1. If Jacob and Monod used only one copy of the lac region, they would not likely have found operator mutants, but instead would have found more i^- mutations.

2. a. You would not expect the addition of lactose to induce b-galactoside permease z^-y⁺ mutants; lactose canot be leaved to form allolactose, which is the required inducer.  The addition of allolactose will induce b-galactoside permease in these mutants.

b. You would not expect the addition of lactose or allolactose to induce b-galactoside permease z⁺y^- mutants; the permease gene is defective.

c. Cells synthesize low levels of b-galactosidase and permease even when there is no lactose in the medium so that the system can be induced should it be exposed to lactose.   Without low levels of the enzymes, no lactose could be brought in initially and no allolactose could be made.

3. a. The control of the system is most likely to be negative repressible.  According to the table, the most frequent class of R mutations (R^C) leads to constitutive expression; therefore, R is a repressor that represses production of S, the structural gene, and its loss (R^c) causes constitutive expression.  R^- causes constitutive repression (it is a super repressor).

b.

In R⁺S^-/ R⁺S^- merodiploids, both structural genes are defective, so the enzyme will never be synthesized.  In R^CS⁺/ R⁺S^- merodiploids, there is one functional R gene and one functional S gene; enzyme will be produced in the absence of pro.  In R^CS⁺/ R^-S^- merodiploids, R^- will prevent enzyme synthesis; i.e. it acts as a trans dominant.

4. a. Epistasis is when the phenotype produced by the mutation of one gene is seen and prevents the phenotype produced by mutation in a second gene.

b. i. z^- is epistatic to i^-, as without the z gene there is nothing to turn on or repress.

ii. o^c is epistatic to i^S, as o^C prevents the binding of any repressor to the operator.

iii. crp^- is epistatic to o^C ; without crp, the RNA polymerase can't bind.

5. a. (i) Without the regulatory gene to help induce production of the enzyme, the enzyme will be absent with or without the presence of the inducer.  In merodiploids (r^-p⁺o⁺s⁺/r⁺p⁺o⁺s^-), the enzyme production is inducible. (ii) Without the promoter, enzyme production will always be absent (both in the mutant and in the merodiploid r⁺p^-o⁺s⁺/r⁺p⁺o⁺s^-).

b. (i) Without the regulator repressor, enzyme production will be constitutive; in merodiploids (r^-p⁺o⁺s⁺/r⁺p⁺o⁺s^-) enzyme production is still repressible. (ii) Point mutations in the operator will prevent repressor binding, causing enzyme production to be constitutive in both the mutant and the merodiploid (r⁺p⁺o^Cs⁺/r⁺p⁺o⁺s^-). (iii) Point mutations in r will have a variable effect, depending on the type of mutation.  Some mutations will cause the repressor to lose function, leading to constitutive enzyme production, whereas others may cause the repressor to bind irreversibly to the operator, causing enzyme production to always be absent.  In merodiploids, the first type of mutation would allow enzyme production to be repressible, while in the second case enzyme production would still be permanently repressed.

6. a. Catabolyte repression is the system used by bacteria to turn off lac and other glucose-substitute operons in the presence of glucose.  If glucose is absent, cAMP levels in the cell increase, and cAMP binds CAP (also known as CRP).  cAMP changes the conformation of CAP, allowing it to bind to DNA in lac and araC operons and recruit RNA polymerase for transcription.  However, when glucose levels are high, cAMP is shunted out of the cell, preventing it from complexing with CAP.  Without cAMP, CAP cannot bind to DNA, and RNA polymerase is not recruited to the operons.

b. The genetic elements required for catabolyte repression are cap (crp), the gene encoding adenylate cyclase, and the CAP-binding site of the promoter.

c. A is most likely the crp gene, as it’s a recessive mutation that prevents induction even in the presence of increased cAMP.  B is most likely the gene encoding adenylate cyclase, as enzyme production can still be induced by exogenously adding cAMP.   C, as a cis-dominant mutation, is likely to be the CAP binding site. 

7. Mutagenize bacteria and grow in the presence of lactose, glucose, and X-gal; look for blue cells.  Mutations causing defects in catabolite repression (such as CAP) or mutations affecting lacZ activity/expression will be isolated.  To differentiate between these possibilities look at catabolite repression in the arabinose system (see problem #6 above - this also describes some of the genes.)

8. a. For negative control, the corepressor would bind to the repressor and the complex would bind to DNA (at the operator) and turn off transcription. For positive control the repressor would be a positive factor that is needed for transcription; adding the corepressor (col) would remove it from the DNA.

b. In the negative repressible scenario, loss of function of the repressor would cause the enzyme to be expressed constitutively.  For positive repressive control, loss of function of the regulatory gene would prevent expression.

c. For biosynthetic operons, positive repressible control would likely lead to cell death.  Biosynthetic operons usually express proteins that are necessary for survival; if presence of the protein would cause the cells to stop producing it, then the cells would die.

d. Attenuation is a type of regulation in which the presence of elevated levels of product reduces the rate of transcription of its mRNA.  In a positive repressible system, this type of regulation wouldn’t be needed, as presence of the product already eliminates its transcription.

9. a. cis-dominant mutations work on the same strand of DNA, e.g. the o^c mutation; trans-dominant mutations works through a protein (or RNA) intermediate and so can effect expression elsewhere in the genome.

b. The lac operon produces a polycistronic mRNA and has operator and promoter elements (where the operator binds a repressor to disrupt the promoter).  The typical eukaryotic gene is not polycistronic, often encodes a spliced mRNA, and has enhancer and silencer (negative elements) that are not physically near the start of transcription.

10. a. False, catabolite repression involves the loss of cAMP.

b. True, attenuation causes early termination of transciption.

c. False, mutations that affect antitermination are cis dominant.

11. a. You would expect to get both types of mutations at about the same frequency.  On one hand, you will get loss of lacZ expression through mutations in the lacZ gene that knock out its function.  On the other hand, you will get see constitutive lacZ expression through mutations that cause a loss of function in the repressor. 

b. You would expect i^- to arise more frequently than o^C mutations, because i is a larger target for mutagenesis than the operator.

c. You would expect i^- mutations to arise more frequently than i^S mutations, because more sites on the gene can be mutated to produce a loss of function (i^S requires mutations in a very specific site).

12. trp repressor is an example of negative repressibility.  This involves the repressor itself, tryptophan as a corepressor, and an operator region in the trp operon to which the repressor will bind.  The effect of the binding of the repressor/corepressor complex to the operator is to turn off the synthesis of new mRNA from the operon.

Control by the araC protein on the arabinose operon is an example of positive inducible control.  The binding of araC protein/arabinose (the inducer) complex to the promoter activates transcription at this operon. 

13. Should the trp repressor not be acting, attenuation (slowing of the translation of the leader peptide with the subsequent formation of the 3:4 hairpin and termination of the message) will prevent complete transcription from the trp operon.

14. a. Immediately after mating, b-galactosidase levels will increase because of the absence of lac repressor in the F^- cells; the levels will eventually go down. (PaJaMo experiment)

b. There will be no increase of b-galactosidase levels, because there will be repressor present in the F^- cell (and the wild-type repressor is trans-dominant).

c. In diploids cap⁺ will be dominant to cap^-.  cap⁺ have catabolite repression (express lacZ in absence of glucose only and presence of the inducer), cap^- cannot turn on the lac operon, and cap⁺/cap^- is will have catabolite repression as seen in wild-type.  CAP is a trans-acting factor.

d. If CAP could bind DNA in the absence of cAMP, it would make lacZ, etc. in the presence of glucose, but only if lactose were around to remove the lac repressor.

e. i. m is a physiological suppressor, because it is allele non-specific and gene specific.  n is an informational suppressor (allele specific; gene non-specific).

ii. Polar means that a mutation in one gene affects a downstream gene.  If n is a tRNA nonsense suppressor, then the nonsense mutation in lacY could cause a polar effect on lacA because a polycistronic mRNA is made, and ribosomes would have difficulty initiating lacA translation.