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

Transcription

1 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.

2 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.

3 A Model For Control of Floral Organ Type sepal petal stamen carpel B (AP3, PI) A (AP1, AP2) C (AG)

4 Cloning the ABC genes gene method cloned protein identity AG cloned by TDNA insertion MADS-box transcription factor There are 22 MADS-box transcription factors in Arabidopsis thaliana. AP3

5 Deficiens Mutant of Antirrhinum (snapdragon)

6 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?

7 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 (Deficiens hybridized to a clone from an Arabidopsis cosmid library. RFLPs Identifying that clone mapped to the AP3 locus).

8 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 ap3 and 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).

9 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).

10 Cloning the ABC genes gene method cloned protein identity AP1 AP2 cloned by homology to AG. cloned by TDNA insertion MADS-box transcription factor AP2 transcription factor DEFICIENS was cloned first followed by AG

11 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, transcription factor activates genes in response to growth factor hormones ARVKIKMEFIDNKLRRYTTFSKRKTGIMKKAYELSTLTGTQCLLLVASETGHVYTF MINI CHROMOSOME MAINTENANCE1, yeast, transcription factor regulates mating type ERRKIEIKFIENKTRRHVTFSKRKHGIMKKAFELSVLTGTQVLLLVVSETGLVYTF What do these genes have in common?

12 Plant Type II MADS-domain protein structure NH2 N MADS I K C COOH Region of homology shared between all MADS domain transcription factors

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

14 Plant Type II MADS-domain protein structure NH2 N MADS I K C COOH Region of homology shared between MADS domain transcription factors Region of homology shared between many plant MADS domain transcription factors

15 (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)

16 Protein alpha helix

17 Interaction of amphipathic alpha helices

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

19 SRF DNA Binding CC[A/T] 6 GG

20 Prediction for MADS floral organ identity genes Floral organ-identity MADS genes are DNA binding proteins that interact with other polypeptides.