Pedigree Analysis

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Pedigree Analysis Developmental Genetics
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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

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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)

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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

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08_16_anterior_posteri.jpg
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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.

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08_17_4.gene.reg.prot.jpg
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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.

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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

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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.

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

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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

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08_15_Reg. proteins.jpg
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Combinatorial control

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

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08_24_chromatin.state.jpg
Inactive genes are kept inactive by regulatory
proteins bound to the DNA
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08_25_eye.on.leg.jpg
Activation of the wrong regulator can have
devastating effects
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Pedigree Analysis

• Great Primer on the lab section of the website
• Link