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G Protein Coupled Receptors
Animals
plasma membrane
signal transduction pathway, i.e.;
• cAMP
• phospholipase C
• cGMP phosphodiesterase
• MAPK
activated receptor
system
cycles
something happens
signal transduction pathway;
• cAMP
• phospholipase C
• cGMP phosphodiesterase
• MAPK
something else happens
something happens
Mammals
~ 4 Orthologs
> 23 Paralogs
Why Arabidopsis?
Why me?
Only one prototypical Ga protein.
Gene Families
Why Ga?
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?
Nature
Ti-Plasmid
T-DNA
Agrobacterium
Plant Cells
Hormone Opines
genes
genes
Lab
T-DNA
Out: Ti genes, opine genes,
In: DNA of choice.
Selectable Markers
Reporter Genes
Genes
from
~500,000
seeds
60,000 mutants
Set-Up
DNA Pooling
Maintain lines as pools of seed.
Seeds (9)
Germinate and grow seeds in liquid culture.
Seedlings (225)
Extract DNA,
DNA (225)
Super Pool DNA,
1
2
3
4
5
PCR Screen
6
…30
Super
Pools
(2000)
PCR Strategy
Reaction:
T-DNA
Product:
Reaction:
Product:
T-DNA
T-DNA Insertion Confirmation
G
• Blot gel and hybridize
with a WT probe.
• Band isolate and cycle
sequence PCR
fragment.
wt
Sequence T-DNA Insertion Sites
T-DNA
Reaction:
Product:
Sequence using the
PCR primer from the
T-DNA sequence.
T-DNA
Unknown
GTP
GPA1: \\GCAATGTGTTATTAAGTTGTCT --- ATGCTCTC--GAAAATTTTCGCCACTGGAAAT//
GPA2: \\TGTCTAAGCGTCAATTTGTTTA --- GGGCTCTCTCT--- ACCTGCTCAGGAGCACCTTTAC//
Probability of Finding an Insert
in a Specific Gene
p = 1-(1-f)n
p = probability of insertion event
f = 1-(Genome/Size of Gene)
n = number of T-DNA inserts
thousands of inserts
Finding Random Insertion
Mutants
• Use PCR based approach to identify sequence with foreign
DNA inserted into genes of interest.
Knockology
KO
GTP binding sites.
Effector domains.
Gbg sub-units binding site.
Receptor interaction domain.
T-DNA Mutants
tagged
seed line
Genetic Analysis
tt x TT (wt)
isolate
homozygous
mutant
2x
backcross
to wildtype
phenotype
analysis
Tt
T-DNA
Segregation
T
t
T
TT
Tt
t
Tt
tt
F2
Genetic Analysis
F2 Segregation
T
t
T
TT
Tt
t
Tt
tt
T
t
T
TT
Tt
t
Tt
tt
T
t
T
TT
Tt
t
Tt
tt
1:2:1
1 wt : 2 het
1 wt : 1 het
Not Lethal
Lethal
Gametophyte
Lethal
Arabidopsis Gantlet Project
http://thale.biol.wwu.edu/
Seed/seedlin
g
Mucilage
Germination time
Cotyledon size
Cotyledon shape
Cotyledon color
Time first true leaves
Petiole length
Leaf size
Leaf shape
Leaf color
Leaf margins
Stem length
Stem color
Root length
Root branching
Adventitious roots
Root gravity
Shoot gravity
Etiolated growth
De-etiolation
Nutrition/Seedling
0.1X MS, +/- Sucrose
0.01X MS, +/- Sucrose
Ca SO4 (0.2 mM) Minimal Media
Nutrient Dropouts: Base media (all)
minus one:Ca(NO3), K2SO4, MgSO4,
FeEDTA, KH2PO4, H3BO3, MnSO4,
CuSO4, ZnSO4, NaMoO
Vegetative/Seeds
Initial health in soil
Leaf Anatomy
Time to bolting
No. of leaves
when bolts
Bolt length
Bolt gravity
Stem color
No. of flowers/bolt
Flower morphology
Anther length
Silique length
No. of siliques
No. of seeds
Leaf senescence
Stop flowering
Silique shattering
Seed size
Seed shape
Seed color
Seed wrinkles
Physiology/Seedling
Hormones: ABA, Auxin,
Questions
Brassinolide, Cytokinin, GA
Name?
Calcium , (low w/EGTA) (high)
Quest?
Dimethylglutaric Acid (1) pH 4.0 – 6.0
Air-speed velocity of an unladen swallow?
NaCl
How do you spell Guantlet?
Osmoticum (Mannitol, Sorbitol, PEG, Sucrose)
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.
Genotype?
Southern Analysis
Genomic DNA,
Cut with SpeI,
Probe with 3’ GPA.
~ 11 Kb
wt
gpa1 gpa1gpa2 gpa2
het hom het hom
SpeI
SpeI
…not drawn to scale.
Probe
~ 5 Kb
SpeI
Transcript?
Northern Analysis
Extract total RNA
Use complementary DNA to probe for mRNA
wt
truncated
Protein?
Western Analysis
Proteins extracted
Antibody was raised to the Nterminus of the gene.
Antibodies specific to
GPA proteins facilitate
probing for the
presence of the protein.
Conclusions
GPA1 operates in controlling cell
proliferation, probably by modulating G1
control.
May be involved in auxin signal reception?
KO
MAPK?
something happens
GPA1
What Else?
GPA1, What Else II?
Brassinolide
Since 2001
Plant Physiol, June 2002, Vol. 129, pp. 897-907