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

Principle of Fluorescence Cell with fluorescent molecule

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

Aequorea victoria

Green Fluorescent Protein (GFP)

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

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

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

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

Light Microscope

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

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

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Model for “Septin Ring” Formation

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

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

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

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

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

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

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

Three Cycles of PCR Pg. 349

Three Cycles of PCR Pg. 349

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

CDC10 Gene Sequence

Ethidium Bromide

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

+ Wells 2000 bp 500 bp

+ Wells 2000 bp 500 bp

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

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

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

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

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

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

Ori AmpR pGFP Plasmid HindIII

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

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

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

Transformation of E. Coli plasmid

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

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

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