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1. In broad outlines, describe the assay that is being used in figure 1. What is being measured? What is this measurement designed to reflect? How and why does this work?

[full-credit student answer...]

The assay depicted in Figure 1 is measuring the amount of beta-galactosidase activity in the presence of two proteins, da and sis-b. The lac z gene was placed downstream of the Sxl promoter so that transcription of it would be under control of the Sxl promoter region. The measurement is designed to reflect the effects that da and sis-b have on the Sxl promoter region and further serves to narrow down the region(s) of the promoter necessary for this effect. The works because levels of beta-galactosidase activity are easily detected and transcription/trnaslation of the lac z gene gan be controlled with the Sxl promoter region.

2. Interpret the first line of figure 1B--the data for construct SE3.4K. What does each of the four horizontal bars represent? What do these data suggest?

[full-credit student answer...]

Each horizontal bar represents the amount of beta-galactosidase detected with neither da nor sis-b present (black bar), presence of da (hatched bar), presence of sis-b (shaded bar) and the presence of both da and sis-b (empty bar). These data suggest that the presence of both da and sis-b protein is necessary for complete functionality of the early Sxl promoter.

3. Now compare construct SE3.4K to construct SE385. What does this comparison tell you? What about the comparison of SE385 and SE180?

[full-credit student answer...]

Construct SE385 is the same as construct SE3.4K only missing the region from -386 to -3403. SE385 shows the same relationship between da/sis-b as does SE3.4K except that even more beta-gal is produced in SE385. Perhaps this is due to the loss of a repressor binding site between -385 and -3403. The comparison of these two constructs indicates that the region of DNA with which da/sis-b interacts is most likely not between -385 and -3403. Between SE385 and SE180, however, we see a dramatic drop in beta-gal production in the presence of da/sis-b. This is a very good clue as to the region where this protein complex binds to the promoter to initiate transcription.

4. How do the authors interpret the data for constructs SE385(deltaE) and SE83+E?

[two different full-credit student answers...]

#1) Because the level of beta-gal dropped by 75% when the E box was deleted in SE385deltaE (as compared to SE385), and because construct SE83+E induced more beta-gal production than SE83, the authors concluded that the E box is part of da/sis-b dependent activation of the sxl promoter.

#2) The fact that a deletion of the E-box doesn't completely bring the expression down to the basal level suggests there are other mechanisms by which sis-b and da activate expression. However, it also supports the hypothesis that the da/sis-b proteins directly activate the early sxl promoter by binding to the E-box, since transcription levels are greatly reduced when the E box is deleted. The SE83+E construct contained an E box just upstream of the promoter. The authors suggest by comparing an SE83 with an E box to that with a mutant E box that since transcriptional induction is observed in the former and not the latter, the E box plays a direct part in transcriptional activation by sis-b and da.

5. (2 pts) Your textbook discusses gel mobility shift assays (such as the ones presented in figure 3, figure 5, and figure 6C) on pages 346 -347. Consult this information, and then describe the data shown in figure 3B of the paper. What does this experiment show? Why is this significant?

[full-credit student answer...]

The gel mobility shift assay in figure 3B shows that da/sis-b directly binds to the E box as well as to other fragments upstream of the E box in the -280 to -385 region. The da/sis-b protein complex binds to Scl E, S3, and S6 with decreasing affinity, but does not bind at all to a mutated E box or to S2. These three elements [the E box, S3, and S6] are likely to all take part in the in vivo activation of the Sxl early promoter by the da/sis-b heterodimer.

6. (2 pts) What does figure 6B show? What do these data suggest about the mechanism of action of the dpn protein?

[full-credit student answer]

Figure 6B shows a DNase footprinting experiment where fragments of the -84 to -180 region of the Sxl early promoter were mixed with the dpn protein. Regions of the DNA are protected from DNaseI in the presence of dpn. This indicates that dpn represses activation by binding directly to a DNA sequence upstream of the early Sxl promoter and not by rendering da/sis-b nonfunctional.

7. (2 pts) What is the underlying biological question addressed in this paper? Given that the sis-b gene is on the X chromosome of Drosophila, what do the authors' data suggest about this underlying question?

[full-credit student answer]

The underlying biological question in this paper asks what molecular mechanisms regulate expression of Sxl, and thus determine sex in Drosophila, and how this corresponds to the X:A ratio. Because sis-b is required in cooperation with da to activate the early Sxl promoter and because females have two X chromosomes containing the sis-b gene, the da/sis-b heterodimer may only be in a high enough concentration in females to activate early expression of Sxl and determine femaleness.

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