1. Jacob and Monod obtained the operator constitutive (o^c) mutants from cells with two copies of the lac region.  Why did they use two copies of the region, i.e. what was wrong with using just one? 

    

2. (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.

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

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

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

    

3. (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.

   1. 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. 

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

4. a. Define epistasis. 

2. 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.

1. i^- and z^-

2. i^s and o^c

3. crp^- and o^c

 

5. 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.

1. 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. 

2. 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.

 

6. 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).

1. What is catabolite repression? 

2. What genetic elements are required for catabolite repression? 

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

 

7. X-gal is a substrate for β-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? 

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

1. 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. 

2. 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? 

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

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

 

9. Compare and contrast the following.

1. cis dominant and trans dominant mutations

2. The structure and mRNAs of the lac operon and a typical eukaryotic gene.

 

10. Answer TRUE or FALSE.  If the statement is TRUE, explain or give a supporting example.  If the statement is FALSE, either correct the statement or give a counterexample.

1. Catabolite repression involves the accumulation of cyclic AMP.

2. Attenuation assures that expression from the trp operon is off when tryptophan is present.

3. Mutations at the trp operon that affect antitermination are most likely to be seen as acting as recessives.

 

11. Which of the following (if either) would you expect to occur significantly more frequently?  In each case explain your answer.

1. Loss of lacZ expression vs. constitutive expression of lacZ expression.

2. i^- vs. 0^c mutations.

3. i^- or i^S mutations

 

12. Explain the nature of control for both negative repressibility and positive inducibility in bacterial systems.  Be sure to describe all necessary components needed for such control and how these components regulate activity.  Give examples from E. coli of both types of regulation.

13. Explain how attenuation assures that expression from the trp operon is off when tryptophan is present.

14. a. A Lac⁺ Hfr (i.e., its genotype is i⁺o⁺z⁺y⁺) is mated to an F^- strain that is i^-o⁺z^-y^-.  In the absence of inducer and glucose what should happen to $-galactosidase levels immediately after the mating (increase, decrease, or stay the same)?  Explain your answer.

2. Describe what would happen if the F^- recipients were i⁺o^cz^-y^-.

3. In diploids, would you expect cap⁺ (also called crp⁺) to be dominant or recessive to cap^-?  Refer to lac expression and describe the phenotype of cap⁺, cap^-, and cap⁺/cap^- bacteria.

4. What would be the effect on lac operon expression of a mutation in cap that caused it to bind to DNA in the absence of cyclic AMP?

5. Several lacY^- mutations have been identified.  In a screen for cells that could transport lactose for one lacY^- line, two suppressor mutations (m and n) were found.  Subsequent study of these suppressors revealed that m could suppress many if not all lacY^- mutations, but none of a large set of lacZ- mutations.  Mutation n suppressed a few of the lacY^- mutations, but also a few lacZ^- mutations and some in the gene for the trp repressor.

1. What types of suppressors are m and n?  Explain your answer.

2. One of the two sets of lacY^- mutations (those suppressed by m and those suppressed by n) are polar (in the absence of the suppressors) .  Explain what is meant by a polar mutation and give your reason for why one set might be polar.

 

Answers