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Developmental Biology > Lecture 12 - Homeotic genes in flower development and the ABC model > Flashcards

Lecture 12 - Homeotic genes in flower development and the ABC model Flashcards

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1
Q

What is the ABC model for organ identity?

A

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)

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2
Q

What genes are involved in the ABC model for organ identity?

A
  • homeotic genes
  • mutatns give homeobotic transformations
  • class a, b, and c
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3
Q

What do class a, b, and c genes normally control?

A 
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)
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4
Q

What results from a class A mutant?

A

Four whorls:
-carpel, stamen, stamen, carpel
Genetically:
c, bc, bc, c

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5
Q

Waht results from a class B mutant?

A

Four whorls:
-sepal, sepal, carpel, carpel
Genetically:
a, a, c, c,

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6
Q

What results from a class C mutant?

A

Four whorls:
-sepal, petal, petal, sepal
Genetically:
-a, ab, ab, a

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7
Q

What is transposon mutagenesis?

A
  • exploit natural transposons to generate insertional mutants
    e. g. in antirrhinum majus
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8
Q

What is a transposon/transposable element?

A

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

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9
Q

What is a trasposase?

A

a class of enxyme ecoded within a transposable element that catalyses the excision and insertion of a transposable element

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10
Q

What is transposon tagging?

A

a method of finding mutations and cloning a gene using the random insertion of transposons

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11
Q

What is the phenotype of a plena mutant?

A

class c mutant

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12
Q

What is the phenotype of an ovulata mutant?

A

a class mutant

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13
Q

How was the molecular basis of ple mutations identified?

A

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
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14
Q

How do you carry out a southern blot?

A
  • 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
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15
Q

What was identified about revertent ple mutations through the southern hybridisation technique? And what was this technique?

A
  • 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)
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16
Q

What is the plena gene from antirrhinum?

A

AGAMOUS

17
Q

What are teh key technique hypothesis and model building to explain the gain of function ovulata mutants?

A
  1. A gain of function of ple might be expected to give a complementary (or opposite) phenotype (ABC model antagonistic function)
  2. The ovulata mutant was semi-dominant to wild type as expected for gain of function mutants (fits with hypothesis)
  3. 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?
18
Q

What experimental technique was used to confirm that ovulata mutants are ple alleles?

A
  • 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)

19
Q

How did genetics confirm that ovulata mutants are ple alleles?

A
  • 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
20
Q

What experiment showed that plena is ectopically expressed in ovulata mutants, and what else did it show?

A
  • 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

21
Q

What did in situ hybridisations reveal about ple, def and flo expression?

A 
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
22
Q

What is the expression the ple transcript in recessive ple alleles?

A

-little or no ple transcript is detected in recessive ple alleles indicating that the phenotype they confer corresponds to the loss of ple activity

23
Q

What occurs in the absense of the ple gene?

A

petal and sepals grow in the place of stamens and carpels

24
Q

Where is ple expressed?

A

whorls 3 and 4

25
Q

What do in situ hybridisation of ovulata meristems probed with ple show?

A

ple is expressed ectopically

26
Q

What alleles of ple can confer the ovulata phenotype?

A

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
27
Q

What is the result of the ple transcript being expressed in all 4 whorls in floral meristems?

A
  • 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
28
Q

What is the model to explain the plena and ovulata phenotypes?

A

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

Developmental Biology (14 decks)

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