arabidopsis experiments forward genetic screen (ethylene insensitive mutants) reverse genetic screen...

ArabidopsisArabidopsis Experiments ExperimentsForward Genetic Screen (Ethylene Insensitive Mutants)Forward Genetic Screen (Ethylene Insensitive Mutants)

Reverse Genetic Screen / PCR Genotyping (HReverse Genetic Screen / PCR Genotyping (H++- ATPase Mutants)- ATPase Mutants)

ArabidopsisArabidopsis

AArabidopsis thalianarabidopsis thaliana is the predominant model organism used by is the predominant model organism used by plant biologists today. Considered a “weed" in nature, this small plant biologists today. Considered a “weed" in nature, this small mustard serves as an experimental subject for everything from root mustard serves as an experimental subject for everything from root growth to flower development in the laboratory. growth to flower development in the laboratory. ArabidopsisArabidopsis has has gained prominence as a model organism for several reasons: gained prominence as a model organism for several reasons: 

Generation timeGeneration time. Seed to seed in about 42 days.. Seed to seed in about 42 days. FecundFecund. One . One ArabidopsisArabidopsis plant yields thousands of seeds. plant yields thousands of seeds. Genome sizeGenome size. 125 . 125 mmegaegabbases. Maize has 5000 mb, tobacco1500 mb.ases. Maize has 5000 mb, tobacco1500 mb. DiploidDiploid. Relatively simple genome.. Relatively simple genome. TractableTractable. Easily worked and amenable to genetic, molecular genetic, . Easily worked and amenable to genetic, molecular genetic,

physiological and biochemical studies.physiological and biochemical studies. Real plantReal plant. Roots, leaves, flowers, seeds, and a full component of . Roots, leaves, flowers, seeds, and a full component of

physiological and biochemical processes. physiological and biochemical processes.

ArabidopsisArabidopsis HistoryHistory

LinnaeusLinnaeus

Yours Truly

Forward vs. Reverse Forward vs. Reverse GeneticsGenetics

…we’ll be using both approaches.…we’ll be using both approaches. Treat thousands of organisms with a mutagen,Treat thousands of organisms with a mutagen,

- random mutagenesis, - random mutagenesis,

Identify an Identify an individualindividual with a with a phenotypephenotype of interest, of interest,

Identify the gene. Identify the gene.

• Treat thousands of organisms with a mutagen (usually),Treat thousands of organisms with a mutagen (usually),

– random mutagenesis, random mutagenesis,

• Identify an Identify an individualindividual with a with a genotypegenotype of interest, of interest,

• Identify the phenotype. Identify the phenotype.

Forward

Reverse

First, Forward GeneticsFirst, Forward Genetics

Experiment #1Experiment #1

Conditional ScreenConditional Screen for for Ethylene Ethylene InsensitivityInsensitivity

EthyleneEthylene“the gaseous “the gaseous

hormone”hormone”

Egyptians gassed figs in Egyptians gassed figs in order to stimulate ripening, order to stimulate ripening,

The ancient Chinese burned The ancient Chinese burned incense in closed rooms to incense in closed rooms to enhance the ripening of enhance the ripening of pears.pears.

In 1864, gas leaks from street In 1864, gas leaks from street lights were observed to stunt lights were observed to stunt plant growth, twist plants, plant growth, twist plants, and abnormally thicken and abnormally thicken stems stems

Dimitry Neljubow (1901) Dimitry Neljubow (1901) showed that the active showed that the active component was ethylene. component was ethylene.

R. Gane (1934) reported that R. Gane (1934) reported that plants synthesize ethylene.plants synthesize ethylene.

HH22C = CHC = CH22

Receptor Receptor enzyme-linked receptorenzyme-linked receptor

…found first in bacteria, then in plants, now in most eukaryotes, including mammals.

Two-component regulators.Two-component regulators.

EthyleneEthylene…promotes fruit …promotes fruit

ripening,ripening,

Ethylene signals the Ethylene signals the transition from transition from unripe to ripe fruits,unripe to ripe fruits,

cell wall cell wall components are components are broken down,broken down,

starches and acids starches and acids are broken down are broken down resulting in resulting in “sweetening” and “sweetening” and aromatic aromatic compounds ,compounds ,

pigmentation may pigmentation may also be induced.also be induced.

EthyleneEthylene…promotes the “triple …promotes the “triple

response”,response”,

……in etiolated seedlings,in etiolated seedlings,

reduced stem reduced stem elongation,elongation,

thicker stem, thicker stem,

horizontal growth,horizontal growth,

May provide the plant May provide the plant with “behavior” that with “behavior” that will provide escape will provide escape from soil from soil impediments.impediments.

EthyleneEthylene…mutant analysis,…mutant analysis,

wild typewild type einein

ein ein (ethylene present),(ethylene present),

……eethylene thylene ininsensitive.sensitive.

wild typewild type

ctr ctr (ethylene absent),(ethylene absent),

……cconstitutiveonstitutive t triple riple rresponse.esponse.

ctrctr

Ethylene Signal Ethylene Signal TransductionTransduction

…negative regulation.…negative regulation.

Tricky Concept(s)Tricky Concept(s)

In the absence of ethylene, the In the absence of ethylene, the enzyme receptor activates enzyme receptor activates CTR1,CTR1,

active CTR1 active CTR1 inhibitsinhibits the triple the triple response, response,

With ethylene present, or the With ethylene present, or the receptor “absent”, or the receptor “absent”, or the CTR1CTR1 gene mutated, gene mutated,

the triple response is activated.the triple response is activated.

ein, etr*, etc,

…blocks pathway.

ethylene,

…or ctr mutant,

no ethylene

…no triple response.

active

inactive

induces transcription,erf: ethylene response

factor.

?

Wedsnesday’s To Do, Wedsnesday’s To Do, #1#1

ems-treated seedsems-treated seeds

Sterilizing/PlantingSterilizing/Planting GerminatingGerminating

Breaking DormancyBreaking Dormancy  

HH22O/Imbibition, O/Imbibition, 

OO22/Aeration,  /Aeration,  

Cold/Prechilling Cold/Prechilling "stratification” "stratification” 

Inducing GerminationInducing Germination  

LightLight

ACC: 1-aminocyclopropane-1-carboxylate

Conditional ScreenConditional Screen

Grow on ACC,Grow on ACC,

……in the dark in the dark (etiolated).(etiolated).

Score for mutants,Score for mutants,

Transfer to 0.5X MS Transfer to 0.5X MS (Murisige and (Murisige and Skoog) media (-Skoog) media (-ACC),ACC),

Grow in light.Grow in light.

NotNotthis this Class.Class.

What Next?What Next?

Backcross to wild-type,Backcross to wild-type,

what might the F1 and what might the F1 and F2 tell us?F2 tell us?

Complementation Complementation tests?tests?

dominantdominant

recessiverecessive

Thought Experiments…Thought Experiments…

Second, Reverse Second, Reverse GeneticsGenetics

Experiment #2Experiment #2

PCR genotypingPCR genotyping of a of a t-DNA t-DNA mutantmutant

Wednesday’s To DO, Wednesday’s To DO, #2#2

t-DNA insertion mutantst-DNA insertion mutants

Sterilizing/PlantingSterilizing/Planting GerminatingGerminating

Breaking DormancyBreaking Dormancy  

HH22O/Imbibition, O/Imbibition, 

OO22/Aeration,  /Aeration,  

Cold/Prechilling Cold/Prechilling "stratification” "stratification” 

Inducing GerminationInducing Germination  

LightLight

Proton Pumps Proton Pumps in plantain planta

Stemstransport; sucrose hormones Leaves

stomata (gas exchange)sucrose transport

Antherscell elongation

Pollentip growth

Embryo/SeedsloadingRoots

root hair growthmineral uptake

Arabidopsis

Adapted from Biochemistry and Molecular Biology of Plants, pp. 115

H+ (protons) ATP synthase

ATP hydrolase (ATPase)

Transporters

- carriers, - channels.

Arabidopsis Arabidopsis GenomeGenome

~125 Mb (Megabases, million base pairs),~125 Mb (Megabases, million base pairs),

Rice: 420 Mb, Human: 3 Gb,Rice: 420 Mb, Human: 3 Gb,

25,498 genes from 11,000 gene families,25,498 genes from 11,000 gene families, Rice: 32,000 - 50,000, Human: 25,000 - 66,000.Rice: 32,000 - 50,000, Human: 25,000 - 66,000.

PCR GenotypingPCR GenotypingI.I. T-DNA Insertion MutantsT-DNA Insertion MutantsII.II. Hs Mt_DAN Hypervariable RegionHs Mt_DAN Hypervariable Region

Proton Pumps Proton Pumps in plantain planta

Stemstransport; sucrose hormones Leaves

stomata (gas exchange)sucrose transport

Antherscell elongation

Pollentip growth

Embryo/SeedsloadingRoots

root hair growthmineral uptake

Arabidopsis

Gene Location FunctionAHA1 whole plant ?AHA2 root cortex ?AHA3 phloem ?AHA4 root endodermis nutrient uptakeAHA5 whole plant ?AHA6 - ?AHA7 - ?AHA8 - ?AHA9 anthers ?

AHA10 seeds ?AHA11 hypocotyl ?AHA12 - psuedogene

Arabidopsis H+-ATPase

Gene Family

Phylogenetic Family Tree(ClustalW --> Phylip: protdist, fitch)

Baxter et al. , Plant Physiol, 123, (2003)

Reverse GeneticsReverse GeneticsFunctional GenomicsFunctional Genomics

Gene DNASequence

Gene Disruption PhenotypeAnalysis

Function

MutateDNA Sequence

DevelopmentPhysiology

Cell Biology

Agrobacterium

Plant Cells

NatureTi-Plasmid T-DNA

HormonesOpines

Lab

Selectable MarkersReporter Genes

Genes

Out: Ti genes, opine genes,

In: DNA of choice.

T-DNA

wtwtplantplantchromosomechromosome

Ti PlasmidTi Plasmid(from (from agroagro))

hormone genes (i.e. auxins)hormone genes (i.e. auxins)

opalineopaline

nopalinenopaline

virulencevirulencegenesgenes

virulencevirulencegenesgenes

hormone geneshormone genes

opaline, nopalineopaline, nopaline

neoplastic transformationneoplastic transformation

Agrobacterium tumefaciensAgrobacterium tumefaciensTi Plasmid (Ti Plasmid (TTumor umor iinducing)nducing) Mother NatureMother Nature

AgroAgrofoodfood

Construct T-DNAConstruct T-DNA

selection genesselection genes

virulencevirulencegenesgenes

infect plant, infect plant, select(2)select(2) for plants with T-DNA for plants with T-DNA

T-DNA (T-DNA (TTransfer DNA)ransfer DNA)LaboratoryLaboratory

transform, transform, select(1)select(1) for for agroagro with T-DNA with T-DNA

AgrobacteriumAgrobacterium

……if the T-DNA lands in a gene, the gene is disrupted. if the T-DNA lands in a gene, the gene is disrupted.

……can put other genes.can put other genes.

Probability of Finding an Insert in a Specific Probability of Finding an Insert in a Specific GeneGene

thousands of insertsthousands of inserts

p = 1-(1-f)p = 1-(1-f)nn

p = probability of insertion eventp = probability of insertion event

f = 1-(Genome/Size of Gene)f = 1-(Genome/Size of Gene)

n = number of T-DNA insertsn = number of T-DNA inserts

KnockologyKnockology

Plants/Pools DNA/Pools

Set-UpSet-UpDNA PoolingDNA Pooling

Seeds (9)

Seedlings

(225)

DNA (225)

1 2 3 4 5 6 …30SuperPools(2025)

Germinate and grow seeds in liquid culture.

Extract DNA,

Super Pool DNA,

Maintain lines as pools of seed.

PCR Screen

94o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Denature Step~30 seconds

~65o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Annealing Step~30 seconds

72o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

Synthesis~1 minute/kb

PCR

PCR StrategyPCR Strategy

5’ 3’

PPolymerase olymerase CChain hain RReaction (PCR),eaction (PCR),

with oligonucleotide primers with homology with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers.the DNA sequence between the primers.

Your geneReaction:

Product:Your gene amplified

Reverse Genetic PCR StrategyReverse Genetic PCR Strategy

T-DNAReaction:

Product:

Reaction:

Product: none.

PCR Screens for PCR Screens for MutantsMutants

PCR StrategyPCR Strategy

T-DNAReaction:

Product:

T-DNAReaction:

Product:

Find the Find the PlantPlant

You are ~hereYou are ~here

T-DNA T-DNA MutantsMutants

Genetic Analysis Genetic Analysis

taggedseed line

taggedseed line

isolate homozygous

mutant

isolate homozygous

mutant

backcrossto wildtypebackcrossto wildtype

2x

phenotype analysis

phenotype analysis

tt x TT (wt)

Tt

T-DNASegregation

TT Tt

Tt tt

T t

T

t

F2

PCR GenotypingPCR Genotyping

L t T

5’ 3’

5’ 3’heterozygote

L t T

5’ 3’

5’ 3’homozygotewt

L t T

5’ 3’

5’ 3’

homozygotemutant

Genetic AnalysisGenetic AnalysisF2 Segregation F2 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

Mitochondrial DNAMitochondrial DNA

- 16, 569 bp,16, 569 bp,

- multiple copies per mt,multiple copies per mt,

- 100 - 1000 mt per cell,100 - 1000 mt per cell,

- 37 genes;37 genes;- 22 oxidative phosphorylation,22 oxidative phosphorylation,- 13 tRNA, 13 tRNA, - 2 rRNA,2 rRNA,

- Mitochondrial Control Region.Mitochondrial Control Region.

Mitochondrial Control Region

• control region, – single promoter on each strand

initiates transcription,– ori,

• D-loop,– replication loop topography,

• hypervariable region,– mutation rate 10x greater than

genome.

Mitochondrial Control Region

• Hair follicle DNA extraction,

• PCR,

• Sequencing (at Cold Spring Harbor),

• Sequence analysis here at WWU.

Link Out

This Week?This Week?“hypervariable”“hypervariable”

Wednesday (probably)Wednesday (probably) Mt-DNA isolation/PCR (maybe Fri.),Mt-DNA isolation/PCR (maybe Fri.), analyze EMS mutants on ACC screen.analyze EMS mutants on ACC screen. Wasps?Wasps?

Friday (maybe, maybe Weds.)Friday (maybe, maybe Weds.) Isolate aha3-1 DNA and PCR genotype?Isolate aha3-1 DNA and PCR genotype? other aha3-1 activity,other aha3-1 activity, Wasps?Wasps?