Transcript Cloning the ABC genes

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
sepal
petal
1
2
stamen
carpel
4
3
B (AP3, PI)
A (AP1, AP2)
C (AG)
Cloning the ABC genes
gene
method cloned
protein identity
AG
cloned by TDNA insertion
MADS-box transcription factor
AP3
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
AG
AP3
cloned by TDNA insertion
protein identity
MADS-box transcription factor
cloned by homology to
MADS-box transcription factor
DEFICIENS
• (Deficiens hybridized to a clone from an Arabidopsis cosmid library.
RFLPs Identifying that clone mapped to the AP3 locus).
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 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
MADS-box transcription factor
AP3
cloned by homology to
DEFICIENS
MADS-box transcription factor
PI
cloned by homology to
GLOBOSA, a Class B gene
from Antirrhinum. GLO was
cloned by homology to DEF.
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
MADS-box transcription factor
AP2
cloned by homology to
AG.
cloned by TDNA insertion
AP2 transcription factor
DEFICIENS was cloned first followed by AG
AG Blast results
homology over 56 aa
sequence
AGAMOUS Arabidopsis, Class C, floral organ identity gene
NH2-GRGKIEIKRIENTTNRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSSRGRLYEY-COOH
DEFICIENS Antirrhinum, Class B, floral organ identity gene
ARGKIQIKRIENQTNRQVTYSKRRNGLFKKAHELSVLCDAKVSIIMISSTQKLHEY
SERUM RESPONSE FACTOR, human, activates gene in response to growth
factor hormones
ARVKIKMEFIDNKLRRYTTFSKRKTGIMKKAYELSTLTGTQCLLLVASETGHVYTF
MINI CHROMOSOME MAINTENANCE1, yeast, regulates mating type
ERRKIEIKFIENKTRRHVTFSKRKHGIMKKAFELSVLTGTQVLLLVVSETGLVYTF
What do these genes have in common?
Plant Type II MADS-domain protein
structure
NH2
N
MADS
Region of homology
shared between all
MADS domain
transcription factors
I
K
C
COOH
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
NH2
N
MADS
Region of homology
shared between
MADS domain
transcription factors
I
K
Region of homology
shared between
many plant MADS domain
transcription factors
C
COOH
(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://www.uic.edu/classes/phyb/phyb516/TM2.jpg
http://kbrin.a-bldg.louisville.edu/~rouchka/CECS694/Lecture14_files/image007.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.