Question

I need help with part B. This was the entire problem with all given information.

16. Suppose we want to locate upstream control regions in a certain gene. We have used an exonuclease to chew the gene from the 5 end. Below is the result of several runs, where we have let the exonuclease chew for different periods of time. 1 2 3 4 5 Experiment 1, no exonuclease exposure Experiment 2, 5 min. exonuclease exposure Experiment 3, 10 min. exonuclease exposure Experiment 4, 15 min. exonuclease 2 kb exposure 1650 b Experiment 5, 20 min. exonuclease exposure 1000 b = a. This gel has two controls: one for the experiment, and one for the 500 - gel itself. What are the controls? What does each control show? (2 points) • The gel control is on the 100 bl far left. This is a ladder that is used to show where the bands are and how many base pairs each band indicates The experiment control is in the lane labeled 1. This contained the experiment that had no exonuclease exposure. This acts as a control so all other experiments can be compared to it to show any changes between exonuclease exposure. ILI b. We then cloned the fragments into a plasmid containing the coding region for a reporter gene, and then transfected cells with copies of the plasmid. The following results for reporter expression were obtained: Relative Fluorescence Units (RFU) Plasmid 1 1.000 Plasmid 2 0.993 Plasmid 3 0.658 Plasmid 4 0.648 Plasmid 5 -0.003 (Plasmid n was created from fragment n, from experiment n) Estimate the location of each control region and the promoter complex. Using the gel and fluorescence data (numerical data), explain your rationale. (3 points)

Answer 1

B) plasmid 1 showed maximum flouroscence reading of 1 which means it's has all the regulatory components required for complete expression of gene whose size of 3500bp

Plasmid 2 also showed almost same fluoscence reading reading so till here no regulatory sequence lost and size is ~2700bp

Plasmid 3 showed decrease in flouroscence reading means some component of regulatory genes is loss by exonuclease activity. Since decrease in 0.66 fro 1.0 so we can conclude that enhancer sequence has lost which inhance the gene expression.now size of this plasmid is 2000bp so enhances must be located between 800bp to 1500bp from 5' end of DNA or from starting point.

Plasmid 4 size is1300bp, does not showed any change in flouroscence means no regulatory gene is present 1500-2200bp

Plasmid 5.size 500bp, Flouroscence reading is 0 which means that important regulatigy gene loss which is promotor so now RNA polymerase cannot bond and hence no RNA synthesis so no protein formation so reading is zero.

Promotor region of 800bp while 2200 nucleotide 3000 nucleotide from 5' position or from starting point.

Hope it's clear..thanks