Question:

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

Light Microscope

Question:

Where is the Cdc10 protein located in a yeast cell?

Saccharomyces cerevisiae (Yeast)

Eukaryotic cell15 million bp DNA~ 6000 genesComplete genome sequence known!

Model for “Septin Ring” Formation


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


mRNA



Transcription

Translation

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

CDC10-For

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Rev

5’ – GTGGTGAAGCTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

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


Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

CDC10 GeneSequence

Ethidium Bromide


Purify genomic DNA

~ 6500 genes

Lab #1

15 million bp

PCR

2000 bp

500 bp

Wells

+

2000 bp500 bp

Wells

+

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’

CDC10 ORF DNA from PCR

GTGGTGAAGCTTATGTCCATCGAAGAACACCACTTCGAATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAAAGCTTCACCACTGACGACGACGATCTTTCGAAGTGGTG

5’3’ 5’

3’

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 ORF DNA from PCR

HindIII

Ori

AmpR

pGFP Plasmid

HindIII

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

Ori

AmpR

pGFP Plasmid

HindIII

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 orf

Pg. 344

Construction of a Recombinant DNA Plasmid

(insert)

Transformation of E. Coli

plasmid


plasmid

ColdCaCl2

Pg. 344

(Ampicillin sensitive)

(AmpR)

(LB growth medium with ampicillin)

DNA Cloning

pCDC10-GFP

PlasmidPurification (Lab #5)

Bacterial Transformation(Lab #4)

Pg. 344

(AmpR)


DNA Cloning


Pg. 344

(Ampicillin sensitive)

(AmpR)

DNA Cloning

pCDC10-GFP

(LB-amp Plate)

(LB-amp)

E. coli

Cell Wall CellMembrane Cytoplasm

(chromosome, plasmids)

Restriction Endonuclease Reaction

HindIII (37o C.)

5’

5’

3’

3’

5’

5’3’

3’3’

3’5’

5’

ACGGGGAATTCACGCGGAGAATTCAATGGGAATCGTGGATGCCCCTTAAGTGCGCCTCTTAAGTTACCCTTAGCACCT

ACGGGGTGCCCCTTAA

AATTCACGCGGAG GTGCGCCTCTTAA

AATTCAATGGGAATCGTGGA GTTACCCTTAGCACCT

5’

5’

5’ 5’

5’

5’

5’

5’

3’

3’

3’

3’

3’

3’

3’

3’

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’

Pg. 344

(AmpR)


DNA Cloning


Pg. 344

(AmpR)


DNA Cloning


pCDCGFP orpGFP?

Ori

AmpR

pCDCGFP Plasmid

CDC10GFP

HindIII HindIII

Biol 273 Morning Section Lab #7 Results

Biol 273 Afternoon Section Lab #7 Results

pCDCGFP

Yeast Cells

ObserveCdc10-GFPLocalization

(Lab #7) (Lab #8)

GFP Tagging of Cdc10

mRNA



Transcription

Translation

pCDCGFP

Yeast Cells

ObserveCdc10-GFPLocalization

(Lab #7) (Lab #8)


plasmid

Transformation of Yeast

Linear plasmid

Ori

AmpR

pCDCGFP Plasmid

CDC10GFP

SelectableMarker

“Targeting”sequence

Yeast Chromosome

Chromosome Integration

“Targeting” Locus (RPS10)

Ori

AmpR

pCDCGFP Plasmid

CDC10GFPStuI

URA3 ACT1p-CDC10GFP

Linear pCDCGFP Plasmid

RPS10 RPS10

Yeast Chromosome


URA3 ACT1p-CDC10GFPLinear pCDCGFPPlasmid

RPS10

Yeast Chromosome with CDC10-GFP and URA3 Genes!


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

Yeast Transformation Plate

Lab #7

URA3 Transformants

Cells with theCDC10-GFP “fusion” Gene

ACT1p CDC10GFP

mRNA (CDC10-GFP orf)

Transcription/RNA Processing (nucleus)

Translation (cytosol)

Cdc10-GFP Protein

G AAAAAA

Expression of CDC10-GFP “Fusion” Gene

Integrated inYeast Chromosome

Localization of Cdc10-GFP in the Cell

Septin Proteins of Yeast

*

Lipid BilayerBinding Domain

Model for Septin Polymerization

Yeast Cells

Yeast Mitotic Cell Cycle

~ 3 Hours

G2

Septin Dynamics in theYeast Mitotic Cell Cycle

Septins marksite of budformation

Septin ring atthe mother/bud neck

Septin ringsplits into two

SeptinsDe-polymerize

G2

Light Microscope

(10X)

(10-100X)

ExcitationWavelength(e.g. 490 nm)

EmissionWavelength(e.g. 510 nm)

GFP

Question:

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