Question

What control elements regulate expression of the mPGES-1 gene?

The promoter of a gene includes the DNA immediately upstream of the transcription start site, but expression of the gene can also be affected by control elements. These can be thousands of base pairs upstream of the promoter, grouped in an enhancer. Because the distance and spacing of these control elements make them difficult to identify, scientists begin by deleting sections of DNA that contain possible control elements and measuring the effect on gene expression. In this exercise, you will analyze data obtained from DNA deletion experiments that tested possible control elements for the human gene mPGES-1. This gene codes for an enzyme that synthesizes a type of prostaglandin, a chemical made during inflammation.The researchers hypothesized that there were three possible control elements in an enhancer region 8–9 kilobases upstream of the mPGES-1 gene. Control elements regulate whatever gene is in the appropriate downstream location. To test the activity of the possible elements, researchers synthesized four types of DNA molecules (“constructs”):

One DNA construct had an intact enhancer region.

One DNA construct had possible control element #1 deleted from the enhancer region.

One DNA construct had possible control element #2 deleted from the enhancer region.

One DNA construct had possible control element #3 deleted from the enhancer region.

In all constructs, the enhancer region was located upstream of a “reporter gene,” a gene that would be expressed at the same time as mPGES-1 and whose mRNA product could be easily measured experimentally.The researchers then introduced each DNA construct into a separate human cell culture, where the cells took up the artificial DNA molecules. After 48 hours, the amount of reporter gene mRNA made by the cells was measured. Comparing these amounts allowed the researchers to determine if any of the deletions had an effect on expression of the reporter gene, mimicking the effect that deletions would have had on mPGES-1 gene expression. (The mPGES-1 mRNA itself couldn’t be used to measure expression levels because the cells in culture have their own mPGES-1 gene with an intact enhancer region. Therefore, the cells produced mPGES-1 mRNA regardless of which DNA construct they took up.)

Part A - Understanding the experimental design

The diagrams show the intact DNA sequence (top) and the three experimental DNA sequences. A red X indicates the possible control element (1, 2, or 3) that was deleted in each experimental DNA sequence. The area between the slashes represents the approximately 8 kilobases of DNA located between the promoter and the enhancer region. The horizontal bar graph shows the amount of reporter gene mRNA that was present in each cell culture after 48 hours relative to the amount that was in the culture containing the intact enhancer region (top bar = 100%).

What was the independent variable in this experiment?

What was the independent variable in this experiment?

A. the relative level of reporter gene mRNA

B. the length of time that the cells were incubated

C. the possible control element that was deleted

D. the distance between the promoter and the enhancer

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Part B

What was the dependent variable in this experiment?

What was the dependent variable in this experiment?

A. how many of the artificial DNA molecules were taken up by the cells

B. the distance between the promoter and the enhancer

C. the relative level of reporter gene mRNA

D. the length of time that the cells were incubated

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Part C

What was the control treatment in this experiment?

What was the control treatment in this experiment?

A. the temperature, pH and salt concentration of the incubation medium

B. the construct that had no DNA deleted from the enhancer

C. the construct that resulted in the lowest amount of reporter mRNA

D. the reporter gene

Answer 1

Independent variable, dependent variable and control:

• Independent variable is that variable which can be manipulated or changed by researchers during an experiment. In our case, researchers manipulated with deletion in various regions of the enhancer.
• Dependent variable is that variable which is the ultimate outcome or result of the experiment. Its outcome depends on the experimental design and choice of independent variables. In our case, level of expression of reporter gene was the ultimate outcome/result.
• control refers to the experimental set up without any manipulations. In our case, the DNA construct without any deletions in enhancer region is the control.

Answer A- option C : the possible control element that was deleted

Answer B- option C : relative level of reporter gene mRNA.

Answer C- option B: the construct that had no DNA deleted from the enhancer