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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.

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Pedigree Analysis & Developmental Genetics

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  1. Pedigree Analysis& Developmental Genetics

  2. 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

  3. 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)

  4. 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

  5. 08_16_anterior_posteri.jpg

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

  7. 08_17_4.gene.reg.prot.jpg

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

  9. 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

  10. 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.

  11. Isolate the different regions from the promoter and test in embryo. 08_18_reporter.gene.jpg

  12. 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

  13. 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’

  14. 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

  15. 08_15_Reg. proteins.jpg

  16. Combinatorial control • Not just by how much are genes regulated but also when. • It is akin to an orchestra….

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

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

  19. Pedigree Analysis • Great Primer on the lab section of the website • Link

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