You have cloned the promoter of a mouse gene that is induced by testosterone, a
ID: 66842 • Letter: Y
Question
You have cloned the promoter of a mouse gene that is induced by testosterone, a steroid hormone, and have a tube of the relevant hormone-responsive transcription factor. Describe how you could: identify binding sites for transcription factors on the promoter, using method(s) discussed in class. test if regulation of the transcription factor involves aliening its sub cellular location, assuming that you have become expedience in separating cell extracts into nuclear and cytoplasm fractions. test if the transcription factor must be to bind the promoter, assuming that you have tubes of the protein kinas that the transcription factor, a protein phosphates that the transcription factor, and ATP (the phosphate donor in the kinas reaction). You are studying the mushroom gene ZIP. which you have cloned. ZIP is not transcribed in young mushrooms, but is transcribed in old mushrooms. Previous studies had suggested that the two genes encoding HLH transcriptional factors GOO and DEE are required to express ZIP. Describe an assay that you could use to test whether the transcription of ZIP is regulated by changes in chromatin structure during mushroom aging. Assuming that the sequences of the genes encoding GOO and DEE are known, describe how you might use the yeast two-hybrid method to test if those two proteins physically interact. Include in your answer a description of components of the vectors and yeast strains that you will use.Explanation / Answer
1a. It can be identified using DNA foot printing assay- which restricts it from digestion with restriction enzyme. electrophoretic mobility shift assay - Which changes the mobility of the DNA bound with protein in an given electric field.
1b. The transcription factor can be cloned into different organelles using site specific genes. The level of mRNA in different organelles can be determined using RT PCR and it can be identified whether the regulation involves subcellular location or not.
1.c Two vials containing protein kinase and not containing protein kinase with the DNA can be incubated for the reaction. When the sample from both the vials (with and without protein kinase) can be run on electrophoretic mobility shift assay. Change in the presence of protein kinase indicates that the transcription factor requires phosphorylation. Change in the absence of protein kinase from control DNA indicates that it does not require phosphorylation.
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