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)

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

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

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

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

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

* Septin Protein Family

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

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

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

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

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

Taq DNA Polymerase

Pg. 202 DNA Synthesis Primer

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

CDC10 Gene Sequence (non-template strand sequence)

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

Three Cycles of PCR Pg. 349

Agarose Gelidium comeum (kelp)

Ethidium Bromide

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

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

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

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

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

Ori Amp R pGFP Plasmid HindIII

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

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

Transformation of E. Coli plasmid

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

Ori Amp R pGFP Plasmid HindIII

Ampicillin Inhibits cell wall synthesis

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

Transformation of E. Coli plasmid Log Phase Growth Cold (4 o C) CaCl 2