Download Pedigree Analysis & Developmental Genetics

Pedigree Analysis
&
Developmental Genetics
The Story of ‘Eve’
 This example illustrates why gene regulation is
fundamental to development
 The Players
 Drosophila embryo
 Even-skipped gene
 Regulatory proteins
 Bicoid
 Hunchback
 Giant
 Kruppel
The setup
 In Drosophila, the fertilized egg initially
performs many rounds of mitosis without cell
division - resulting in a cell with many nuclei
 The embryo is 400 um long and 160 um wide
 It has anterior (that will eventually develop
into the head) and a posterior (that will
develop into the abdomen)
Act 1
Removing the cytoplasm from the
anterior will result in the failure to
develop a head
Replacing this cytoplasm with
some taken from the posterior of
another embryo will result in an
embryo with two tails
08_16_anterior_posteri.jp
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Act 2
Labeling of the 4 proteins - Bicoid,
Hunchback, Giant, Kruppel with
fluorescent dyes reveals that they
are not randomly located inside
the cell.
08_17_4.gene.reg.prot.jp
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Act 2 continued
The nuclei are therefore bathed in
differing concentrations of these
four proteins - depending on their
position in the cell.
Act 3
Even-skipped - ‘EVE’
It is a master regulatory gene
whose product is a master
regulatory protein
This genes’ promoter region has
the ability to bind to all four of the
regulatory proteins
Act 4 The Reporter
 A reporter gene is an artificial DNA construct
used to reveal information
 Lac Z reporter gene is used extensively
 GAL (b-galactosidase) - Hydrolyzes colorless
galactosides to yield colored products.
 Attach different parts of the promoter region
of the ‘eve’ gene to LacZ to determine if that
region is bound and activated by proteins.
Isolate the different regions from the promoter and test in
embryo.
08_18_reporter.gene.jpg
The same STRIPE 2 region actually has binding sites for all
four of regulatory proteins - Bicoid and Hunchback are
activators of this region and Giant and Kruppel are repressors.
08_19_eve.stripe.2.jpg
Curtain
 The regulatory region of ‘Eve’ extends more
than 20,000 bp
 It is thought to bind more than 20 different
proteins
 It is very sensitive to the position of the gene
(nucleus) within the developing giant cell
 The different concentrations of the different
proteins impact on the expression of ‘Eve’
Gene Expression
Regions huge distances from the gene
have an effect on the activation (or
repression) of that gene
These regions bind proteins that then
interact, by DNA looping, with the local
promoter regions
A combination effect is seen - and thus
the term combinatorial control is used
08_15_Reg. proteins.jpg
Combinatorial control
Not just by how much are genes
regulated but also when.
It is akin to an orchestra….
Inactive genes are kept inactive by regulatory proteins bound
to the DNA
08_24_chromatin.state.jp
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Activation of the wrong regulator can have devastating effects
08_25_eye.on.leg.jpg
Pedigree Analysis
Great Primer on the lab section of
the website
Link