Experimental biology
Description > Manipulation > Understanding
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- 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
- Available methods
- ,,Trendy topic"
- Serendipity
Arabidopsis thaliana
- Small, fully sequenced genome
- Easy genetics (diploid/self-polinater)
- 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?
- "Monte Carlo" candidate gene approach
- Functional complementation
- From the protein back to the gene
- Expression
- Forward genetics
"Monte Carlo"
o Homology to known factors
(trimeric G-proteins)
o Interesting domains
(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
o 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 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
,
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.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
Gene verification
o Multiple alleles
o Transposone reversion
o Complementation
QTL
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
QVVGWPPVRSYRK
BDL
MP
S
Homo and hetero-
dimerisation
Protein
stability
Homo and hetero-
dimerisation
AuxRE binding
DBD MR III IV
IVIIIIII
ARF
Aux/IAA
bdl mutation
Hardtke and Berleth 1998
CRE1 ­ cytokinin receptor
Phenotype analysis
o Visual evaluation
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
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
- Ferrocyanid + Ferrocyanid
GUS ­ ß-Glucuronidase
Green Fluorescence Protein
Cell identity markers
Actin Tubulin
Subcellular structure markers
In situ mRNA/protein localisation
o Probe preparation
o Fixation
o Embedding
o 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 YFP
o Genetic interactions
o Upstream and downstream
ClassicalClassical transcriptiontranscription factorfactor
1. DNA Binding domain
2. Activation domain
DNABinding
Activation
+
Bait Prey
Prey
lexA Protein VP16 Protein
Activation Activation
DNA-
binding
Yeast two hybrid
pSH18-34
genomisch
lacZlexA Operator
lexA Operator
pSH18-34
genomisch
lacZlexA Operator
lexA Operator Leu2
Leu2
DNA-
binding
Bait Activation
Bait
Summary for Y2HSummary for Y2H Prey
Prey
ActivationDNA-
binding
pSH18-34
genomisch
lacZlexA Operator
lexA Operator
pSH18-34
genomisch
lacZlexA Operator
lexA Operator Leu2
Leu2
Bait
Prey
(HIS3)
(TRP1)
(URA3)
lexA-BaitADH Promotor
VP16-PrayGal1 Promotor
EGY48: Mutant for HIS3, TRP1, URA3 und LEU2
Galaktose
Activation
Activation
DNA-
binding
DNA-
binding
Bait Prey
ConditionsConditions forfor Y2HY2H--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
Beads GST GST-GNOMGNOM Beads GST GST-Cyp5Cyp5
1-246
anti-GNOM
GST-Cyp5 and GST-GNOM1-246 bind GNOM from
Arabidopsis protein extract
Interaction of GNOMInteraction of GNOM in vivoin vivo
DCB 1xHA
Genomic Fragment DCB 3xmycEndogenous Promotor
ATG
ATG
TAA
TAA
anti-myc
antianti--HAHA
lysat
depl.
lysat
mycmyc--
beadsbeads
Cytosol S100
165 kD
165 kD
Immunoprecipitation with anti-myc beads
Genomic Fragment Endogenous Promotor
Split-Ubiquitin Split-YFP
Split-YFP
o Protoplast transfection
Genetic interactions
c-Myc
P72S
Promoter (bp)
2,400 Yes
1,500 Yes
1,000 Yes
500 No
150 No
Activity
promoter::GUS
Upstream - Promotor analysis
(yeast one hybrid)
Promotor analysis
­ yeast one hybrid
Downstream targets
o expression profiling
o 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
DR5 35S min GUS 35S pA
5´ CCTTT TGTCTC 3´
9x inv.
DR5: Ulmasov et al., 1997
- Auxin + Auxin
DR5::GUS
DR5::GFP Auxin Reporter
EmbryosRoot Root + Auxin
DR5rev 35S min GFP 35S pA
5´ CCTTT TGTCTC 3´
9x inv.
anti-IAA AB
Laser ablations
Transient transfection
Protoplasts
GUS GUS + Diphteria Toxin
Onion epidermis cells
GUS GUS + Diphteria Toxin
GUS + IPT (cytokinin biosynthesis)
Laser capture