Question I: human error or a key observation?

The first time progeny production was examined as function of cloned alpha3 C gene induction; the titer of the 90-minute time point was significantly higher when the cloned gene was induced. However, in a second experiment, the opposite result was obtained: induction significantly lowered progeny production. Two explanations are possible: 1) An error was made during one of the experiments or 2) both results are valid; no errors were made in the course of either experiment.

 If the second alternative is correct, it demonstrates that the system may contain an additional level of complexity. When investigating the regulation of biological processes, initial models often contain a simple on/off switch. However, evolutionary success can involve the ability to respond to subtle changes in macromolecular concentrations. Allosteric regulated enzymes, such as aspartate carbamoyl transferase, well illustrate this phenomenon. This enzyme’s activity is exquisitely sensitive to miniscule changes in substrate concentrations within a particular concentration range. Thus, it is quite possible that subtle differences in C protein concentrations could have a similar effect on progeny production.

 A. Technical differences that could affect C protein concentrations.

 1. How could the IPTG half-life affect C protein levels in experiments conducted on different days?

 2. Provide another technical source that could affect induction levels.

 3. If conducting another experiment, describe the conditions under which infections should be conducted. These conditions should eliminate, or at least minimize, technical variations and directly address the effects of induction conditions, and by extension C protein levels, as a function of progeny production.

 B. Theoretical/physiological consideration.

 1. MOI could affect replicative form DNA concentrations, C protein concentration and RF-DNA:C protein ratios. The first experiment was conducted at a high MOI (5-10), whereas the second experiment was conducted at an extremely low MOI. How could differences in MOI affect progeny production in cells expressing the cloned alpha3 C gene?  Remember replicative form DNA serves two functions in single-stranded DNA virus infection.

 2. If conducting another experiment, describe the conditions under which infections should be conducted. These conditions should eliminate, or at least minimize, technical variations, circumvent the need to calculate precise MOI values (relative MOI values are good) and directly address the effects of MOI as a function of progeny production.

 C. The inhibition of cloned alpha3 C gene expression was first defined in plating assays. Expression typically resulted in the formation of pinprick plaques that over time developed a much larger morphology. Assuming that error did not cause the differences between the low and high MOI experiments, how may the kinetics of plaque development relate to the results of the liquid culture infections?

Question II: model building.

 In our initial model, protein C and ssB act as antagonists. Explain this model. Explain how the phenotypes of the alpha3 C resistance mutants support it. In scientific writing, figures often accompany text. These illustrations need not be elaborate but serve as visual aid that simplifies complex text. Include a diagram with your answer.

 Question III: genetic selections.

 A. Two genetic selections were conducted with cells over-expressing the cloned ssB gene. In the first selection, wild-type alpha3 was. In a simple model, these newly selected mutants should contain one mutation. In what proteins and/or genetic elements (origins, promoters, Shine-Dalgarno sequences) would one expect these mutations to reside and why?

 B. In the second selection, mutants resistant to the over-expression of the alpha3 C gene were used. This is a much more complex selection as the parental strain is not wild-type. The selection may produce double mutants or the selection may produce strains that are genetically identical wild-type. Explain why these two outcomes may be possible.

Question 4: miscellaneous.

 A. In vitro vs. in vivo experiments

 1. In vitro and in vivo approaches have advantages and drawbacks, briefly contrast these advantages and disadvantages.

 2. When reconstituting a biochemical process in vitro, biochemists often use crowding agents. What are these entities, what do they do and why are they needed?

B. SDS-PAGE

 1. SDS-PAGE reliably separates proteins by molecule mass. This technique equalizes charge:mass ratios between proteins. How is this accomplished?

 2. Effective separation first requires stacking the sample between two matrices, the stacking and separation gels. Explain how the chemical differences between the stacking and separation gels accomplish this.