Question

YFG is a transcription factor that binds to the promoter of RIP, a gene that encodes for a

protein that facilitates cellular secretion. YFG is required for RIP expression and

reduction of RIP levels are linked to neuromuscular disorders, such as muscular

dystrophy (MS). You discover 3 different point mutations in YFG in 3 different MS

patients. You predict these mutations interfere with the stability of YFG. To test this, you

create a luciferase reporter gene vector and transfect this vector into a muscle cell line.

The table below shows the measurements of luciferase enzyme activity after the addition

of luciferin

YFG in cell line	Units of luciferase activity
WT YFG	500
YFG w/ nt177 G->C	75
YFG w/ nt356 A->T	320
YFG w/ nt598 G->A	490

1. Draw the design of your luciferase reporter vector below:
2. What do the results in the table above mean and what are “units of luciferase activity”? What does higher luciferase activity indicate?

1. C. You suspect that there might be variation between your different cell lines leading to inaccurate results. Specifically, you think some cell lines may be expressing YFG at higher levels, which artificially is causing an increase in units of luciferase activity. This is preventing you from determining how the mutations are affecting luciferase production. How would you set up a control to solve this problem?

Answer 1

the upper table shows that the mutation which gives more luciferase activity expressing more the gene of interest which is only possible when the promoter is bound by transcription factor YFG.

SO less amount of luciferase activity like 75, in this case the G to C mutation very dangerous and hugely affected the interaction of YFG with promoter.

Whereas, G to A don't affected the interaction so much.

Luciferase activity has no unit. It is measured in amount of light.

Higher luciferase activity means higher binding between promoter and transcription factor.

C) then you can use the amount of YFG AS A CONTROL respectively.

And then you can deduce the results accordingly with reference with amount of YFG