Methods in Molecular Biology

Molecular Biology Bio4751 Spring 2003Gary A. Bulla, PhD

a. Northern

1. Lyse with detergent, 2. Phenol extract (removes proteins)3. Precipitate RNA4. Load onto agarose gel5. Transfer RNA to nylon membrane6. Add radioactive DNA or RNA to detect individual species

Nylon membrane

probed with labeled a1-antitrypsin RNA, then

tubulin DNA

RNA quantitation

b. RNase protection1. Lyse with detergent 2. Phenol extract (removes proteins)3. Precipitate RNA4. Incubate with radioactive antisense RNA 5. Degrade single strand RNA with RNases6. Load on 8% PAGE

AAAAAmRNA

Radioactive antisense RNA

HybridizeRNase treat

1. Heat , PAGE2. Expose to film

RNA quantitation

Primer extension analysis

Prevent reinitiation

TATAA

RNA pol II holoenzymeHistone/DNA ratios

Primer

Also used to identify locations where transcription starts

c. Primer extensionRNA quantitation

mRNA

+dNTPs

Heat, PAGE, probe

Enhancer

Western Transfect C ells

35S amino acid, immunoprecipitate

electrophoresis

Molecular Biology Bio4751 Spring 2003Gary A. Bulla, PhD

Cell extract PAGE (+ SDS)

Rx---

Transfer to nylon membrane

Incubate with anti-Rx

Anti-myc

Alkaline peroxidase

Anti-Anti-

Alkaline peroxidase

Western (protein detection)

Anti-Rx

A. Immunoblot

B. Radiolabeling

Genetically Modified FoodsIncludes frost-resistant tomatoes

Disease-resistant sweet potatoes

Muscle-rich cattle

…..and many others

• Zambia’s government rejected 1000s of tons of corn from US because it may contain some GM kernels

•Approx 2.9 people at risk of starvation from drought-induced famine since 2001

•35,000 will die by 2003 if food not provided (WHO) •GM corn produces a bacterial toxin that is toxic to insects•GM corn used world-wide for 6 years without adverse effects (FDA)

Last month-

How can we detect measure gene activation?

DNA

RNA

Protein

Northern

Western

RNase Protection

How do we examine DNA-protein interactions?

Mad

Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)

DNaseI protection

How do we examine protein-protein interactions?

GST pull-down

EMSA Supershift

Co-immunoprecipitation

Primer extension

Photo-crosslinking

How can we measure promoter activity?

CAT-261 +44

1AT

Answer- Link it to a gene that is easy to monitor

Luciferase

B-galactosidase

(Chloramphenicol acetyl transferase)

CAT1AT

+ HNF1

CAT

-261 +44

Control- TK-CAT

+ + +-- -

1AT

Acetylated

Un-acetylated

CAT assay

Lyse cells, mix with 14C-acetyl CoA, extract and apply to thin layer chromatography

migration

Chloramphenicol

Chloramphenicol

Protein-DNA interactions

EMSA (Gel Shift)

Photo-crosslinking

DNAseI footprinting

fos jun

TATATRE

J=junF=fosM=myc (another bZIP protein)C= bZIP domain only

Fig. 12.31- Fos and jun binding to a TREObserve- • jun or fos cannot bind alone (lanes 1-3)• Jun+ fos does bind (lane 4)• only bZIP domain (C) is required for binding (lanes5, 10 , 11)• another bZIP factor (myc) fails to allow fos or jun to bind (lanes 14-15)

EMSA (Gel Shift assay)- 4% PAGE (non-denaturing)-To detect protein-DNA interactions

- Usually transcription factor binding

Labeled DNA + protein15 min.

PAGE

Unbound DNA

DNA-protein

complexes

TheoryExpose to film

80V, 3hr

Note- complexes migrate according to protein size

Footprinting by DNaseI and Cu++

Observe- •TFIID binds poorly•A + D binds strongly•B doesn’t enhance binding of D+A

Experiment

TFIID, A and/or B added to DNA

treat with DNaseI or Cu++

polyacrylamide gel electrophoresis(PAGE)

DNase I digestion products

32P

DNaseI footprintingFig. 11.4

DNAseI footprinting

Fig. 6.25

Fig. 6.24

• To identify proteins which bind DNAPhotocrosslinking)

Fig. 11.15- TAF 250 and 150 bind promoter DNA

TFIID + P-32 labeled promoter DNA which contains bromodoexyuridine (BrdU)

UV irradiate (causes BrdU to be crosslinked to proteins it contacts)

nuclease

SDS-PAGE

DNA* * * * * * * *

UV, nuclease

**

Photocrosslinking)

Health stupidity reigns supreme

• Magnet therapy

• electronic ab exercisers- called “pump fiction” by Federal Trade Commission -ftc/gov/opa/2002/05/projectabsurd.htm

• Acupuncture-No proven benefit in controlled studies

• Chiropractic medicine- only useful for lower back pain. Period.

To make a lie into a truth-

“Say it loud, say it often” G. Gordon Liddy

All are largely accepted based upon “wart phenomenon”

How can we detect measure gene activation?

DNA

RNA

Protein

Northern

Western

RNase Protection

How do we examine DNA-protein interactions?

Mad

Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)

DNaseI protection

How do we examine protein-protein interactions?

GST pull-down

EMSA Supershift

Co-immunoprecipitation

Primer extension

Photo-crosslinking

Protein-protein interactions

Method- Epitope Tagging Ligate a small peptide onto a protein, introduce that protein into cells, then lyse the cells, and use antibodies raised against the small peptide to bind the protein plus any proteins interacting with that protein.

Gene of interest

ATG TAA

Poly-Adenylation sequence

PromoterFLAG epitope

(7-9 amino acids)

TAA

ATG

Epitope-tagged protein3A, slide 5

How do we determine identify protein-protein interactions?

Figure 10.13

Method- Epitope Tagging

RNA polymerase II structure- yeast has 12 subunits

MadSin3A

HDACFlag

Example- Epitope tagging experiment

Ac

Epitope-tagged histone deacetylase (HDAC2) to generate FLAG-HDAC2

Introduce FLAG-HDAC2 + Mad1 plasmids into cells

Prepare cell extracts

Immunoprecipitate with anti-FLAG Ab

PAGE

Probe with anti- SinA or anti-Mad1

Transfer to membrane

FLAG

HDAc

How do we determine identify protein-protein interactions?

Co-immunoprecipitation

Observe- FLAG alone doesn’t interact with Sin3A or Mad1 (lanes 1-3)HDAC2 interacts with Sin3A (Lane 4)Mad1, but not mutant Mad1pro, interacts with Sin3A (lanes 5 and 6)

Fig. 13.38- Evidence for ternary complex involving HDAC2. Sin3A and Mad1

9E, slide 46

MadSin3A

HDACFlag

AcEpitope tagging experiment results

Anti-FLAG

FLAG

CBP

HNF1 myc

Anti-myc

Alkaline peroxidase

M2 High Sensitivity Capture Assay

96-well format

CMV PolyAFLAG CBP

CMV PolyAC-myc HNF1Co-transfect Cos7 cells

Another clever assay for protein-protein interaction

Assay- 1. separate nuclear protein on SDS-PAGEimpregnated with histones2. incubate gel with tritium -labeled AcCoA, wash away

Fig. 13.33 Activity gel assay for HAT activity

H4

Ac

AcAc

AcH3

H2BH2A

**

**

How do we determine whether a protein is a histone acetyltransferase (HAT)?

All nuclear proteins

How do we identify methylated DNA?

Methylation analysis: The results of MspI and HpaII cleavage are compared by Southern analysis

CCGG CCmGG CCGG

DNA probe

Msp

I

Hpa

II

•Digest genomic DNA with enzyme pair •Load onto agarose gel•Southern transfer•probe with 32-P DNA

Inactive Active

DNAseI DNAseI

Remove proteins Remove proteins

Cut with restriction enzyme

6kb 4kb 5kb 3kb

6kb4kb

5kb3kb

How does one find “open” vs “Closed” DNADNase sensitivity assay

Fig. 13.31 Dnase I hypersensitivity of an active gene

Remove protein, Isolate DNA

Treat with DNaseI

Isolate chromatin

Digest with BamHI

Southern blot

Agarose gel

MSB= non-expressing cells

Ovalbumin Probe: -globin

Transcription run-off assay• To monitor transcriptional activity of a gene

Measure transcription directly. Thus post-transcriptional processing in not a concern

Figure 5.27

8B, slide 18

375 nt transcript

Note- Each lane contains RNA pol II + TFIIA,B,E and F

Fig. 11.18- TBP alone can’t respond to Sp1

SP1

TATAA

bh= bacterially derived human TBPvh=virus derived human TBP

TBP

Transcription run-off assay