Molecular Biology of the Cell, Fifth Edition_ the Problems Book - John Wilson & Tim Hunt

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Figure 7-28 Binding sites for repressors

and activators of Eve stripe 2 (Problem

7-67)

fragments from the two strains after growth on raffinose or galactose, and then precipitate them with HA

antibodies. You strip the precipitated chromatin of protein and analyze the DNA by quantitative PCR to

measure the amounts of specific DNA segments in the precipitates. The ratio of immunoprecipitated

chromatin from galactose-grown cells to that from raffinose grown cells (Gal/Raf) is shown for several

segments around the galactose promoter in Figure 7-27B.

A. Which of the tested fragments would you have expected to be enriched if SAGA behaved as a

coactivator? Explain your answer.

B. Does SAGA meet the criteria for a coactivator? Is it physically present at the promoter, and does its

recruitment depend on an activation signal?

7-67

The protein encoded by the Even-skipped (Eve ) gene of Drosophila is a transcriptional regulator

required for proper segmentation in the middle of the body. It f irst appears about 2 hours after

fertilization at a uniform level in all the embryonic nuclei. Not long after that, it forms a pattern of seven

stripes across the embryo. Each stripe is under the control of a separate module in the promoter, which

provides binding sites for both repressors and activators of Eve transcription. In the case of stripe 2, there

are multiple, overlapping binding sites for activators and repressors (Figure 7-28). Two activators,

Hunchback (Hb) and Bicoid (Bcd), and two repressors, Giant (Gt) and Kruppel (Kr), are required to give

the normal pattern. The binding sites for these proteins have been mapped onto the 670-nucleotide

segment shown in Figure 7-28; deletion of this upstream segment abolishes Eve expression in stripe 2.

The patterns of expression of Hunchback, Bicoid, Giant, and Krtippel in the embryo are shown in Figure

7-29. It seems that Eve expression in stripe 2 occurs only in the region of the embryo that expresses both

activators, but neither repressor: a simple enough rule.

To check if this rule is correct, you construct a β-galactosidase reporter gene driven by a 5-kb

upstream segment from the Eve promoter. (This segment also includes the controlling elements for

stripes 3 and 7.) In addition to the normal upstream element, you make three mutant versions in which

several of the binding sites in the Eve stripe-2 control segment have been deleted. (Note, however, that

because many of the binding sites overlap it is not possible to delete all of one kind of site without

affecting some of the other sites.)Construct 1. Deletion of all the Kruppel-binding sites

Construct 2. Deletion of all the Giant-binding sites

Figure 7-29 Expression of repressors and

activators of Eve stripe 2 in the

Drosophila embryo (Problem 7-67)

Molecular Biology of the Cell, Fifth Edition_ the Problems Book - John Wilson & Tim Hunt

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