Lecture 12 - Homeotic genes in flower development and the ABC model Flashcards
What is the ABC model for organ identity?
Overlap between different activities generates disctinct combinations for each whorl
-A and C fucntions have complimentary expression patterns (without A, C replaces A and vv)
What genes are involved in the ABC model for organ identity?
- homeotic genes
- mutatns give homeobotic transformations
- class a, b, and c
What do class a, b, and c genes normally control?
Class a -sepals and petal Class B -petal and stamens Class C -stamens and carpels
A - sepal ab - petal bc - stamen C - carpel (A and C have mutually antagonistic functions)
What results from a class A mutant?
Four whorls:
-carpel, stamen, stamen, carpel
Genetically:
c, bc, bc, c
Waht results from a class B mutant?
Four whorls:
-sepal, sepal, carpel, carpel
Genetically:
a, a, c, c,
What results from a class C mutant?
Four whorls:
-sepal, petal, petal, sepal
Genetically:
-a, ab, ab, a
What is transposon mutagenesis?
- exploit natural transposons to generate insertional mutants
e. g. in antirrhinum majus
What is a transposon/transposable element?
a discrete DNA sequence element that can move from one location in the genome to another, often iunder the control of a transposase encoded by the element itself
What is a trasposase?
a class of enxyme ecoded within a transposable element that catalyses the excision and insertion of a transposable element
What is transposon tagging?
a method of finding mutations and cloning a gene using the random insertion of transposons
What is the phenotype of a plena mutant?
class c mutant
What is the phenotype of an ovulata mutant?
a class mutant
How was the molecular basis of ple mutations identified?
Southern blots of wild-type, ple mutant, and reverant geenomic DNA cut with EcoRI
- probed at low stringency with an AGAMOUS gene from arabidopsis
- most bands are the same size
- but key bands are of different sizes (different in the ple mutant)
- through southern hybrisation
- AGAMOUS is a class c gene
How do you carry out a southern blot?
- take genomic DNA and cut with restriction exyme into smaller fragments
- run a southern blot of 2.5mg/5mg
- produces a smear, bands with different intensities in the smear
- repeat with the fragments
- treat gel with acid to remove purines in the DNA
- this forms ssnicks in the DNA
- add alkaline to denature teh DNA (ssDNA)
- return gel to neurtral solution
- add nitrole membrane on top of DNA and piece of tissue (sucks liquid out of gel can transfers ssDNA to the membrane)
- fix DNA on the position it was on the gel
- can apply probes to the membrane which are typically labelled with radioactivity
- can produce strands of DNA with radiactivity
- then hybridise (at 55*C) the probe to the nylon membrane
- put against film and develop
- can see where probe binds to DNA and can identify the particular position of the of band and find reciprocal pieces of DNA
What was identified about revertent ple mutations through the southern hybridisation technique? And what was this technique?
- southern blots of wild type, ple mutants a dnreventant genomic DNA cut with EcoRI
- probed at high stringency with a subclone of the ple locus
- confirms that the PLE gene carries a transposon insertion in the mutant alleles
- REVERTANTS= have a wild type band
- this links the reversion of wild type to a moecular event (the location of the transposon)
What is the plena gene from antirrhinum?
AGAMOUS
What are teh key technique hypothesis and model building to explain the gain of function ovulata mutants?
- A gain of function of ple might be expected to give a complementary (or opposite) phenotype (ABC model antagonistic function)
- The ovulata mutant was semi-dominant to wild type as expected for gain of function mutants (fits with hypothesis)
- plants carrying the ple-625 allele sometime gave progeny with the ovulata mutant (at a higher frequency than would be expected) indictating that the ovulata phenotype might result from alterations at ple
Most obvious conclusion is that ovulata mutants are a class mutants is not consistent with the evidence in 2 and 3 Are they ple alleles?
What experimental technique was used to confirm that ovulata mutants are ple alleles?
- ple probes were used on a southern blot of genomic DNA from ovulata mutants
- the ovulata mutant isolated from the transposon mutagenesis experiemtn was shown to carry a Tam3 transposon insertion in the large intron of ple
- the ovulata phenotype segregated with the insertion confirming that this phenotype was caused by an allele of ple
- three ovulata mutants that arose from the unstable ple-625 line were analysed and shown to have alterations at the ple locus
Confirms that ovulata mutants are ple alleles (although they have the opposite phenotype)
How did genetics confirm that ovulata mutants are ple alleles?
- plena has a particularly large 2nd intron with a transposon insertion
- ovulata genotypes are generated by the same transposon insertion as ple-625 but in the opposite orientation
What experiment showed that plena is ectopically expressed in ovulata mutants, and what else did it show?
- ran a northern blot of the total RNA from leaves or inflorescences probed with the 3’ end of the ple cDNA clone
- for influorescenes RNA was extracted from WT, two different plena mutants (ple-624 and ple-625) and two ovulata mutants (ple-627 and ple-649)
- in the plena phenotype, transposon activity altered the levels of expression the the gene whereas in the ovulata mutant there was ectopic expression
Also did a northern blot of the total RNA from leaves, bracts and floral whorls from wild type and the ovulata mutant ple-627 probed with the 3’ end of the ple cDNA
- in WT only expressed in whorls 3,4
- consistently expressed in the ovulata mutant
This is most likely to be responsible for the ovulata phenotype
What did in situ hybridisations reveal about ple, def and flo expression?
ple (c class)expressed in stamen and carpets not whirls 1 and 2 def (b class) expressed whirls 2 and 3, expressed earlier than ple flo (floral meristem identity gene) expressed early
What is the expression the ple transcript in recessive ple alleles?
-little or no ple transcript is detected in recessive ple alleles indicating that the phenotype they confer corresponds to the loss of ple activity
What occurs in the absense of the ple gene?
petal and sepals grow in the place of stamens and carpels
Where is ple expressed?
whorls 3 and 4
What do in situ hybridisation of ovulata meristems probed with ple show?
ple is expressed ectopically
What alleles of ple can confer the ovulata phenotype?
semindominant alleles of pkle
- representing a gain of ple activity
- in these mutants ple transcript is found in all tissues - flowers, leaves, stems and roots
What is the result of the ple transcript being expressed in all 4 whorls in floral meristems?
- gain of expression in whorl 1 promotes carpel development
- in whorl 2 it promotes stamen production
- ectopic expression of ple leads to the activation of the c function, so that sex organs develop in the place of perianth organs
What is the model to explain the plena and ovulata phenotypes?
WT
-represoor binds to second introns and represses the activity in whorls 1 and 2, but in whorls 3 and 4 it is translated
plena
-a stop codon is inserted into the transcripti which leads to same activity as the repressor, blocking transcript in whorls 1,2,3 and 4
ovulata
-oppositely oritentated transposon insertion means that the repressor cannot bind so that 1, 2, 3, and 4 are expressed
