DNA mRNA Protein
Transcript of 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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Detection of proteins
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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Detection of proteins
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**