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G Protein Coupled Receptors

Animals

signal transduction pathway, i.e.;• cAMP• phospholipase C• cGMP phosphodiesterase• MAPK

activated receptor

something happens

system cycles

plasma membrane

signal transduction pathway;• cAMP• phospholipase C• cGMP phosphodiesterase• MAPK

something happenssomething 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.

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

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?

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?

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.

Conclusions

GPA1 operates in controlling cell proliferation, probably by modulating G1

control.

May be involved in auxin signal reception?

something happensMAPK?

KO

GPA1What Else?

GPA1, What Else II?Brassinolide

Plant Physiol, June 2002, Vol. 129, pp. 897-907 Since 2001