Question

Remember: Crx is a key retinal transcription factor. Crx binds regulatory elements called CBRs (Crx-binding regions).

You use the DNA sequences from the patients to create plasmids for a luciferase assay.

In a luciferase assay you add your promoter and any regulatory regions of interest to a vector containing the gene for luciferase. You then transform bacteria with the vector (you make bacteria take up the vector). The transcription of luciferase is controlled by the promoter/regulatory region you added to the vector (the region that normally regulates GADD expression).

After letting the bacteria grow for a few days you can extract any protein made in the cells and check for luciferase activity. You mix luciferase substrates with the protein you extracted from the cells and if there is luciferase present the reaction luciferase catalyzes will produce light (luciferase is what makes fireflies “glow”).

The luciferase assay results are below. Note, results are relative to wildtype/unmutated:

CBR1 mutation (Patient 1): 70%

CBR2 mutation (Patient 2): 60%

CBR1 and CBR2 mutation (Patient 3): 20%

If you were to measure GADD protein levels in the three patients, what would you expect to see?

1. Lower than normal/wildtype levels of GADD protein.

2. More than normal/wildtype levels of GADD protein.

Are these mutations gain-of-function mutations, or loss-of-function mutations?

1. Gain-of-function.

2. Loss-of-function.

What type of transcription factor is Crx?

1. Silencer.

2. Enhancer.

3. Activator.

4. Repressor.

5. Core promoter.

6. Retinal specific RNA polymerase.

Answer 1

Question 1: Answer is - 1. Lower than normal/wildtype levels of GADD proteins

CBR1, CBR2 & both CBR1-CBR2 mutation leads to 70%, 60% & 20% expression of GADD relative to wildtype respectively. So, all of the mutation don't lead to 100% expression. Thus, mutations cause lowering of expression.

Question 2: Answer is loss of function.

Loss of function mutation leads to lesser expression of GADD.

Question 3: Answer is activator.

Crx is not part of core promoter. Because if it is a part of core promoter then mutation of both CBR1 & CBR2 would lead to no expression of GADD. But, there is 20% expression of GADD. So, Crx must be activator.