Biology 4410
Sample Questions
3. In the early 1960s, Anfinsin and his colleagues conducted
experiments on the folding of the enzyme ribonuclease S (RNase S).
Starting with purified RNase S, they unfolded the protein using
denaturing agents such as urea. Some of their results are
summarized below.
When the RNase S was added to a solution containing urea and
mercaptoethanol, the protein was unfolded and lost all of its
enzymatic activity.
If the urea and mercaptoethanol were removed slowly by the
process of dialysis, the enzyme regained its full activity.
If only the urea was removed, but the protein remained in the
presence of mercaptoethanol, the enzyme did not regain its
enzymatic activity.
(a) Based on these experiments, what conclusions can you reach
about the requirements for the proper folding of RNase S? In other
words, where is the information for the proper folding to be found?
(5 pt)
(b) What did the mercaptoethanol do, and why did it have to be
removed for the protein to regain its activity? (5 pt)
(c) Do these experiments support the “molecular chaperone” model of
protein folding? Briefly explain why or why not. (5 pt)
(d) Based on the above experimental approach, design an experiment
to test the hypothesis that molecular chaperones can affect the
rate of folding of RNase S. Be sure to make predictions about how
the presence of molecular chaperones should affect the rate of
folding. (5 pt)
5. Restriction endonucleases are site-specific endonucleases found in
many bacteria. Each one binds to a a short, highly specific
sequence of double-stranded DNA. For example, EcoR1 recognizes
the sequence
G A A* T T C
C T T A* A G
Restriction endonucleases are believed to serve bacteria by
destroying bacteriophage (bacterial virus) DNA when it enters the
cell. Bacteria protect their own DNA by methylating (attaching a
methyl group to) certain specific bases within the recognition
sequences of the bacterial DNA. The EcoR1 methylase enzyme
methylates the adenine base of the adenosine nucleotides,
indicated by asterisks in the above diagram. In this process, a
methyl group (CH3-) is substituted for a hydrogen on the nitrogen
in the #6 position on the adenine base.
a. Assume that the EcoR1 endonuclease recognizes its recognition
sequence by the mechanisms of protein-DNA recognition discussed
in class and in the textbook. How could methylation of a nucleotide
base interfer with the ability of the enzyme to recognize its
sequence? Be as specific as possible in your answer. (5 pt)
b. Under conditions of alkaline pH, the specificity of EcoR1 is reduced
so that only the internal tetranucleotide sequence is recognized:
AATT
TTAA
Based on the mechanism of protein-DNA recognition, how can this
result be explained? (5 pt)
7. Renaturation kinetics studies (cot) played a key role in our early
understanding of eukaryotic DNA composition.
a. Does the renaturation kinetics curve of calf thymus DNA fit the
mathematical model (“idealized curve”) from renaturation kinetics
theory? Briefly explain. In your explanation, include appropriate
information about the structural organization of eukaryotic DNA.
(5 pt)
b. Consider the following experiment.
DNA and mRNA were isolated separately from a culture of
eukaryotic cells and purified. The mRNA was labeled with a
radioactive isotope. The DNA was denatured by heating and mixed
with the labeled mRNA. Then, the mixture was cooled to allow the
mRNA and the DNA to hybridize.
Denatured DNA
____________ _____________
****************
***************
Labeled mRNA DNA-mRNA hybrid (double-stranded)
At timed intervals, samples were analyzed to determine the amount
of labeled mRNA that had hybridized to DNA. The results were
plotted on a cot curve (with co in this case being the initial
concentration of labeled mRNA). In the plot, cot was plotted on the
x-axis, and the fraction of single-stranded mRNA remaining was
plotted on the y axis.
Sketch the cot curve plot that you would predict in this experiment,
and briefly explain the rationale for your prediction. (You may
assume the same theoretical basis as for DNA renaturation
kinetics.) (5 pt)
2. According to the model of the synthesis of plasma membrane
integral proteins in the endoplasmic reticulum and Golgi
apparatus, one would predict that the carbohydrate attached to
plasma membrane glycoproteins is located on the exterior domain
of the proteins, and not on the cytoplasmic domain or the
transmembrane domain.
(a) Briefly explain how the model of membrane protein synthesis
would lead to this conclusion. (5 pt)
(b) Design an experiment to test the prediction (that the carbohydrate
is located only on the exterior domain of the plasma membrane
proteins), using erythrocyte plasma membrane as the experimental
system. You may assume that you have a reagent that tests
specifically for carbohydrates. (5 pt)
3. Fab fragments are antibody fragments containing the antigen
binding sites from the original antibody molecules. They are made
by treating antibody molecules with the proteolytic enzyme papain.
Each Fab fragment contains only one antigen binding site per
molecule. (A diagram is found on pg 1209 of Alberts.)
(a) In class, we talked about an experiment to demonstrate the lateral
mobility of integral membrane proteins by observing “patching and
capping” after treating cells with fluorecently labeled antibody.
What results do you predict should be seen if fluorescently-labeled
Fab fragments were used instead of whole antibody molecules in
the experiment? (5 pt)
(b) Would this result (using Fab instead of whole antibody molecules)
support, contradict, or have no bearing on the conclusions that we
reached in class regarding lateral mobility in the patching and
capping experiment? Briefly explain. (5 pt)
1. Succinylcholine is a chemical analog of acetylcholine. It is used by
surgeons as a muscle relaxant because it produces a type of flaccid
paralysis by blocking the nerve impulse at motor neuron end
plates.
(a) If succinylcholine is a chemical analog of acetylcholine, why do you
think it causes muscles to relax and not contract as acetylcholine
does? In your answer, you must relate your explanation specifically
to the process of cell-cell signaling that takes place at the motor
neuron end plates. Also, suggest an experiment to test your
hypothesis. (A hint and note: this question has nothing to do with
actin and myosin. Keep it simple and don’t get sidetracked.) (5 pt)
(b) Care must be taken in the use of succinylcholine because some
individuals have an adverse reaction. In most cases, the patient
recovers from the succinylcholine paralysis after a short time. In
cases with sensitive patients, the individual recovers abnormally
slowly from the drug, with life-threatening consequences. It is
known that abnormal sensitivity to this drug can be inherited.
Considering that it is an analog of acetylcholine, suggest a
molecular explanation of the sensitivity to succinylcholine in
sensitive patients. If your explanation is correct, then how could a
physician treat a sensitive patient to whom succinylcholine has
been accidentally administered? (5 pt)
5. Cyclic AMP (cAMP) plays an important role as a second messenger
in cell signaling processes using G-protein-linked receptors.
Therefore, it is important for a cell to carefully regulate the
intracellular concentration of cAMP. An interesting illustration of
the role that cAMP plays in the physiology of the whole organism
comes from studies of Drosophila melanogaster. There is a
mutation in Drosophila called dunce. Flies that are homozygous for
dunce have a reduced amount of an enzyme called cyclic AMP
phosphodiesterase. In fact, dunce flies have only about half the
amount of cAMP phosphodiesterase as normal wild-type flies.
Cyclic AMP phosphodiesterase acts to break down cAMP, reducing
its level in the cells. Researchers developed a learning test in which
flies were presented two metallic grids, one of which was electrified.
If the electrified grid was painted with a strong-smelling chemical,
normal flies learned quickly to avoid the grid even when it was no
longer electrified. Mutant dunce flies, on the other hand, never
learned to avoid the smelly grid.
(a) Assume that Drosophila cells have a similar mechanism for the
regulation of glycogen metabolism as mammalian cells, and that
Drosophila cells are capable of responding to epinephrine in a
manner similar to mammalian cells. In separate experiments, cells
from either wild-type or dunce flies were treated for a short time
with epinephrine; then, the epinephrine was removed. The rates of
glycogen synthesis and glycogen breakdown were measured at
timed intervals after the epinephrine treatment. How would dunce
cells differ from wild-type cells? Briefly explain your answer. (5 pt)
(b) Caffeine is a phosphodiesterase inhibitor. What effect do you
predict that caffeine would have on the learning performance of
normal, wild-type flies. Briefly explain your answer. (5 pt)
6. Microtubules are formed by the polymerization of tubulin dimers.
The process can be studied by pulse-chase radiolabeling
experiments. In these experiments, a suspension of microtubules
is treated for a brief time with radiolabeled tubulin dimers. Then,
the radiolabeled tubulin is removed and replaced with
nonradioactive tubulin. Samples are removed at timed intervals
following the radiolabel pulse and analyzed by autoradiography, a
microscopic technique in which the location of the radioactivity is
visualized.
The diagram shows the results of two separate pulse-chase
experiments on microtubule assembly. In the first experiment,
microtubules were maintained in suspension under conditions in
which the average microtubule length remained constant. In the
first experiment, only the microtubules and an appropriate
concentration of tubulin monomers were suspended in buffer. In
the second experiment, the conditions were identical except that
isolated centrosomes were also added to the suspension. In the
diagrams, the position of the radiolabeled regions is indicated by
the shaded areas. Also, note that the length of the microtubules
increases over time in the second experiment.
Pulse-chase experiment on Microtubule Assembly
(A) Microtubule assembly in the absence of centrosomes
Time after
radiolabel pulse
(A) Microtubule assembly in the presence of centrosomes
Time after
radiolabel pulse
(a) In the model of microtubule assembly, one end of a microtubule
(the plus end) polymerizes at a rapid rate, while the other end of
the microtubule (the minus end) depolymerizes at a rapid rate. Do
the data in experiment (A) support this model? Briefly explain why
or why not. If possible, indicate which end of the microtubule
shown in experiment (A) is the plus end, and which end is the
minus end. (5 pt)
(b) What do the data in experiment (B) suggest about the function and
the mechanism of action of centrosomes? (5 pt)
(c) In other experiments on the in vitro assembly of tubulin into
microtubules, researchers have found that tubulin must be
present in solution at a certain critical concentration before it will
form microtubules. In the absence of centrosomes, the critical
concentration of tubulin for microtubule formation is about 15M.
In the presence of centrosomes, the critical concentration is about
3M. Explain this difference. (5 pt)