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Flashcards in Paper questions Deck (18):
1

Questions to ask about in vivo expression patterns

 

 

How do we know the order in which patterning genes act in development?

  • Knock out mutants allow us to see, in a one by one way, what happens when certain genes are knocked out. 
     
  • In this way, we can see what happens when certain developmental proteins are knocked out and how this affects patterning of development. 

2

Questions to ask about in vivo expression patterns

 

How do we isolate stripe 2 eve expression, and why is that important?

  • You can isolate stripe 2 eve expression by
    • Cloning the eve stripe 2 gene into a plasmid
    • Within the plasmid, include truncated promoter containing 1.7 kb of eve 5'-flanking sequence.
      • This is sufficient to drive the expression of a reporter gene (lacZ) within the limits of stripe 2.
  • Stripe 2 eve is important as previous promoter fusion analyses and genetic studies have provided considerable information about the cis and trans components that participate in its localized expression.
    • Potential trans-regulators of stripe 2 have been identified on the basis of examining the distribution of eve protein in all known segmentation mutants
    • These studies, as well as the tight linkage of the wild-type expression patterns, suggest that the gap genes hunchback (hb), giant (gt), and Krfippel (Kr) are the most likely candidates for regulators of stripe 2 expression.

3

shiQuestions to ask about in vivo expression patterns

 

 

How were potential trans-acting regulators of stripe 2 identified?

4

Questions to ask about in vivo expression patterns

 

 

What would happen to the domain of expression of eve stripe 2 in giant or Kruppel mutant embryos?

5

Questions to ask about in vivo expression patterns

 

 

Do these vivo expression patterns indicate direct interaction between Kr and gt and the eve stripe 2 regulatory region?

6

How was CAP activation produced.controled?

  • Adenyl cyclase, which is regulated by glucose. 
  • So they used glucose to control CAP activation.
  • Low Glucose -> 
  • High Glucose -> 

7

Are these interactions direct?

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8

Questions to ask about DNA footprinting analysis

 

Do the sites show differing affinity for their respective trans-acting factors?

9

Questions to ask about DNA footprinting analysis

 

Do the sites for different trans-acting factors overlap?

10

Questions to ask about DNA footprinting analysis

 

 

What could be the explanation for differential affinity?

11

Regulator binding sites in the eve stripe 2 regulatory region

 

What does the tight linkage of these sites suggest?

(Hint: compare bcd and Kr binding site sequences)

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12

What is happening at each red arrow?

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13

Using reporter genes to dissect regulatory sequences

 

In relation to the paper, what is each arrow pointing to?

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14

Questions to ask about CAT assays

 

What is Kr9 and gt B4?

What do these experiments show?

What does Figure 6 have to do with this?

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15

Discussion

 

How strict is this developmental hierarchy in Drosophila development? (Example?)

16

Discussion

 

What contributes to sharpening the borders of gene expression? (Example?)

17

Discussion

 

How do stripe initiation elements function autonomously of each other?

18

Discussion

 

What is the mechanism by which positive and negative regulators interact? (competition vs. quenching)