Experimental biology
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- Anatomy- Anatomy
- Physiology (spray and pray)
- Chemistry (identification of signals)
- Biochemistry (protein isolation/structure)
- Genetics (genes/mutants)
- Cell biology (subcellular structures)
- Molecular biology (gene manipulation)
Choice of research topic?
- Gene/Gene family
- Biological process
- Signaling pathway
- Model system- Model system
- Available methods
- „Trendy topic“
- Serendipity
Arabidopsis thaliana
- Small, fully sequenced genome
- Easy genetics (diploid/self-polinater)
- Short vegetation time- Short vegetation time
- No large space requirement
- Simple organ and tissue structure
- Many established tools and facilities
(transformation, libraries, databases)
How to get your favorite gene?
- “Lottery” candidate gene approach
- Functional complementation
- From the protein back to the gene
- Expression
- Forward genetics
“Lottery”
o Homology to known factors
(trimeric G-proteins)
o Interesting domains
(kinases, phosphatases)(kinases, phosphatases)
o „Other“ reasons
(serendipity)
Functional complementation
Protein > gene
o Ligand binding (affinity
chromatography, azidolabeling;
ABP1, NPB, Zm-p60)
o Enzyme activity (CKX, NOS)
o Complex members
Proteomics approacheso Proteomics approaches
(phosphoproteomics, differential
display)
- Microsequencing
- Blast search:
amino acid > nucleotide
- Search for a gene
Protein > gene
o Ligand binding (affinity
chromatography,
azidolabelling; ABP1, NPB,
Zm-p60)
o Enzyme activity (CKX, NOS)
o Complex members ()o Complex members ()
o Proteomics approaches
(phosphoproteomics,
differential display)
- Microsequencing
- Blast search:
amino acid > nucleotide
- Search for a gene
Expression pattern
o Enhancer/Gene-trap libraries
o Differential display
substractive hybridisation
microarray
Gene and enhancer trap libraries
Microarray
Expression map of Arabidopsis root
Forward genetics
EMS mutagenesis
Mutant screen at seedling level
Patterning mutant types
Fatty acids
Sterols
Signalling
Vesicle traffic
Activation tagging - YUCCA
Second site mutagenesis - suppressors
Suppressors of CLV3 overexpression
Second site mutagenesis - enhancers
Chemical genetics
Zouhar et al., 2004
QTL
Gene verification
o Multiple alleles
o Transposone reversion
o Complementation
Towards a gene role
o Loss of function: Reverse genetics
o Gain of function: Ectopic expression
o Mosaics
o Sequence manipulations
o Phenotype analysis
o Biochemical function
Loss of function
o Reverse genetics/TILLING
o Antisense and RNAi approaches
o Immunomodulation
o Repression domain
o Titration
Reverse genetics
– indexed mutant
libraries
TILLING
TILLING
Gain of function
o Overexpression
o Tissue specific expression
o Conditional expression
o Protein stabilisation
CaMV 35S Promotor
GENE of interest
Two component system for gene expression
The hidden function
of WUSCHEL
Mosaics – Cre/Lox
Sequence manipulation
o Site-directed mutagenesis
o Domain deletions and swaps
o Chimeric proteins
rop GTPases mutants
AUX/IAA and ARF proteins
BDL
Homo and hetero-Protein
IVIIIIII
Aux/IAA
QVVGWPPVRSYRK
MP
S
Homo and hetero-
dimerisation
Protein
stability
Homo and hetero-
dimerisation
AuxRE binding
DBD MR III IV
ARF
bdl mutation
Hardtke and Berleth 1998
Phenotype analysis
o Visual evaluation
o Ultrastructure (EMS)o Ultrastructure (EMS)
o Use of markers
o Treatments
Biochemical function
o Protein activity
o Yeast complementation
o Xenopus oocytes
0
0,5
1
1,5
2
2,5
3
relativeGDP/GTP
exchangerate
+GNOM
ARFalone
o Xenopus oocytes
ARF
+GNOM
ARF
+GNOM
+BFA
ARFalone
Gene Expression and Protein
Localization
o Blots, RT-PCR
o Reporter genes
o In situ mRNA hybridization
o In situ protein localization
o In situ protein activity detection
Blots and RT-PCR
Northern blots RT-PCR
- IAA + IAA
Southern and Western blots
Reporter genes
o Transcriptional fusions
o Translational fusions
o GUS, Luciferase, GFP
o Applications
Transcriptional fusion
GUS – ß-Glucuronidase
GUS – ß-Glucuronidase
- Ferrocyanid + Ferrocyanid
Green Fluorescence Protein
Cell identity markers
Actin Tubulin
Subcellular structure markers
In situ mRNA/protein localisation
o Probe preparation
o Fixation
o Embedding
o Sectioningo Sectioning
o Deparafinization
o Treatment with probe
o Removal of unbound probe
o Signal visualization
GUS ProteinmRNA
Analysis of gene expression
Analysis of protein localisation
Friends and associates
o Yeast-two-hybrid
o Split ubiquitin, split YFPo Split ubiquitin, split YFP
o Genetic interactions
o Upstream and downstream
Classical transcription factorClassical transcription factor
1. DNA Binding domain
2. Activation domain
DNABinding
Activation
Yeast two hybrid
+
Bait Prey
Prey
lexA Protein VP16 Protein
Activation Activation
DNA-
binding
pSH18-34
genomic
lacZlexA Operator
lexA Operator Leu2
DNA-
binding
Bait Activation
Summary for Y2HSummary for Y2H Prey
pSH18-34
genomic
lacZlexA Operator
lexA Operator Leu2
Bait Prey
ActivationDNA-
binding
pSH18-34
genomisch
lacZlexA Operator
lexA Operator Leu2
Bait
Prey
(HIS3)
(TRP1)
(URA3)
lexA-BaitADH Promotor
VP16-PrayGal1 Promotor
Galaktose
Activation
DNA-
binding
genomisch lexA Operator
pSH18-34
genomisch
lacZlexA Operator
lexA Operator Leu2
Leu2
EGY48: Mutant for HIS3, TRP1, URA3 und LEU2
ActivationDNA-
binding
Bait Prey
Conditions for Y2HConditions for Y2H--SystemSystem
1. Proteins must be able to localize to the
nucleus
2. Bait construct must not have its own
activation domain
(Autoactivation)
In vitroIn vitro PulldownPulldown--AssayAssay
GST-Cyp5 and GST-GNOM1-246 bind GNOM from
Arabidopsis protein extract
Beads GST GST-GNOMGNOM Beads GST GST-Cyp5Cyp5
1-246
anti-GNOM
Interaction of GNOMInteraction of GNOM in vivoin vivo
DCB 1xHA
Genomic Fragment DCB 3xmycEndogenous Promotor
ATG
ATG
TAA
TAA
Immunoprecipitation with anti-myc beads
Genomic Fragment Endogenous Promotor
anti-myc
antianti--HAHA
lysat
depl.
lysat
mycmyc--
beadsbeads
Cytosol S100
165 kD
165 kD
Immunoprecipitation with anti-myc beads
Split-Ubiquitin
Split-YFP
o Protoplast transfection
Genetic interactions
c-Myc
P72S
Promoter (bp)
2,400 Yes
Activity
Upstream - Promotor analysis
(yeast one hybrid)
1,500 Yes
1,000 Yes
500 No
150 No
promoter::GUS
Downstream targets
o expression profiling
o proteomicso proteomics
o second site mutagenesis
o educated guess
Special methods and tools
o DR5 auxin response reporter
o Transient transfection
o Laser ablations and laser capture
DR5 (Auxin) Response Reporter
5´ CCTTT TGTCTC 3´
9x inv.
DR5 35S min GUS 35S pA
DR5: Ulmasov et al., 1997
- Auxin + Auxin
DR5::GUS
DR5::GFP Auxin Reporter
DR5rev 35S min GFP 35S pA
5´ CCTTT TGTCTC 3´
9x inv.
EmbryosRoot Root + Auxin anti-IAA AB
Laser ablations
Transient transfection
Protoplasts
GUS GUS + Diphteria Toxin
Onion epidermis cells
GUS GUS + Diphteria Toxin
GUS GUS + Diphteria Toxin
GUS + IPT (cytokinin biosynthesis)
Laser capture