dna mrna protein

8/14/2019 DNA mRNA Protein

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Modern Methods in Developmental BiologyMastermodul WS 08/09

Matthias Hammerschmidt

Detection of transcripts

Protein-DNA interaction

Detection of proteins

Protein-protein interaction

mRNA

miRNA

TranscriptionGene

ProteinTranslation

today

tomorrow


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Detection of transcripts

Northern Blot

S1 nuclease analysis, RNase protection

RT-PCR (quantitative real-time RT-PCR)

Systematic searches for differential transcription

In situ hybridization (spatial distribution)

Reporter assays(indirect: detection of reporter protein under control of

cis-regulatory elements of gene of interest)

(Differential display, cDNA substraction, differential chip hybridization)


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Northern blot

RNA purification (either total or oligo(dT)-affinity purified -> mRNA)

Separation via Gel agarose electrophoresis under denaturing conditions (glyoxal)

Partial RNA hydrolysis of large molecules via incubation in 0.05N NaOH

Transfer (blotting) and immobilization on nylon- or nitrocellulose membranes

Hybridization with complementary, directly or indirectly labeled DNA probe

Quantification via densitometry in comparison to uniformly present reference RNA

Np63 mRNA

reference mRNA

bmp2

-/-

bmp7

-/-

smad

5-/-

chd-/

-

WT

Chordin

Bmp2/7

Smad5

?


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Nuclease S1 analysis RNase protection

40-80 nt oligonucleotide(5 l abelled via PNK)

- Nuclease S1 fromAspergillus oryzae

- hydrolyzes single stranded RNA and DNA

- also used to map 5 and 3 ends of mRNAs and

introns of genes

- more sensitive (probe more strongly labeled)

- RNA/RNA hybrid more thermostable

- Hydrolysis more robust (optimization of temperature

and enzyme concentration not necessary)

RNA/DNA hybrid protected

from hydrolysis by Nuclease S1

Run-off-transcription of cDNA (100-300 bp)in transcription vector (T7,T3,SP6)

RNA/RNA hybrid protected

from hydrolysis by RN ases


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RNA probe in vitro synthesis

oder Dig-UTP


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RT: reverse transcriptase

(RNA-dependent DNA polymerase)

Tth-DNA polymerase:

from bacterium Thermus thermophilus;can catalyze both stops

RT-PCRRNA purification

DNase digest !


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CT: cycle when signal above threshold line

Semi-quantitative RT-PCR stopped in exponential phase (cycle 30)

(Ethidiumbromide-Agarose-Gel, optionally plus Southern blotting / hybridization)

house-keeping gene

Really

in

exponential / proportional

phase ???

-> look at earlier cycles

-> more sensitive dye


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SYBR-Green

(fluorescent with dsDNA; unspecific;

background with primer dimers etc.)

Real-time RT-PCR

Fluorescent DNA measurement after each cycle:

1.

2. Reporter and quencher-marked probe (specific; multiplex; expensive)


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Stop of PCR after 33 cycles:

Red, blue and pink samples

give identical results, although

100 fold difference in DNA content

-> important to compare at early phase of

exponential phase (CT value)

Calculation of DNA content according to Pfaff:


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Systematic searches for differential transcription

1. Differential Display

2. cDNA substraction (Suppression substractive hybridization)

3. Differential hybridization of oligonucleotide microarrays (Affymetrix chips)

with complex cDNA probes (-> Wolfgang Werr)

IL-2R GAPDH

1 12 2 33

1: unsubstracted tester cDNA

2: unsubstracted driver cDNA

3: substracted cDNA

- PAGE of 3 samples

- when differentially l abelled (Cy3/5),samples can run in one l ane

- In red: products only present in one sample

(Clontech Kit)


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Spatial differentiation: In situ hybridization

DNA in situ hybridization

for genomic DNA analysis / molecular cytogenetics (FISH, CGH):DNA probes; denaturation step; nuclear signal; histone extraction;

low target numbers (2-4 per cell); specimen: tissue sections, single cells

RNA in situ hybridizationmRNA: longer, abundant; miRNA, shorter, more abundant; cytoplasmic signal;

RNA probes (RNA/RNA > RNA/DNA > DNA/DNA); no denaturation step;

addition of formamide to reduce annealing temperature

Specimen: whole mounts or sections

In vitro synthesis of RNA probe:

incorporation of NTPs that are either

- radioactively or fluorescently labelled (direct labelling) or

- coupled with specific haptens (Digoxygenin, fluorescein, biotin; indirect labelling),

haptens are detected via antibodies

- antibodies are either coupled to fluorochromes or- to enzymes (alkaline phosphases, peroxidase, -galactosidase)

products of enzymatic reaction are either

- visibly colored (e.g. X-gal, X-phophate + NBT; colormetric detection) or

- fluorescent (e.g. Fast red; fluorescent dection)


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radioactive on consective sections

double-colormetric in whole mounts

double-fluorescent in whole mounts (LSM)

Eno

crine

pituitarycells

skinionocytes

Opercle

osteoblasts

Shh Bmp4

Epitheliu

m

and

mesenchyme

oftoothb

ud


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In situ RT-PCR

Slides of prostate cancer biopsy

i: spondin2-specific primer pairii: scrambled primers

Stamps et al. (2003). J. Nanobiotechnology 1: 3


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Reporter assays

Reporter proteins: - enzymes (-galactosidase; luciferase)

- fluorescent proteins (GFP, RFP etc.)Reporter cDNAs put under control of cis-regulatory elements

of gene of interest (promoter + enhancers + suppressors; upstream + downstream + introns)

Mimicking of entire expression pattern: BAC recombination

Dissection of elements required for specific domains etc.:

testing of different genomic elements alone or in combinationIn vitro or in vivo (transgenesis; live imaging)

Tg(flk1:eGFP) , Tg(gata1:dsRe

Endothelial cells blood cells)

Zebrafish heart:


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in vivo imaging of the zebrafish lateral line primordium

Tg(cldnB:GFP)

WT

WT

fss


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Identification and dissection of crucial cis-regulatory elements

via transgenesis in mouse

Echelard e t al.(1994), Development 120, 2213-2224

Rowitch et al. (1998), Development 125, 2 735-2745

Wnt1 mRNA Tg(Wnt/5.5kb:lacZ)

Insituhybridization

-galactosidasestaining

Tg(Wnt/3x110bp:lacZ)

Contains consensus binding sequences

for Pax transcription factors

(5.5 kb BglII/BglII fragment)


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Test of identified cis-regulatory elements / binding sites

via Luciferase-reporter assays in zebrafish

- Injection of luciferase reporter constructs together with -galactosidase (for normalization)

and bmp2ornoggin mRNA into zebrafish embryos;

- Preparation of embryonic protein extracts and luciferase activity assays

Bakkers et al. (2002)Dev. Cell 2, 617-627

- Np63 is expressed in ventral ectoderm of zebrafish

embryo: expression is absent in bmp2mutant- Identification of Smad4/5 consensus binding sequences

upstream ofNp63 TATA box

Are they required forNp63 expression?


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Protein-DNA interaction

Electrophoretic Mobility Shift Assays (EMSA)

DNA footprinting

Chromatin-Immunoprecipitation (ChIP)

ChIP on Chips(to search for DNA targets of specific protein)

DNA affinity chromatography (to search for proteins binding of specific DNA)

Yeast-one-hybrid system(to search for proteins binding of specific DNA)

DNA-binding proteins usually have specific protein domains

and specific binding consensus sequences

E-boxes


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Electrophoretic Mobility Shift Assays (EMSA)

Radioactive DNA fragment: annealed complementary oligonucleotides, PCR fragment or restriction fragment

Binding proteins: from purified recombinant protein to crude cell lysate for search of binding proteins

Putative Smad4 binding site ofNp63 promoter

+ recombinant Smad4 protein

160bp regulatory DNA sequence of Ig light gene gene

+ protein extract of cell line

not shown:Supershiftupon incubation withanti-Smad4 antibody

-> analysis via

polyacrylamide-gelelectrophoresis (PAGE)

andautoradiography


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DNA footprinting

polyacrylamide-gelelectrophoresis (PAGE)

with urea as denaturing agent

(like sequencing gels)

plus

autoradiography

Labeling of DNA:

5: Polynucleotide-Kinase3: Fill-in with Klenow or

T4-DNA polymerase

(Label has to be only on one

strand of DNA double strand)

or via primer extensions


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Chromatin-Immunoprecipitation (ChIP)

Although many bound genomic DANN fragments immuno-precipitated,

only those detected that are probed via PCR (primer pairs available)


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ChIP on ChipsSystematic search for DNA target of specific protein


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ChIP on ChipsSystematic search for DNA targets of specific protein

Genomic microarray with 60mer

spanning 2kb promoter regions of

11,000 zebrafish genes in 0.25kb distances

ChIP on chip

with antibody against

K4-trimethylated Histone 3 (H3K4Met3)

(marker for active chromatin)

and with pan-Histone 3 (H3) antibody

Scatter plotsEnriched probes are above diagonal

Control spots in red

-> 4,735 genes occupied by H3K4Met3

(transcriptionally active)

Wardle et al. (2006), Genome Biology 7, R71

Linker ligation and PCR amplification

of precipitated DNA

Differential labelling with Cy3 or Cy5

Co-hybridization of microarrays

0.3kb fragments


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DNA affinity chromatographySystematic search for proteins binding to specific DNA


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Yeast-one-hybrid systemSystematic search for proteins binding to specific DNA

- Target element with specific target DNA

inserted in yeast promoter with low basal

expression of downstream reporter cDNA

(selective: HIS3; colormetric: LacZ)

- Construction of cDNA library and cloning into

vector encoding a fusion of the library ORFsto the Gal4-activation domain (Gal4-AD)

- co-transformation of yeast and growth on

mimimal medium lacking histidine

DNA under investigation

(Clontech Kit)


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Why important to know protein in addition to transcript pattern?

- Proteins are the effectors

- Because of differences in stability, and particularly in the case of secreted proteins,

their spatial and temporal distribution within the embryo can differ from that of the mRNA

- Subcellular distribution can give important insights into function and mechanism

Why nevertheless mostly in situ hybridizations used?

Because most protein analyses require antibodies;

Antibody production takes more time and money than that of in situ hybridization probes

wt

fras1 -/-

fras1mRNA

Fras1protein

A8 A8

A7A7 A6

wt

bcd-/-


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Via specific antibodies:

Via other binding proteins

MALDI-TOF (mass spectroscopy)

Systematic searches for differentially present proteins2D-PAGE + MALDI-TOF -> 2 parallel runs

Differential labelling (e.g. ICAT) -> DIGE / one run

(Proteomics; Wolfgang Werr)

Western blotting; immunoprecipitation, immunohistochemistry / in situ

(GST-pulldown; in situ)


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Production of specific primary antibodies

1. Immunization of mouse, rabbit, guinea pig, goat, chick, carp

with protein / recombinant protein fragment / peptide-> polyclonal antibodies

-> monoclonal antibodies

2. Phage display

Primary antibody(e.g. from mouse);

usually not further modified

Secondary antibody

against Fc region of

primary antibody

(e.g. goat-anti-mouse);covalently coupled to

fluorochrome, hapten

or enzyme)

IgG

IgM (pentameres)


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Polyclonal antibodies

-> Loading of antisera at pH 6.5

-> Elution of high affinity antibodies

at pH 2.5 (glycine / HCl)

bead withcovalentlyattachedantigen

boundspecific

antibodyunspecificantibodies andother proteinspass through

1. Affinity chromatography(ideally with same protein

made via different expression system

e.g. GST-fusion vs. (His)n-fusion

Covalent coupling of protein on

cyanbromide-activated sepharose

To remove less specific antibodies causing background signals:

2. Pre-adsorption, e.g. with mutant or morphant specimen lacking antigen

- Subcutaneous injection of protein/peptide into other species together with

adjuvans (microbial stimulant of innate immune system)

- Test bleedings; testing of antisera (ELISA; Western etc). In case of low signal

compared to pre-immune serum: additional immunization (boosting)

- Sacrificing of animal for final bleeding; usage of entire blood serum (contains many

different antibodies and other serum proteins; titer of antibodies against immunogen

particularly high; also this population very diverse)


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Monoclonal antibodies(Nobel prize for Georges Koehler , MPI Freiburg)

Concept: Selection, isolation and amplification (cloning) of those plasma cells

from spleen that generate very specific and strong antibodyPrerequisit: Immortilization of plasma cells via fusion with tumor cells


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Monoclonal antibodies


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Phage displayFilamentous coat proteins of Ff phage tolerate being fused with foreign protein

without losing their function -> foreign proteins displayed at surface of phage

-> Construction of recombinant phage libraries, with each phage only generating

one random protein, or here, a unique pair of light and heavy chain


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Western blotting: 1- or 2D-PAGE1D: discontinuous SDS-PAGE

(stacking and separating gel)2-dimensional PAGEFirst dimension: isoelectric focusing (IEF)

(urea instead of SDS; separation by intrinsic charge)

Separation in narrow tube with stable pH gradient

Second dimension: narrow tube subjected to SDS-PAGEseparation by size

separation

by size

Variants:

-non-reducing (no MeSH, persistence of S-S compexes)

-Non-denaturating (no SDS, blue native; persistenceof non-covalent complexes)


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Western blotting

1D-PAGE:Comparison wt - morphant with

primary antibody specifically detecting

phosporylated (BMP-activated) Smad5

(C,D: difference in whole mount histochemistry

less clear; background staining)

Silver stain Western

2D-PAGE:Lysates of deviding tabacco cells probed withspecific antibody detecting

phosphorylated threonine residues

(protein pairs 1/2, 3/4 and 5/6 would not have

been separated without IEF; could represent

different phospho-isoforms of same protein)

1,2

3,4

5,6

Western Immunohistochemistry

- Secondary antibody

coupled with HRP

- catalyses chemo-

luminescent reaction(e.g. ECL-System)

- Detection via

autoradiography

or Phospo-Imager

(quantitative)

Nitrocellulosemembrane

Nitrocellulosemembrane


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Immunohistochemistry (in situ)- on sections or in whole mounts

- fluorescent or colormetric

- multiplex: primary antibodies from different origins (mouse,rabbit etc.)secondary antibodies coupled with different enzymes or fluorochromes

1. Colormetric:Primary: mouse-anti-p63

Secondary: goat-anti mouse-biotin

Third: complex of avidin (4 biotin-binding sites) + HRP-(biotin)nHRP substrate: Di-aminobenzidine (brown product)

In situ hybridization: Digoxygenin-labelled hai1 RNA probe

goat-anti-Dig-AP

AP substrate: BCIP + NBT (blue product)

2. Fluoresent:

Mouse-anti-p63

Goat-anti-mouse-Alexa647

Rabbit-anti-GFP

Goat anti-rabbit-Alexa488

Dig-labelledATPase probe

Goat-anti-Dig-AP

AP substrate: Fast Red

Clonal analysis of GFP-labelled epidermal cell -> LSM


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Protein detection with other binding proteins

Neutrophil lysate incubated with PAK1-PBD-GST agarose (pull-down)

Precipitate separated via SDS-PAGE + anti-GST Western blotting

Probing with anti-Rac1 antibody

pos.

ctr

neg.ctr Time after neutrophil stimulation

Example: small GTPases Rac1 and Cdc24

In vitro (Far-Western): In situ:

Ueda et al. (2008)

Biol. Cell 100, 645-657

Activated by outside-in signaling;

active Rac1/GTP binds range of cytoplasmic proteins

to regulate cell proliferation and cytoskeletal rearrangements.

One binding partner: p21-activated kinase (PAK-1)


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Protein detection, identification, sequencing

via MALDI-TOF MSMatrix-assisted laser desorption ionization-time-of-flight mass spectroscopy

Mass spectroscopy used for structure determination of many different organic chemicalsMajor advantage: extremely little material required !

Principle: uniform ionization (charging) of molecule or molecule fragments

-> same accelaration in electric field, velocity inverse proportional,

time of flight proportional to mass of molecule / molecule fragment

-> determination of size of molecule

-> determination of structure / composition of molecule by pattern of fragments

Laser pulses(3-4 nsec)

electrostaticfield

Protontransfer

Time of flight (m/z)


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Protein detection, identification, sequencing

via MALDI-TOF MS

(A)

Measurement of peptide masses

without further fragmentation ->

protein identification

(B)

Measurement of masses of

peptides and fragments ->

protein sequencing


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Detection of protein-protein interaction(for identification and/or confirmation)

Far-Western; Co-Immunoprecipitations

GST pull-downs; affinity chromatography

Yeast-two-hybrid technique

Phage display

Analytic ultracentrifugation, blue native PAGE

Cross-linking

Optical techniques:

(with bifunctional or photo-activateable chemicals)

Bimolecular fluorescence complementation (BiFC; Split-YFP) (->M. Hlskamp)

Fluorescence resonance energy transfer (FRET) (->M. Hlskamp)

Surface plasmon resonance (BIACORE)

(with regular protein phage library)


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Far-WesternInitially used as dot-blot to screen expression libraries with protein probe

Nowadays mainly confirmatory

- Expression of different flagellar proteins from

Treponema orBacillus in E. coli- PAGE of E. coli lysate on membrane

- probed with GST-labelled Bacillus protein YviF

Minor bands degradation products of flagellar proteins

Or (unspecifically) other interacting proteins

Chemo-

luminescence

Limitation:

Some interactions not present

after protein denaturation

(->native instead of SDS-PAGE)


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Co-Immunoprecipitations

(from Staphylococcus aureus;

binds Fc of IgGs; coupled to bead)

Binding between Formin (F) and Src (S) - Mapping of S-binding site in F:

- expression of F, S, or F+S in E. coli; Co-IP of lysate with IgGs against different domains of S

Western blotting with anti-F IgG-> SH3 antibody cannot precipitate F -> SH3 domain of S occupied by F

-> specificity test of working 2-17 antibody by competition with 2-17 peptide

Anti-Src IgG

Anti-Formin

Western blot


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GST pull-downs

bait

prey1prey2prey3

baits

Pull-down of histonesOwn example

Co-IP

prey

Fusion of bait and

glutathionine-S-transferase

glutathionin


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Yeast-two-hybrid techique / screen

e.g. Gal4-e.g. Gal4-

e.g. UAS


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Yeast-two-hybrid techique / screen

Many controls to reduce false positives !!


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Surface plasmon resonance (SPR)

According to supplier also called Biacore


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Surface plasmon resonance

kon konkoff

koff= KD

Major advantages:

little protein required; proteins do not have to be labelled;

quantification of affinity; determination of stoichiometry

Studies of competitive binding versus multiprotein complex formation


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Surface plasmon resonance

Own example:

Chordin and Crossveinless-2 compete for binding of Bmp2

Immobilized on

gold ship:Bmp2

After saturation of Bmp2

with Cvl2

Chordin cannot bind

(no increase at ** ofCcomparable to that in B)

-> Cvl2 and Chordin share

same binding site of Bmp2

A B

D**

dna mrna protein

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