1. Which mutations do you expect to arise more frequently i^- or i^S? Why? What about i^- versus o^c?

2. "By selecting for constitutivity (unrepressed synthesis) in cells with two copies of the lac region, [operator] mutants were found and labeled o^c, for operator constitutive." (Griffiths, p. 339) Why did Jacob and Monod use two copies of the region, i.e. what was wrong with using just one? 

3. X-gal is a substrate for b-galactosidase that turns the cells blue when it is cleaved by the enzyme. How could you use this property to isolate mutants that are defective in catabolite repression (i.e. what conditions woul d you use)? What genes would you expect to be mutated? Would all mutants isolated in your screen actually be defective in catabolite repression? What further tests would you do to be sure? 

4. (From Wood et al. Molecular Biology of Eucaryotic Cells, 1975) Allolactose is an isomer of lactose that is produced by b-galactosidase as an intermediate in the cleavage of lactose to galactose and glucose. It is allolactose, not lactose, that is the natural inducer for the lac operon.

a. Would you expect b-galactoside permease to be induced in a z^-y⁺ mutant upon the addition of lactose? Upon the addition of allolactose? 

b. Would you expect b-galactosidase to be induced in a z⁺y^- mutant upon the addition of lactose? Upon the addition of allolactose? 

c. Can you suggest a reason why cells synthesize low levels of b-galactosidase and permease even when there is absolutely no lactose in the medium?

5. (From Wood et al.) An enzyme required for the synthesis of proline (Pro) in a soil bacterium normally is made only when Pro is absent from the growth medium (see the accompanying table). When mutants with defects in control of this enzyme are isolated, two classes of mutants altered in a regulatory gene R are found with the frequencies and phenotypes shown in the table. S is the structural gene for the enzyme.

a. From the results in the table, what is the most likely nature of the control system (inducible, negative; repressible, negative; inducible, positive; or repressible, positive)? Briefly explain your reasoning. 

b. Complete the table to show the most likely phenotypes of the indicated merodiploids, again very briefly explaining your answers.

6. (E) a. Define epistasis. 

b. Considering the control of lactose utilization in E. coli, state which mutation in the following pairs would be epistatic to the other. Explain your answers.

(i) i^- and z^-

(ii) i^s and o^c

(iii) crp^- and o^c

7. Consider a regulated bacterial system consisting of a regulatory gene (r) a promoter (p), an operator (o), and a structural gene (s), carrying information for an easily measurable enzyme activity. For each of the following questions, indicate whether the enzyme will be constitutively produced, inducible, repressible, or absent under all conditions, in both the mutant indicated and a merodiploid made by introducing the normal regulatory elements (i.e., an episome of the ge notype r⁺p⁺o⁺s^-) into the mutant.

a. If the system is positively controlled by the product of gene r and inducible by some metabolite, what are the ways in which enzyme production can be affected by (i) deletion mutations in r or (ii) deletion of p. 

b. If the system is negatively controlled and repressible, what are the ways in which enzyme production can be affected by (i) deletion mutations in r, (ii) point mutations in o, or (iii) point mutations in r.

8. (E) Three E. coli mutants altered in the machinery of catabolite repression were isolated. Each was tested for the inducibility of the lactose and arabinose operons with and without exogenously added cyclicAMP (cAMP).

 a. What is catabolite repression? 

b. What genetic elements are required for catabolite repression? 

c. What is the most likely explanation for each mutant?

9. (E) Although negative repressibility is seen in the control of biosynthetic operons (e.g. the trp operon), positive repressibility has not been observed.

a. Briefly explain how negative repressibility and positive repressibility would act to control the production of mRNA from an operon for the biosynthetic enzymes for the hypothetical amino acid columbine (col). Be sure to indicate what control proteins are needed, how they act (not only say what the net effect is, but also provide a possible mechanism for the action), and what would happen in the presence and absence of col. 

b. For each of the two cases in (a), what would be the consequence of a loss-of-function mutation in the gene for the control protein? 

c. From your answer in (b), give a possible reason why positive repressibility does not seem like such a great way to control biosynthetic operons. 

d. Briefly define attenuation control. Would attenuation be needed in a positive repressible system? Why?