Part 4: Gene regulation (2)

Question 1

What is enhancer

Answer 1

• It is a part of regulatory sequences that when bound by TFs enhances transcription:
• Position and orientation independent
• Can be as far as 100kb away from promoter
• Regulate time, place and amount of transcription

Question 2

What are boundary elements or insulators?

Answer 2

Molecules that bind enhancer and inhibit its activity

Question 3

Promoter trap

Answer 3

Reporter gene construct lacking promoter, only expressed when is inserted into another gene

Question 4

Enhancer trap

Answer 4

Reporter gene construct that has no enhancer, only expressed when lies next to another gene

Question 5

Use of enhancer and promoter traps

Answer 5

• Identify position of enhancers and promoters functioning during different stages of development
• Identify genes transcribed during different stages of development

Question 6

Describe enhancer/promoter trap experiment

Answer 6

1) Make a construct in the E. coli plasmid and isolate the plasmid DNA
2) Insert into the organism of interest using transposon and nuclease (random insertion)
3) Screen the expression of reporter gene
4) Clone the gene and its regulatory sequence that are close to the trap

Question 7

Which one of the following statements is incorrect?
a) Both enhancer and promoter traps are specially engineered reporter gene constructs
b) Both enhancer and promoter traps are used to
identify genes with specific expression patterns
c) The reporter gene in an enhancer trap is expressed
only if it’s inserted within a gene
d) The reporter gene in a promoter trap is expressed
only if it’s inserted inside a gene
e) Promoter traps can be used to find knockout mutants
of genes with specific expression patterns

Answer 7

c) Enhancer trap can be near the regulatory sequence of the gene, but not necessarily within the gene

Question 8

How can we find the sequence next to the reporter gene?

Answer 8

Restriction digest followed by inverse PCR and DNA sequencing

Question 9

A researcher interested in expressing a foreign
protein in the pancreas finds a mouse in an
enhancer trap population with B-galactosidase only
in the pancreas. To clone the pancreatic specific
promoter she needs to isolate:
a) Genomic DNA from the pancreas
b) Genomic DNA from any tissue
c) mRNA from the pancreas
d) mRNA from any tissue
e) cDNA from the pancreas

Answer 9

Need DNA from any tissue since genome is the same in all cells, RNA will not contain promoter sequence