MIDTERM PRACTICE QUESTIONS

The midterm is approaching, and Craig provided us with a number of old midterm exams. I don't know if they will be up on the website, so I will put a FEW COPIES in my mailbox for you to TAKE, LOOK AT AND/OR PHOTOCOPY, AND BRING BACK (so that others can see have access to them).

My mailbox is located in the biosci (biology) building, outside the zoology office, opposite room 2361. Look below the tag with my lastname (Kalas) on Friday.

Happy studying!

Pam

Quick facts and a couple of practice questions on techniques can be found here

SOME QUESTIONS THAT CAME UP

1) genotypes: we assume that all genes that are not mentioned are wild-type. Wild-type bacteria have "+" alleles for all their genes (and are sensitive to antibiotics)
2) For us, all lacO[c] mutants prevent the LacI repressor from binding.
3) If nothing is mentioned, we assume that there is no glucose in the medium
4) For the extra questions (the ones that had sucrose involved): think about what happens when sucrose is broken down in the cell...
5) Midterm: how are they going to test all the recombinant DNA stuff? You may have to interpret the results of a set of restriction digestions and come up with the map of the plasmid, or a cloning procedure may be described to you and you may have to draw the final product, pretend to restriction digest it, pretend to run the digestion reactions on a gel and draw the result (banding patterns), or...I think Dr Beatty may be posting old midterm questions on the 335 website!

Have a good week-and don't mix up northerns and Southerns when you do problem set 3 (the last question is quite tricky-focus on the first part of the problem set first!)

Cheers

Pam
PS: practice questions can be found here
or by clicking on 'lac operon stuff' (it's under the 'archives and links' column) and following the instruction.
(sorry about the formatting-I am trying to fix it)

P.P.S.: ANSWERS TO EXTRA PRACTICE QUESTIONS WILL BE AVAILBALE OVER THE WEEKEND (FOR NOW I TRIED TO SUMMARIZE OUR ANSWERS TO PROBLEMS SET 2-below)

OUR ANSWERS TO PROBLEM SET2

For question 1a
we have three important pieces of information:
I-"cellobiase is only made in high amounts when cellobiose is present";
meaning that the 'cellobiose-metabolizing system' is inducible, and the cellobiose is its inducer

and

II-"a mutation in a gene that encodes a regulatory protein [...]results in the lack of synthesis of cellobiase enzyme;
meaning that either this mutation makes a regulatory protein thatn is unable to bind the DNA and the system is positively regulated,
OR this mutation makes a regulatory protein that never binds the cellobiose (kind of like lacI[s]) and the system is negatively regulated

and

III-the mutant strain can be complemented in trans with the wild type regulatory protein-encoding gene (i.e. we can put in a plasmid that expresses WT regulatory protein and everything works like WT);
meaning that our system must be positively regulated....why?
Because if it was negatively regulated, then the regulatory mutation would have to work like a 'superrepressor', and we know that 'superrepressor' mutants (mutants where the regulatory protein is always bound to the DNA) are typically dominant to the wild type!
In this case the wild type is dominant, so it must be a positively regulated system (and the mutation is a 'lacI- type' mutation).

Question 1 part b:
if the CelI-binding site is mutated, it can't bind to the promoter region and therefore can never activate the expression of the cellobiase!
Clarification: the "CelI binding site" refers to the DNA sequence to which the CelI binds. In general "proteinX-binding site" always refers to the DNA sequence that protein X binds to.
The part of the CelI protein that binds to the DNA would be called 'CelI DNA-binding domain'.

Question 2a:
Since we know that cellobiase is only produced when cellobiose is there, and that the regulatory protein is an ACTIVATOR, then one would expect that binding of the cellobiose to the activator would increase its affinity for the DNA target sequence. (If it decreased it, presence of cellobiose would have either no effect or a negative effect on the production of cellobiase).

Question 2b:
In this case, it would be hard for the cellobiose to bind the CelI protein (kind of like in the case of lactose and the LacI[s] mutation!), and we know that if it is not bound to the cellobiose, the CelI protein has low affinity for its DNA target. Therefore, CelI would not bind the DNA very often, causing the levels of cellobiase to be always low.