Pedigree Analysis & Developmental Genetics

The Story of ‘Eve’ RThis example illustrates why gene regulation is fundamental to development RThe Players RDrosophila embryo REven-skipped gene RRegulatory proteins RBicoid RHunchback RGiant RKruppel RThis example illustrates why gene regulation is fundamental to development RThe Players RDrosophila embryo REven-skipped gene RRegulatory proteins RBicoid RHunchback RGiant RKruppel

The setup RIn Drosophila, the fertilized egg initially performs many rounds of mitosis without cell division - resulting in a cell with many nuclei RThe embryo is 400 um long and 160 um wide RIt has anterior (that will eventually develop into the head) and a posterior (that will develop into the abdomen) RIn Drosophila, the fertilized egg initially performs many rounds of mitosis without cell division - resulting in a cell with many nuclei RThe embryo is 400 um long and 160 um wide RIt has anterior (that will eventually develop into the head) and a posterior (that will develop into the abdomen)

Act 1 RRemoving the cytoplasm from the anterior will result in the failure to develop a head RReplacing this cytoplasm with some taken from the posterior of another embryo will result in an embryo with two tails RRemoving the cytoplasm from the anterior will result in the failure to develop a head RReplacing this cytoplasm with some taken from the posterior of another embryo will result in an embryo with two tails

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Act 2 RLabeling of the 4 proteins - Bicoid, Hunchback, Giant, Kruppel with fluorescent dyes reveals that they are not randomly located inside the cell.

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Act 2 continued RThe nuclei are therefore bathed in differing concentrations of these four proteins - depending on their position in the cell.

Act 3 REven-skipped - ‘EVE’ RIt is a master regulatory gene whose product is a master regulatory protein RThis genes’ promoter region has the ability to bind to all four of the regulatory proteins REven-skipped - ‘EVE’ RIt is a master regulatory gene whose product is a master regulatory protein RThis genes’ promoter region has the ability to bind to all four of the regulatory proteins

Act 4 The Reporter RA reporter gene is an artificial DNA construct used to reveal information RLac Z reporter gene is used extensively  GAL (galactosidase) - Hydrolyzes colorless galactosides to yield colored products. RAttach different parts of the promoter region of the ‘eve’ gene to LacZ to determine if that region is bound and activated by proteins. RA reporter gene is an artificial DNA construct used to reveal information RLac Z reporter gene is used extensively  GAL (galactosidase) - Hydrolyzes colorless galactosides to yield colored products. RAttach 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.jpg Isolate the different regions from the promoter and test in embryo.

08_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.

Curtain RThe regulatory region of ‘Eve’ extends more than 20,000 bp RIt is thought to bind more than 20 different proteins RIt is very sensitive to the position of the gene (nucleus) within the developing giant cell RThe different concentrations of the different proteins impact on the expression of ‘Eve’ RThe regulatory region of ‘Eve’ extends more than 20,000 bp RIt is thought to bind more than 20 different proteins RIt is very sensitive to the position of the gene (nucleus) within the developing giant cell RThe different concentrations of the different proteins impact on the expression of ‘Eve’

Gene Expression RRegions huge distances from the gene have an effect on the activation (or repression) of that gene RThese regions bind proteins that then interact, by DNA looping, with the local promoter regions RA combination effect is seen - and thus the term combinatorial control is used RRegions huge distances from the gene have an effect on the activation (or repression) of that gene RThese regions bind proteins that then interact, by DNA looping, with the local promoter regions RA combination effect is seen - and thus the term combinatorial control is used

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Combinatorial control RNot just by how much are genes regulated but also when. RIt is akin to an orchestra…. RNot just by how much are genes regulated but also when. RIt is akin to an orchestra….

08_24_chromatin.state.jp g Inactive genes are kept inactive by regulatory proteins bound to the DNA

08_25_eye.on.leg.jpg Activation of the wrong regulator can have devastating effects

Pedigree Analysis RGreat Primer on the lab section of the website RLinkLink RGreat Primer on the lab section of the website RLinkLink