Apetala1 Mutant. Testing the ABC floral-organ identity model: cloning the genes Objectives: To test the validity of the ABC model for floral organ identity.

Apetala1 Mutant

Testing the ABC floral-organ identity model: cloning the genes

Objectives:

To test the validity of the ABC model for floral organ identity we will:

1. Use the model to make predictions concerning the phenotype of double or triple loss-of function mutants and compare with the actual double mutant phenotypes.

2. Clone and sequence the ABC genes. Look for similarities with sequenced genes already in the database.

3. Determine the time and place of expression for each ABC gene and consider whether the expression correlates with the functional domain defined by the loss-of-function phenotype.

4. Test regulatory interactions between ABC genes by examining how the loss-of-function of one gene affects the expression domain of another.

5. Create gain-of-function mutants by generating transgenic plants carrying an ABC gene cDNA under the control of the CaMV35S promoter.

Testing the ABC floral-organ identity model: cloning the genes

Objectives:

To test the validity of the ABC model for floral organ identity we will:

1. Use the model to make predictions concerning the phenotype of double or triple loss-of function mutants and compare with the actual double mutant phenotypes.

2. Clone and sequence the ABC genes. Look for similarities with sequenced genes already in the database.

3. Determine the time and place of expression for each ABC gene and consider whether the expression correlates with the functional domain defined by the loss-of-function phenotype.

4. Test regulatory interactions between ABC genes by examining how the loss-of-function of one gene affects the expression domain of another.

5. Create gain-of-function mutants by generating transgenic plants carrying an ABC gene cDNA under the control of the CaMV35S promoter.

A Model For Control of Floral Organ Type

1

2 3 4

B (AP3, PI)

C (AG)A (AP1, AP2)

sepal petal stamen carpel

Cloning the ABC genesgene method cloned protein identity

AG cloned by TDNA insertion

AP3

MADS-box transcription factor

Deficiens Mutant of Antirrhinum (snapdragon)

AP3 and PI have orthologues in Antirrhinum majus (snap dragon)

• AP3 = Deficiens (Def)• Deficiens was cloned by transposon tagging.• Deficiens encodes a MADS-box transcription

factor.

Cloning the ABC genes

gene method cloned protein identity

AG cloned by TDNA insertion

AP3 cloned by homology to

DEFICIENS• (Deficiens hybridized to a clone from an Arabidopsis cosmid library.

RFLPs Identifying that clone mapped to the AP3 locus).

MADS-box transcription factor

MADS-box transcription factor

AP3 and PI have orthologues in Antirrhinum majus (snap dragon)

• AP3 = Deficiens (Def)• Deficiens was cloned by transposon tagging.• Deficiens encodes a MADS-box transcription

factor.

• PI = Globosa (Glo)• Globosa was cloned by homology to Deficiens.

(Deficiens hybridized to a clone from a floral cDNA library and RFLPs identifying the clone mapped to the position of GLO).

Cloning the ABC genes

gene method cloned protein identity

AG cloned by TDNA insertion

AP3 cloned by homology to

DEFICIENS

PI cloned by homology to

GLOBOSA, a Class B gene

from Antirrhinum. GLO was

cloned by homology to DEF.

MADS-box transcription factor

MADS-box transcription factor

MADS-box transcription factor(GLOBOSA hybridized to a clone from an Arabidopsis floral cDNA library. RFLPs identifying that clone mapped to the PI locus).

Cloning the ABC genes

gene method cloned protein identity

AP1 cloned by homology to

AG.

AP2 cloned by TDNA insertion

DEFICIENS was cloned first followed by AG

MADS-box transcription factor

AP2 transcription factor

AG Blast resultshomology over 56 aa

sequence

AGAMOUS Arabidopsis, Class C, floral organ identity geneNH2-GRGKIEIKRIENTTNRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSSRGRLYEY-COOHDEFICIENS Antirrhinum, Class B, floral organ identity gene ARGKIQIKRIENQTNRQVTYSKRRNGLFKKAHELSVLCDAKVSIIMISSTQKLHEYSERUM RESPONSE FACTOR, human, activates gene in response to growth

factor hormones ARVKIKMEFIDNKLRRYTTFSKRKTGIMKKAYELSTLTGTQCLLLVASETGHVYTFMINI CHROMOSOME MAINTENANCE1, yeast, regulates mating type ERRKIEIKFIENKTRRHVTFSKRKHGIMKKAFELSVLTGTQVLLLVVSETGLVYTF

What do these genes have in common?

Plant Type II MADS-domain protein structure

N MADS I KNH2 COOHC

Region of homology shared between allMADS domain transcription factors

SRF DNA Binding

http://www.bmb.psu.edu/faculty/tan/lab/gallery_protdna.html

The MADS domain binds the core DNA sequence CC[A/T]6GG = CArG box

Plant Type II MADS-domain protein structure

N MADS I KNH2 COOHC

Region of homology shared between MADS domain transcription factors

Region of homology shared between many plant MADS domain transcription factors

(K)eratin domain

AG

NH2QESAKLRQQIISIQNSNRQLMGETIGSMSPKELRNLEGRLERSITRIRSKKNELCOOH

NH2QQNKVLDTKWTLLQEQGTKTVRQNLEPLFEQYINNLRRQLDSIVGERGRLDSELCOOH

Keratin

amino acids with nonpolar side chains: eg. Leucine(L), Methionine (M) Isoleucine (I), Tryptophan (W), Glycine (G), Valine (V)

amino acids with polar side uncharged chains: eg. Serine (S), Threonine (T), Asparagine (N)

Protein alpha helix

http://kbrin.a-bldg.louisville.edu/~rouchka/CECS694/Lecture14_files/image007.jpg

http://www.uic.edu/classes/phyb/phyb516/TM2.jpg

Interaction of amphipathic alpha helices

http://www.uic.edu/classes/phyb/phyb516/TM2.jpg

Two Proteins each with an amphipathic alpha helix can interact to form a coiled-coil

http://myhome.hanafos.com/~s9euno/fig3/fig3-9.gif

SRF DNA Binding

http://www.bmb.psu.edu/faculty/tan/lab/gallery_protdna.html

Prediction for MADS floral organ identity genes

Floral organ-identity MADS genes are DNA binding

proteins that interact with other polypeptides.