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

There are 22 MADS-box transcription factors in Arabidopsis thaliana.

AP3

MADS-box transcription factor

Deficiens Mutant of Antirrhinum(snapdragon)

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

• The deficiens mutant (def) has a phenotype similar to ap3 and pi

• Deficiens was cloned by transposon tagging.• Deficiens encodes a MADS-box transcription

factor.

How can we use this information to find the AP3 or PI gene sequence?

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 counterparts in Antirrhinum majus (snap dragon)

• The deficiens mutant (def) has a phenotype similar to ap3 and pi

• Deficiens was cloned by transposon tagging.• Deficiens encodes a MADS-box transcription factor.

• The globosa mutant (glo) has a phenotype similar to ap3and pi

• Globosa was cloned by homology to Deficiens. (Deficiens hybridized to a clone from an Antirrhinum majus 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 wascloned 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 genesgene method cloned protein identity

AP1 cloned by homology toAG.

AP2 cloned by TDNA tagging

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, transcription factor activates genes in

response to growth factor hormonesARVKIKMEFIDNKLRRYTTFSKRKTGIMKKAYELSTLTGTQCLLLVASETGHVYTF

MINI CHROMOSOME MAINTENANCE1, yeast, transcription factor 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

AGNH2QESAKLRQQIISIQNSNRQLMGETIGSMSPKELRNLEGRLERSITRIRSKKNELCOOHNH2QQNKVLDTKWTLLQEQGTKTVRQNLEPLFEQYINNLRRQLDSIVGERGRLDSELCOOHKeratin

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

amino acids with polar, uncharged side-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.htmlCC[A/T]6GG

Prediction for MADS floral organ identity genes

Floral organ-identity MADS genes encodes DNA binding

proteins that interact with other polypeptides.