apetala1 mutant. testing the abc floral-organ identity model: cloning the genes objectives: to test...
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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.