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