BiolChem 473

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Biol/Chem 473
Schulze lecture 5 Eukaryotic gene regulation
Early Drosophila development
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Maternal genes

• Required before the embryo starts transcribing
  its own genes
• Mother expresses these genes during oogenesis
  (mum packs a lunch)
• Localization of maternal gene products is the
  first step in the cascade

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Gap genes

• The gap genes are expressed by the zygote
  (activated in the syncytial blastoderm)
• Their expression patterns are regulated by the
  maternal gene products, and they regulate each
  other
• They divide the embryo into broad, unique domains
  containing different combinations of
  transcription factors

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Pair-rule genes

• Pair-rule genes are expressed just before
  cellularization in patterns of 7 stripes (half
  the number of segments in a wild type larva)
• Some pair-rule genes respond to patterns already
  laid down by the maternal and gap genes, while
  others are regulated by other pair-rule genes
• Pair rule gene expression is very dynamic!
  (pretty pictures)

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Segment polarity genes

• Segment polarity genes are expressed at the onset
  of gastrulation
• They are expressed in 14 segment bands, refining
  the patterns of all the previous genes in the
  hierarchy. Maternal, gap and pair-rule gene
  expression begins to fade away

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Stripe boundaries are precisely defined
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Control regions upstream of pair-rule genes are
complex
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ASSIGNED PAPERTranscriptional regulation of a
pair-rule stripe in Drosophila
Small, S. et al., Genes Development (1991) 5
827-839
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Main players

• Even-skipped (eve) is a homeo-box containing
  transcription factor.
• Hunchback (hb) has multiple Zn fingers.
• Bicoid (bic) is a homeo-box containing
  transcription factor.
• Kruppel (Kr) contains a Zn finger.
• Giant (gt) contains a Leucine zipper.

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is red
• hunchback protein is green
• overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is red
• Kruppel protein is green
• overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is green
• giant protein is red
• Overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2
High levels of gt
High levels of Kr
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Wild type expression patterns of putative
regulators of eve stripe 2
High levels of gt
High levels of Kr
What predictions arise from this picture?
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eve-lacZ gene fusion
Posterior end of embryo (where germ line will
form)
This is a transgene gets injected into the
embryo (P transformed embryos), integrates into
the genome and is expressed stably along with all
the other genes. BUT this transgene will express
lacZ in an even-skipped-stripe 2 dependent manner!
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eve-lacZ gene fusion
This is a transgene gets injected into the
embryo (P transformed embryos), integrates into
the genome and is expressed stably along with all
the other genes. BUT this transgene will express
lacZ in an even-skipped-stripe 2 dependent manner!
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Questions to ask about in vivo expression patterns

• How do we know the order in which patterning
  genes act in development?
• How were potential trans-acting regulators of
  stripe 2 identified?
• What would happen to the domain of expression of
  eve stripe 2 in giant or Kruppel mutant embryos?
• Do these in vivo expression patterns indicate
  direct interaction between Kr and gt and the eve
  stripe 2 regulatory region?

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Are these interactions direct?
Sequencing lanes (Ye Olde Fashionde Maxim Gilbert)
Control lanes
Footprints represent regions in the eve upstream
regulatory region that are protected by the
bicoid protein. What does this imply?
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Questions to ask about DNA footprinting analysis

• Do the sites show differing affinity for their
  respective trans-acting factors?
• Do the sites for different trans-acting factors
  overlap?
• What could be the explanation for differential
  affinity?

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Regulator binding sites in the eve stripe 2
regulatory region
eve skipped promoter
What does the tight linkage of these sites
suggest? (Hint compare bcd and Kr binding site
sequences)
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Regulator binding sites in the eve stripe 2
regulatory region
eve skipped promoter
Bicoid binding sites are solid circles
hunchback binding sites are shaded circles
Kruppel binding sites are hatched boxes
giant binding sites open boxes
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Using reporter genes to dissect regulatory
sequences
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Using reporter genes to dissect regulatory
sequences
bcd OR hb OR Kr OR gt protein coding sequence
CAT in this paper
ACT5 promoter
Hsp70 TATA in this paper
Schneider cells (cultured fly cells) in this paper
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CAT assays
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Recipe for CAT assay

• CAT chloramphenicol acetyltransferase
• Perform assay in cell system (transform/transfect
  cells etc)
• Extract proteins
• Mix with hot (radioactively labeled)
  chloramphenicol
• Add substrate acetyl coenzyme A
• If CAT has been produced by your extract, it will
  transfer acetyl groups from Acetyl coenzyme A to
  chloramphenicol
• Thin layer chromatography to separate out the
  products
• Autoradiography to measure amount of acetylated,
  radioactive chloramphenicol (which is
  proportional to how much CAT was made which is a
  reflection of how well the CAT reporter was
  activated).

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Organic Chickenwire figure
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(No Transcript)
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CAT assay results Table 2
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Questions to ask about CAT assays

• What is Kr9 ? What does this experiment control
  for?
• How does figure 6 support the implication of this
  control?

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Discussion

• How strict is this developmental hierarchy in
  Drosophila development? (Example?)
• What contributes to sharpening the borders of
  gene expression? (Example?)
• How do stripe initiation elements function
  autonomously of each other?

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Control regions upstream of pair-rule genes are
complex
General principle regulatory regions of
eukaryotic genes are complex the promoter of any
given gene has to integrate a large amount of
combinatorial inputs that will define its
activity depending on the context of the cell in
which the gene resides. (Are bicoid levels high
or low? What about Kruppel? Etc. )
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Exam stats Prody
Average 40/60
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Exam stats Schulze
Mean 25/40 Median 26/40