Pedigree Analysis & Developmental Genetics. The Story of ‘Eve’ RThis example illustrates why...
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Transcript of Pedigree Analysis & Developmental Genetics. The Story of ‘Eve’ RThis example illustrates why...
Pedigree Analysis&
Developmental Genetics
Pedigree Analysis&
Developmental Genetics
The Story of ‘Eve’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
This example illustrates why gene regulation is fundamental to development
The Players Drosophila embryo Even-skipped gene Regulatory proteins
Bicoid Hunchback Giant Kruppel
The setupThe 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)
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 1Act 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
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.jpg08_16_anterior_posteri.jpg
Act 2Act 2
Labeling of the 4 proteins - Bicoid, Hunchback, Giant, Kruppel with fluorescent dyes reveals that they are not randomly located inside the cell.
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.jpg08_17_4.gene.reg.prot.jpg
Act 2 continuedAct 2 continued
The nuclei are therefore bathed in differing concentrations of these four proteins - depending on their position in the cell.
The nuclei are therefore bathed in differing concentrations of these four proteins - depending on their position in the cell.
Act 3Act 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
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 ReporterAct 4 The Reporter
A reporter gene is an artificial DNA construct used to reveal information
Lac Z reporter gene is used extensively GAL (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.
A reporter gene is an artificial DNA construct used to reveal information
Lac Z reporter gene is used extensively GAL (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.
08_18_reporter.gene.jpg08_18_reporter.gene.jpgIsolate the different regions from the promoter and test in embryo.
08_19_eve.stripe.2.jpg08_19_eve.stripe.2.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.
CurtainCurtain
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’
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 ExpressionGene 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
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.jpg08_15_Reg. proteins.jpg
Combinatorial controlCombinatorial control
Not just by how much are genes regulated but also when.
It is akin to an orchestra….
Not just by how much are genes regulated but also when.
It is akin to an orchestra….
08_24_chromatin.state.jpg08_24_chromatin.state.jpg
Inactive genes are kept inactive by regulatory proteins bound to the DNA
08_25_eye.on.leg.jpg08_25_eye.on.leg.jpgActivation of the wrong regulator can have devastating effects
Pedigree AnalysisPedigree Analysis
Great Primer on the lab section of the website
Link
Great Primer on the lab section of the website
Link