Question

You are conducting an experiment identifying enhancers that regulate the expression of a gene that codifies for a protein that participates in cell division. The gene is called Mitosis Regulatory Protein A or MRPA. The complete DNA sequences for the MRPA promoter and coding region have been identified. However, it is unknown if MRPA has enhancers regulating its transcription. To answer this question your lab first produced a transgenic cell line where GFP has been inserted as a reporter gene. Then different cell lines were obtained, each one with mutations in specific genomic regions, upstream from MRPA. You ran standardized experiments measuring GFP activity in each cell line by triplicate. The following table shows the location of the genomic region mutated, GFP activity, measured as photons per second per milligram of cells, and statistically significant groups (i.e. different letters indicated significant differences among experimental groups):

GFP Activity deviation 48 + 5 Experimental Groups (mean t standardStatistical Groups Normal cells without GFP (Negative Control) Transfected Cells with construct containing GFP and 6000 base pairs of the genomic region upstream of MRPA (see diagram below) 1186 t 76 Genomic Location of Mutations, in base pairs Cell Lineupstream (+) from MRPA original locatiorn 10 50 100 150 500 600 1000 - 1100 2500 - 2600 5000 - 5250 59 t 12 1315 t 113 657 + 42 1127 + 85 947 + 97 359 + 52 3 4 6 MRPA gene substituted by GFP Genomic Region of 6000 base pairs upstream of MRPA GFP 0 +6000 Putative Regulatory Sequence a) How many enhancers were you able to identify with these set of experiments? Explain your answer. b) Assuming that the enhancer(s) identified are all the regions that regulate the transcription of MRPA, design an experiment to estimate the basal transcription activity for this gene. Hint: think in the experiments implemented to understand the functioning of the lac operon in E. coli and how the mutants were combined.

Answer 1

a) 3 enhancers were identified because only in 3 cases(mutations in upstream regions ie in cell line 1, 3, 6) there was a significant drop in the the GFP activity. The mutations in these regions lead o decrease in the GFP activity showing that these 3 regions were regulating the transcription of the gene.

b) Create a cellline with the all the identified enhancers in the muutant form and measure the GFP activity and this would corresspond to the basal activity.