Molecular Biology Final Exam - spring 1998

ALL EXAMS ARE DUE AT 5 pm on MAY 8, 1998.

I will be out of town all day May 8 so do not wait until Friday to ask questions.

There is no time limit on this test. You may find it easier to take this test over several days, though if you are confident in your molecular skills, you could wait until May 7. However, I predict it will take many of you a bit longer to think of all the answers (just some friendly advice). You are not allowed to use your notes, the WWW, any books or journals, nor are you allowed to discuss the test with anyone until all exams are turned in at 5 pm on MAY 8, 1998. You may use a calculator, a ruler. The answers to the questions must be typed, though you may want to supplement your text with hand drawn figures (write neatly for any labels in your figures).

Please do not write your name on any page other than this cover page.
Staple all your pages (INCLUDING THE TEST PAGES) together when finished with the exam.
Name (please print):

Write out the full pledge and sign:

How long did this exam take you to complete (excluding typing)?


Questions based on figures from page A
8 pts.
1) Use the photo of the agarose gel and the graph paper provided here to calculate the molecular weight of the two bands in lane A as marked. Notice the molecular weights on the left side of 1.6, 1.0, 0.51, 0.40, 0.34, and 0.30 kb. You must draw a correct graph to get credit. Write your answers here:

Top band = _________

Bottom band = __________


8 pts.

2) Look at figure 2C and 2D. tk is a weak promoter and SV40 is the 3' UT portion that allows the CAT mRNA to be spliced and processed properly.
a) What is Oct-4? What is 6W?

b) What do we learn about the location of 6W?

6 pts.
3) Look at Figure 3 A - F. In E and F we see the plasmids used in this experiment. p6WtkCAT is the reporter plasmid from figure 2. pE1A is a piece of the Adenovirus that encodes the E1A protein. pCMV-Oct4 plasmid uses a strong viral promoter (CMV) to drive the expression of Oct4.
a) What do we learn about Oct-4 and E1A as demonstrated by these CAT assays (A-D)?

b) Why do you think panel A looks so funny?

Questions based on figures from page B
16 pts.

4) Figures 2 - 4 deal with the Drosophila gene oskar (osk ). Look at all 3 figures before you answer any questions. These should be short answers.
a) When is osk transcribed in Drosophila (assume the positive control was positive for figure 2)?

b) What role does osk play in Drosophila development?

c) Briefly describe the localization of osk in wt embryos as shown in figure 4A - C. Stage 5 is at about 2.5 hrs while stages 2 and 3 are in the 0 - 2 hrs time period.

d) What would you predict the phenotype to be for figure 3 if this shows an in situ hybridization for osk of a stage 3 embryo?

8 pts.
5) What can you tell me about this epitope tag?

Questions based on figures from page C
6 pts.

6) Interpret these results from figure 4. PAB II is not a polymerase.

Questions based on figures from page D
8 pts.

7) Figures 2, 3 and the abstract are all related. What aspects of these two blots indicates the mutant genotypes? Notice that figures 2 and 3 are different kinds of blots so there might be more than one answer to this question.

10 pts.
8) Figure 4 shows the results of an experiment where we are monitoring a truncated protein called SC which is a mutant form of a plasma membrane receptor but it has had its transmembrane portion deleted. This mutant protein has about 800 amino acids while the full-length would have had about 1000 amino acids. WT means that of the remaining 800 amino acids, the sequence is wild type, while 669t has an amino acid substitution at position 669, and
D 655-668 is a short deletion mutation. The assay takes advantage of the fact that the cells used in this study form a tight monolayer of cells which secrete some proteins up, away from the attachment side (apical), and other proteins are secreted down (basolateral) towards the attachment side. It is possible to collect these two separated media (apical and basolateral) because the cells are grown on a fine mesh filter instead of solid plastic and the cells and mesh can be lifted out of the culture disk at any time.
Interpret these results.

Questions based on figures from page E
12 pts.

9) Figure 1 shows a series of related panels related to a strain of knockout mice with the p27 gene being tested.
a) What are the genotypes for mice 1 - 5 in panel B?

b) Describe the phenotypes from panels C and D and hypothesize an explanation.

c) Does this knockout mouse use the cell-specific mechanism for deleting a gene?

8 pts.

10) p53 has long been known to be involved in cancer as a tumor suppressor. In figure 2, we learn a new aspect about p53 - it is also an exonuclease. In this experiment, a 30mer oligonucleotide was end labeled either on the 3' end (left lanes) or the 5' end (right lanes) and incubated with 100% pure p53. Which end does p53 attack? Explain your answer. This one will take some time to think about but the answer should be short. Also, don't worry if you cannot read all of the legend, there is nothing important missing that I have not told you above, but if you want to try to read it you can.

Questions based on figures from page F
8 pts.

11) And the final question on this final is some what related to the paper we just finished on BMP and tld. Its great how our discussion of cloning the tld receptor was so close to what actually was done!

Ignore the top part (labeled EARLY) of panel A.

First, the frog (Xenopus ) homolog for tld was cloned. Then in flies, the tld receptor was cloned and called mad. This figure was performed with the frog homologs to mad (or Xmad1 and Xmad2). Two Xmad genes were cloned. E marks the lane where an entire embryo was used. The wedges indicate how much of the appropriate gene was injected into the frog eggs.

There are various mRNAs being used here to figure out which part of the embryo is being induced with the two Xmads. M. actin = back muscles Globin = ventral tissue N-CAM = notochord/CNS

a) Interpret these results. You do not need to describe in detail what is in each panel, simply summarize your conclusions for all panels.

b) What do we learn about Xmads 1 and 2 that might lead to future research in flies and possibly humans?

2 pts.
c) Final easy two points to bring the total up to 100 pts. How many bands did you see on your zooblot from lab?

Now go out into the world and clone, sequence, etc. and publish your results so I will have test questions for future classes!

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