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)

ExcitationWavelength(e.g. 490 nm)

EmissionWavelength(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 OverviewIsolation of CDC10 gene Open Reading Frame

Purification of Genomic DNA from yeastPolymerase Chain Reaction (PCR)

Construction of CDC10-GFP “fusion” gene

Restriction endonuclease/LigaseCloning 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 cell15 million bp DNA~ 6000 genesComplete genome sequence known!

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

Taq DNAPolymerase

Pg. 202 DNA Synthesis

Primer

CDC10-Forward

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Reverse

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

CDC10 GeneSequence

(non-template strand sequence)

First Cycle of PCR

Pg. 349

(94o C.) (52o C.)(72o C.)

CDC10

For

Rev

5’5’ 3’3’

3’

3’ 5’

5’

Three Cycles of PCR

Pg. 349

Three Cycles of PCR

Pg. 349

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

Agarose

Gelidium comeum (kelp)

Ethidium Bromide

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6500 genes

Lab #1

15 million bp

PCR

+

+

Restriction Endonuclease Reaction

HindIII (37o C.)

5’

5’

3’

3’

5’

5’3’

3’3’

3’5’

5’

Ligation Reaction

“Compatible” ends

DNA Ligase + ATP (15o C.)

HindIII recognition site is reconstituted

5’

5’3’

3’3’

3’5’

5’

3’

3’

5’

5’

1. Annealing

2. Phosphodiesterbond formation

Pg. 344

Construction of a Recombinant DNA Plasmid

(insert)

CDC10-For

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Rev

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

GTGGTGAAGCTTATGTCCATCGAAGAACACCACTTCGAATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAAAGCTTCACCACTGACGACGACGATCTTTCGAAGTGGTG

5’3’ 5’

3’

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 ORF DNA from PCR

HindIII

Ori

AmpR

pGFP Plasmid

HindIII

Ori

AmpR

pGFP Plasmid

HindIII

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 orf

ACT GCT GCT GCT AGA AAG CTT ATG TCT AAA GGTHindIII Site

- Thr - Ala - Ala - Ala - Arg - Lys - Leu - Met - Ser - Lys - Gly -

Cdc10 GFP

5’ 3’

pCDC10-GFP Plasmid

CDC10 orf GFP orfACT1pHindIII HindIII

Pg. 344

(Ampicillin sensitive)

(AmpR)

(LB growth medium with ampicillin)

DNA Cloning

pCDC10-GFP

PlasmidPurification (Lab #5)

Bacterial Transformation(Lab #5)

Pg. 344

(AmpR)

(LB growth medium with ampicillin)

DNA Cloning

PlasmidPurification (Lab #6)

Ori

AmpR

pGFP Plasmid

HindIII

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 orf

Transformation of E. Coli

plasmid

Ori

AmpR

pGFP Plasmid

HindIII

Pg. 344

(Ampicillin sensitive)

(AmpR)

DNA Cloning

pCDC10-GFP

(LB-amp Plate)

(LB-amp)

Transformation of E. Coli

plasmid

Log Phase GrowthCold (4oC)CaCl2

E. Coli

Cell Wall CellMembrane Cytoplasm

(chromosome, plasmids)

Ori

AmpR

pCDCGFP Plasmid

CDC10GFP

HindIII HindIII

1000 bp

Morning section

1000 bp

Afternoon section

Transformation of Yeast

Linear plasmid

Ori

AmpR

pCDC10GFP Plasmid

CDC10GFP

SelectableMarker

“Targeting”sequence

Yeast Chromosome

Chromosome Integration

“Targeting” Locus (RPS10)

Ori

AmpR

pCDC10GFP Plasmid

CDC10GFPStuI

URA3 ACT1p-CDC10GFP

Linear pCDC10GFP Plasmid

RPS10 RPS10

Yeast Chromosome

Chromosome Integration

URA3 ACT1p-CDC10GFPLinear pCDCGFPPlasmid

RPS10

Yeast Chromosome with CDC10-GFP and URA3 Genes!

Chromosome Integration

URA3 ACT1p-CDC10GFP

Uridine MonophosphateOrotidine Monophosphate

URA3 Gene EncodesOrotidine Decarboxylase

(RNA synthesis)

Orotidine Decarboxylase

ura3 mutant can NOT makeUridine Monophosphate

Orotidine Monophosphate

Yeast ura3 Mutant

Orotidine Decarboxylase

Question:

Where is the Cdc10 protein located in a yeast cell?

Yeast Transformation Plate

Lab #9

URA3 Transformants

Cells with theCDC10GFP “fusion” Gene

ACT1p CDC10GFP

mRNA (CDC10-GFP orf)

Transcription/RNA Processing (nucleus)

Translation (cytosol)

Cdc10-GFP Protein

G AAAAAA

Expression of CDC10GFP “Fusion” Gene

Integrated inYeast Chromosome

Observe Localization of Cdc10-GFP in Yeast Cells

Pg. 10

Light Microscope

(10X)

(10-100X)

ExcitationWavelength(e.g. 490 nm)

EmissionWavelength(e.g. 510 nm)

GFP

490 nm 510 nm

Nikon TE200 Inverted Microscope

Septin Proteins of Yeast

*

Lipid BilayerBinding Domain http://en.wikipedia.org/wiki/Septins

TEM

Yeast Cells

Yeast Mitotic Cell Cycle

~ 3 Hours

G2

Cdc10 Protein in theYeast Mitotic Cell Cycle

Cdc10 markssite of budformation

Cdc10 ring atthe mother/bud neck

Cdc10 ringsplits into two

Cdc10De-polymerizes

G2