (Lab #7) (Lab #8)

62. Transformation of E. Coli plasmid

63. Transformation of Yeast Linear plasmid

64. Ori Amp R pCDCGFP Plasmid CDC10GFP Selectable Marker “Targeting” sequence

65. Yeast Chromosome Chromosome Integration “Targeting” Locus (RPS10)

66. Ori Amp R pCDCGFP Plasmid CDC10GFP StuI

67. URA3 ACT1p-CDC10GFP Linear pCDCGFP Plasmid RPS10

68. Yeast Chromosome Chromosome Integration URA3 ACT1p-CDC10GFP Linear pCDCGFP Plasmid RPS10

69. Yeast Chromosome with CDC10-GFP and URA3 Genes! Chromosome Integration URA3 ACT1p-CDC10GFP

70. Uridine Monophosphate Orotidine Monophosphate URA3 Gene Encodes Orotidine Decarboxylase (RNA synthesis) Orotidine Decarboxylase

71. ura3 mutant can NOT make Uridine Monophosphate Orotidine Monophosphate Yeast ura3 Mutant Orotidine Decarboxylase

72. Yeast Transformation Plate Lab #7 URA3 Transformants Cells with the CDC10-GFP “fusion” Gene

73. ACT1p CDC10GFP mRNA (CDC10-GFP orf) Transcription/RNA Processing (nucleus) Translation (cytosol) Cdc10-GFP Protein G AAAAAA Expression of CDC10-GFP “Fusion” Gene Integrated in Yeast Chromosome Localization of Cdc10-GFP in the Cell

74. Septin Proteins of Yeast * Lipid Bilayer Binding Domain

75. Model for Septin Polymerization

76. Yeast Cells

77. Yeast Mitotic Cell Cycle ~ 3 Hours G2G2

78. Septin Dynamics in the Yeast Mitotic Cell Cycle Septins mark site of bud formation Septin ring at the mother/bud neck Septin ring splits into two Septins De-polymerize G2G2

79. Light Microscope (10X) (10-100X)

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