A student is interested in the transcriptional regulation of BOG1 (the bogum syn
ID: 78388 • Letter: A
Question
A student is interested in the transcriptional regulation of BOG1 (the bogum synthetase gene). Northern hybridization analysis shows that BOG1 is strongly expressed in cells if Zubrium (Zub) is absent in the media and is turned off when Zub is present. She contructs a lacZ fusion gene with BOG1 (i.e. BOG1-lacZ activity and observes a pattern of expression of the lacZ reporter gene in the presence and absence of Zub that is similiar to the Northern hybridization results. She then decides to make site-directed mutations in three sites of the BOG1 gene promoter (A,B and C) and then tests for lacZ expression. Mutant sites are highlighted in black boxes in Figure 2 below.
She also individually clones a wild type copy of each site, A, B and C into heterologous reporter promoters with and without the "upstream activator sequence/enhancer" (UAS) (please refer to the lower panel diagrams in Figure 2) and then measures lacZ expression.
A. What can she conclude about the function (activator or repressor, constitutive or inducible) regions A,B,C in -Zub and +Zub conditions? Should she do any further analysis of the regulatory elements in region A, B or C of the BOG1 promoter? If so, how should she do this and what should she be looking for?
B. She isolated several mutants called rob (rob1, rob2 and rob 3) that affect proper regulation of the BOG1 gene. She transforms the heterologous reporter constructs (please refoer to clones 12-14 in Figure 3 below) separately into the rob1, rob2 and rob3 mutant strains and measures expression of the lacZ gene in the absence of Zub (-Zub) and presence of Zub (+Zub) in te medium. The results are shown below. What can you conclude about the function of the proteins encoded by the rob1, rob2 and rob 3 genes? Where do they function and what is their activity?
C. Should she continue her screen to identify other mutants in order to detect additional regulatory proteins that affect BOG1 expression? Justify your answer. If she should do additional screens, indicates which clone (#1-#14) she should use to find the regulatory proteins that regulate BOG1 expression. In your answer, indicate which clones she should use, and under what conditions (-Zub or +Zub) to perform the screen, and whether she should look for increased expression or loss of expression of the BOG1 gene under these conditions.
Level of lacz Activi BOG 1-lacZ fusion Clone Mutant Site A B C TATA -Zub +Zub Lac 1000 0.1 100 1.0 Lacz Lecz 100 1.0 100 Lacz 0.1 a,b H Lacz 100 100 a,C 100 1.0 b,c 100 1.0 Lacz a,b,c 10 10 Lacz Heterologous UAS-less vector Site -Zub +Zub TATA vector Lac2 0.1 0.1 0.1 Lacz 10 10 B Lacz 10 0.1 11 10 Heterologous UAS vector -Zub +Zub Site UAS TATA vector 100 100 12 Lacz 1000 10 13 CB 1000 10 14 Lacz 1000 1000 Figure 2. Experiments to study regulation of the BOGI ene.Explanation / Answer
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