today g protein coupled receptors animals signal transduction pathway, i.e.; camp phospholipase c...
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Today
G Protein Coupled Receptors
Animals
signal transduction pathway, i.e.;• cAMP• phospholipase C• cGMP phosphodiesterase• MAPK
activated receptor
something happens
plasma membrane
cycles
something else happens
~ 4 Orthologs
> 23 Paralogs
Mammals
Why Arabidopsis?Why me?
Only one prototypical G protein.
Gene Families
Why G?Why did my collaborators care?
Looking for the Auxin Receptor,
– auxin is a plant hormone implicated in multiple physiological pathways.
G Protein Coupled Receptors
Auxin Functions
gravitropism/thigmatropism, etc.
perception of light
general signal transduction
embryogenesis
development
cell growth and differentiation
“oncogenesis”
etc.
Auxin Receptor =
G Protein Coupled Receptor?
Maybe Reverse Genetics can supply an answer?
Homologous Recombination Range
• Yes...
– mice, many well characterized mammalian cells,– bacteria, – yeast, (remember the bar code deletion project),
• No (maybe)...
– C. elegans (no),– Arabidopsis (done once, not repeated),– Drosophila (shown in principle, not repeated often),– the rest?
Agrobacterium
Plant CellsNature
Ti-Plasmid T-DNA
Hormonegenes
Opinesgenes
Selectable MarkersReporter Genes
Genes
Lab
Out: Ti genes, opine genes,
In: DNA of choice.
T-DNA
from ~500,000
seeds
Set-UpDNA Pooling
Seeds (9)
Seedlings (225)
DNA (225)
1 2 3 4 5 6 …30SuperPools(2000)
Germinate and grow seeds in liquid culture.
Extract DNA,
Super Pool DNA,
Maintain lines as pools of seed.
PCR Screen
60,000 mutants
PCR Strategy
T-DNAReaction:
Product:
T-DNAReaction:
Product:
T-DNA Insertion Confirmation
• Blot gel and hybridize with a WT probe.
• Band isolate and cycle sequence PCR fragment.
G wt
Sequence T-DNA Insertion Sites
T-DNA Unknown GTP
GPA1: \\GCAATGTGTTATTAAGTTGTCT --- ATGCTCTC--- GAAAATTTTCGCCACTGGAAAT//
GPA2: \\TGTCTAAGCGTCAATTTGTTTA --- GGGCTCTCTCT--- ACCTGCTCAGGAGCACCTTTAC//
Sequence using the PCR primer from the T-DNA sequence.
T-DNAReaction:
Product:
Probability of Finding an Insert in a Specific Gene
thousands of inserts
p = 1-(1-f)n
p = probability of insertion event
f = 1-(Genome/Size of Gene)
n = number of T-DNA inserts
Finding Random Insertion Mutants
• Use PCR based approach to identify sequence with foreign DNA inserted into genes of interest.
Knockology
KO
G sub-units binding site.
GTP binding sites.
Receptor interaction domain.Effector domains.
Genotype?
wt gpa1het
gpa1hom
gpa2het
gpa2hom
Southern Analysis
Genomic DNA,
Cut with SpeI,
Probe with 3’ GPA.
~ 5 Kb
ProbeSpeI SpeI
SpeI
~ 11 Kb
…not drawn to scale.
Transcript?
wt
truncated
Northern Analysis
Extract total RNA
Use complementary DNA to probe for mRNA
Protein?
Western Analysis
Proteins extracted
Antibodies specific to GPA proteins facilitate probing for the presence of the protein.Antibody was raised to the N-
terminus of the gene.
T-DNA Mutants Genetic Analysis
taggedseed line
taggedseed line
isolate homozygous
mutant
isolate homozygous
mutant
backcrossto wildtype
backcrossto wildtype
2x
phenotype analysis
phenotype analysis
tt x TT (wt)
Tt (R / D)
T-DNASegregation
TT Tt
Tt tt
T t
T
t
F2
Genetic AnalysisF2 Segregation
1 : 2 : 1
TT Tt
Tt tt
T t
T
t
Not Lethal
1 wt : 2 het
TT Tt
Tt tt
T t
T
t
Lethal
1 wt : 1 het
TT Tt
Tt tt
T t
T
t
GametophyteLethal
Arabidopsis Gantlet Projecthttp://thale.biol.wwu.edu/
Seed/seedlin
gMucilage
Germination timeCotyledon size
Cotyledon shapeCotyledon color
Time first true leaves
Petiole lengthLeaf size
Leaf shapeLeaf color
Leaf marginsStem lengthStem colorRoot length
Root branchingAdventitious roots
Root gravityShoot gravity
Etiolated growthDe-etiolation
Vegetative/Seeds
Initial health in soilLeaf AnatomyTime to boltingNo. of leaves
when boltsBolt lengthBolt gravityStem color
No. of flowers/boltFlower morphology
Anther lengthSilique lengthNo. of siliquesNo. of seeds
Leaf senescenceStop flowering
Silique shatteringSeed size
Seed shapeSeed color
Seed wrinkles
Physiology/SeedlingHormones: ABA, Auxin,
Brassinolide, Cytokinin, GACalcium , (low w/EGTA) (high)
Dimethylglutaric Acid (1) pH 4.0 – 6.0NaCl
Osmoticum (Mannitol, Sorbitol, PEG, Sucrose)
Nutrition/Seedling0.1X MS, +/- Sucrose
0.01X MS, +/- SucroseCa SO4 (0.2 mM) Minimal Media
Nutrient Dropouts: Base media (all) minus one:Ca(NO3), K2SO4, MgSO4, FeEDTA, KH2PO4, H3BO3, MnSO4,
CuSO4, ZnSO4, NaMoO
QuestionsName?Quest?
Air-speed velocity of an unladen swallow? How do you spell Guantlet?
De-etiolated Plants?
De-etiolated Plants?cell size vs. organ size
Mitosis?cyc1At-CDB-GUS
Overexpression?
Transgene:
GPA1 driven by an inducible promoter,
- dexamethason,
In “WT” plants.
GPA1 function?BY-2 Cells
…over-express GPA1 in cell culture lines,
- measure G1 to G2 transitions,
- measure cell plate formations.
Conclusions
GPA1 operates in controlling cell proliferation, probably by modulating G1
control.
May be involved in auxin signal reception?
something happensMAPK?
KO
Turns out agb1 (-sub-unit) phenotype identical to gpa1,
…what do you think that means?
GPA1What Else?
GPA1, What Else II?Brassinolide
Plant Physiol, June 2002, Vol. 129, pp. 897-907 Since 2001
> 4 Orthologs
> 23 Paralogs
Mammals
Assman, Science (310), 2005
FOR WEDNESDAY...
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