QuestionDetails: To analyze how the promoter regulates transcription levels ofth
ID: 4885 • Letter: Q
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QuestionDetails: To analyze how the promoter regulates transcription levels ofthe human actin gene, you fuse the actin promoter sequence to theGFP (green fluorescence protein) gene, and transform into yeastcells. You also create a series of deletions within the promoter,which remove increasingly larger portions from the 5’ end.For each of the constructs, you determine transcription levels bymeasuring the amount of fluorescence given off. The results aresummarized below. 1.) What conclusion can you make about how the expression ofactin is regulated?2.) While doing the above experiment, your spectrophotometer stopsworking, and you cannot determine the transcription levels usingfluorescence. Describe another method you can use to test theabundance of mRNA produced from each construct. QuestionDetails: To analyze how the promoter regulates transcription levels ofthe human actin gene, you fuse the actin promoter sequence to theGFP (green fluorescence protein) gene, and transform into yeastcells. You also create a series of deletions within the promoter,which remove increasingly larger portions from the 5’ end.For each of the constructs, you determine transcription levels bymeasuring the amount of fluorescence given off. The results aresummarized below.
1.) What conclusion can you make about how the expression ofactin is regulated?
2.) While doing the above experiment, your spectrophotometer stopsworking, and you cannot determine the transcription levels usingfluorescence. Describe another method you can use to test theabundance of mRNA produced from each construct.
Explanation / Answer
The actin cytoskeleton is adynamic structure, present throughout all eukaryotic cells.Processes such as cell growth, signaling, transport, and celldivision depend on an intact and functional actinnetwork.
Numerous studies have usedexpression of GFP, linked to the actin binding domain of actinbinding proteins, talin, plastin, and fimbrin to visualize theactin cytoskeleton in living.
The expression of GFP fusedto actin binding domains is a commonly used and convenient methodto study the actin cytoskeleton in live cells, the effect of theexpression of such proteins on actin organization anddynamics
the actin binding domainused in GFP-mTalin is derived from a mammalian actinbinding protein, it does bind to plant F-actin, and therefore it islikely that it competes with other actin binding proteins for abinding site.
Actin-GFP FusionLocalization in Vivo. The grossappearance,in cells taken from exponentially growing cultures, of theGFP fluorescence in actin-(Ala)lo-GFP transformants is stronglyreminiscent of that observed in fixed cells by standard fluorescentmethods. As expected, we observe asymmetricallocalization of the punctate fluorescent patches similar to thatobserved for the cortical actin patches by immunofluorescence andrhodamine phalloidin labeling during the cell cycle.
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