There is no time limit on this test. You may find it easier to
take this test in two hour blocks over several days, though if you are confident
in your molecular skills, you could wait until Sunday night. However, I
predict it may take you a bit longer to think of all the answers
(just some friendly advice). You are not allowed to use your notes, any
books or journals, nor are you allowed to discuss the test with anyone until
all exams are turned in at 9:30 am on Monday, Feb. 3. While you are taking
the exam, you may not study for any science classes that will help you with
this exam (e.g. Organic Chem.) I do not know which classes may present problems
so talk to me if you are unsure. You may use a calculator. 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)?
Average for 1997 = 85%
You might find it more fun to take this test while humming (Dunt, dunt, dunt, dunt, dunt, dunt...) the tune to Mission Impossible.
20 pts.
1) (Good news, bad news.) You bought a ticket to London for spring break
and the plane is over booked (bad news). You get bumped to first class (good
news) and the man sitting next to you looks a lot like Tom Cruise (really
good news). He is reading what looks like a secret file. On this file is
the following:
A) This not-so-smart-agent needs help. Write down the DNA sequence shown
here in the proper 5' to 3' orientation.
5' ACA TGC TCC CAT GGT 3'
B) He tells you that the sequence you have written down is the template
strand for an mRNA. There are no introns, but he needs you to write down
the mRNA sequence in the proper 5' to 3' orientation.
5' ACC AUG GGA GCA UGU 3'
C) Now he asks you to translate the mRNA using the table provided to
you at the bottom of this page. Start with the 5' most mRNA nucleotide and
then write down the amino acid sequence beginning with the N-terminus.
THR MET GLY ALA CYS
D) Now he asks you to see if you can find an open reading frame. You
must tell him yes or no and explain why you have answered this way.
NO, SINCE THERE IS NO STOP CODON. HOWEVER, YOU DO POINT OUT THAT THERE IS
A START CODON (AUG) THAT INDICATES THAT AN OPEN READING FRAME DOES EXIST
SOMEWHERE IN THIS TRANSCRIPT BUT THE SEQUENCE YOU HAVE BEEN GIVEN IS TOO
SHORT.
15 pts.
2) You are having lunch at a cafe, and you see the following on your
menu. Please convert these letters to the full names of the amino acids:
| G I N G | T W | S E L F | D E S T R C T | I N | P H I V E | S E C |
| glycine | threonine | serine | aspartic acid | isoleucine | proline | serine |
| isolucine | tryptophan | glutamate | glutamate | asparagine | histidine | glutamate |
| asparagine | leucine | serine | isoleucine | cysteine | ||
| glycine | phenylalanine | threonine | valine | |||
| arginine | glutamate | |||||
| cysteine | ||||||
| threonine |
A) find out if the antibody's epitope is also present on acetylcholinesterase
isolated from flies, mice, worms, and humans and if so, how big each version
of acetylcholinesterase is.
+ control: known and purified acetylcholinesterase in a lane by itself-
control: a tissue known to lack acetylcholinesterase, such as yeast cells
a lane for each of the species with total protein loaded. I would choose
muscle tissue for mice and humans.
a lane of MW markers
Run these samples on an SDS-PAGE, transfer to nitrocellulose, probe with
the alleged monoclonal antibody. Detect the antibody.
You should see a band in the + control lane and none in the - control lane.
The + control lane will tell you the approximate size to expect in the other
species. Note where the bands are in each lane and calculate the MW for
each species' form of acetylcholinesterase, using the MW markers.
B) since all of these species do express acetylcholinesterase, clone
the cDNA that encodes the fly version of acetylcholinesterase. (It has never
been cloned from flies, though it has from the other species.)
Two choices, either use the other species' cDNA as a probe for a cDNA library
(lambda gt10), or use the antibody to probe an expression library (lambda
gt11).
But either way will involve these steps:
isolate purified fly mRNA
make cDNA
make cDNA library in lambda library (see above)
make your probe (see above)
screen the appropriate library
isolate lambda phage DNA
sequence the cDNA insert to confirm you have the right clone
9 pts.
4) Below, you see two northern blots. One for Free Bacterial Intake
protein, and one for actin, the universally expressed protein. What can
you deduce from these data? Explain how your made your deduction.
Actin controls: they worked in all lanes; it appears that each lane has
about the same amount of total RNA loaded except human which appears to
have more RNA in that lane since the actin band is more intense.
Flies: the FBI mRNA has a molecular weight of about 1.6 kb. It appears to
be the least abundant of all FBI mRNA.
Worms: appears to have two alleles of FBI, one about 1.1 kb and one about
1.3 kb in length. These two forms are collectively about twice as abundant
as the fly FBI mRNA.
Mice: the molecular weight is about 0.9 kb and appears to be the most abundant
FBI mRNA due to the thickness of the band being largest.
Humans: molecular weight is about 1.3-1.4 kb. Its relative abundance is
difficult to judge since there is more total RNA in this lane than in any
of the others. But it is not the most abundant form since this band is the
same thickness as the mouse FBI band.
12 pts.
5) When you return to your hotel room, you find a balance, a pH meter, some chemical bottles, various pipets, a calculator and the following directions:
A) make solution with a final volume that is 225 ml of a 0.35 M NaCl,
5% v/v acetic acid. Tell me all the volumes and all reagents. Formula weights:
NaCl = 58.5, Tris = 121; acetic acid = 58; EDTA = 372; BamH I and Hind III
= 103 each.
4.6 g NaCl + 11.25 ml acetic acid and water up to 225 ml
B) A stock solution is 2.59 mg/ ml of DNA and you want to make 100 µl
that is 1 µg/ µl. How do you do this?
use 38.6 µl of stock DNA and 61.4 µl water
C) Set up a restriction digestion of 1 µg the DNA above and cut
it in 15 µl final volume with the enzymes
Bam HI and Hind III.
1 µl DNA
1.5 µl 10X buffer
< 0.75 µl Bam
< 0.75 µl Hind III
11 µl water
D) Make 250 ml of 10X TE which is 100 mM Tris (pH 8.0) and 10 mM EDTA.
3.025 g Tris in about 200 ml
0.93 g EDTA
pH with HCl
water up to 250 ml
10 pts.
6) How could you subclone the following 2 fragments (no need for exact
volumes; just the strategy)? Clone each of them into separate plasmids and
convince me that you know which is which. The boxes represent segments of
DNA that terminate with the indicated restriction sites. There are two segments
of DNA that are nonfunctional (blank) but the rest of the DNA segments have
been labeled. Your task is to clone two fragments into a typical cloning
plasmid. The two fragments you should clone are: fragment 1 is from 400
to 1600, and fragment 2 is from 4400 to 5200.
FRAGMENT 1:
Digest plasmid with Bam and gel purify the 1200 bp fragment. This will be
a 50/50 mixture of DNA. Ligate this into a new plasmid (that does not contain
lac Z) cut with Bam, transform into bacteria, and plate on medium
that would allow for the detection of ß-galactosidase (colonies turn
blue). Digest DNA to confirm the size of insert and that there are not two
inserts.
FRAGMENT 2:
Digest plasmid with Sal and purify the 1800 bp fragment. Then digest this
with Bam and purify the only 800 bp fragment. Ligate into Bam cut plasmid
(that does not contain tetR), transform into bacteria and plate
onto tetracylcine containing media. Select colonies that grow.
10 pts.
7) John Majors (#2 in pedigree) is heterozygous for a rare genetic disease
of compulsive secret telling. You can see a family pedigree and a series
of RFLPs below.
A) Which band is associated with the diseased allele? How
do you know this?
The upper band since the two suffering with this disease (4 and 6) are
homozygous for the disease allele and homozygous for the upper band.
B) Which grandchild should not be trusted with secrets?
I would not trust child number 8. He is homozygous for the upper band.
However, he may not suffer from this disease since he is not shaded in.
This would be due to recombination between the RFLP marker and the disease
locus.
10 pts.
8) A top covert operative almost lost his life when he refused to drink
the customary milk drink that all international spies drink. Design a method
to generate a hypoallergenic cow's milk. Since many people develop allergies
to a milk protein called casein, how could you produce casein-free milk
using transgenic technology?
I would make a transgenic cow that expresses casein anti-sense RNA. To
do this, I would take the wild-type casein promoter and ligate it onto the
anti-sense strand of DNA for casein (see diagram).
10 pts.
9) Finally, you return home. You are glad to be away from international
spies and the doorbell rings. (Dunt, dunt, dunt, dunt, dunt, dunt...) The
secrete service has been following your vacation moves, and wants some advice.
It appears that a biological weapon has been discovered in the White House.
It is a normal protein except a single amino acid has be changed from cysteine
to methionine. After looking at the structures below, tell the agent what
possible effect this substitution might have on the protein.
Two major possible disruptions: 1) the loss of a cystein may lead to
a disrupted disulfide double bond. This would alter secondary, tertiary,
and quaternary structure of the wild-type protein and since form gives function,
the mutation could have an altered property. 2) There is the loss of a hydrophilic
R group and the addition of a hydrophobic R group. This could also disrupt
secondary, tertiary, and quaternary structure of the wild-type protein.
© Copyright 2000 Department of Biology,
Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: macampbell@davidson.edu