Problem Solving Tests in MCB (Full)

PROBLEM-SOLVING TESTS IN
MOLECULAR CELL BIOLOGY
József Szeberényi
University of Pécs
Medical School
2015.
Technical assistance
Zita Árvai
Mónika Vecsernyés
With the permission of the International Union of Biochemistry and

Molecular Biology.
Supported by a grant from the European Union (TÁMOP-4.1.1.C-

13/1/KONV-2014-0001).
CONTENTS
Electrophoretic behavior of hemoglobin variants 1.
Real-time polymerase chain reaction 5.
Targeted gene disruption 10.
The effect of bisulfite treatment on genomic DNA 15.
Analysis of the cell cycle by flow cytometry 18.
α1-Antitrypsin deficiency: An example for a protein folding disease 22.
Vectorial transport in intestinal epithelial cells 32.
Expression cloning of the erythropoietin receptor 36.
The yeast two-hybrid system 41.
Analysis of the mechanism of action of an apoptosis inducing compound 45.
The mechanism of action of a human papilloma virus oncoprotein 51.
Signal transduction in Philadelphia chromosome positive leukemia cells 56.

1
ELECTROPHORETIC BEHAVIOR OF HEMOGLOBIN

VARIANTS
Terms to be familiar with before you start to solve the test
Hemoglobins * erythrocytes * reticulocytes amino acids *  and  globin chains *

electrophoresis * isoelectric point * sickle cell anemia
The experiment
Hemoglobin A (HbA), the predominant adult hemoglobin in our red blood cells is a
vital protein responsible for the transport of oxygen. It is synthesized in immature
reticulocytes and carries out its function in mature erythrocytes. It consist of two  and two 
globin chains and one hem molecule bound to each of these subunits. Hemoglobinopathies
represent a large group of inherited diseases caused by mutations in  and  globin genes.
Two of the most common hemoglobin variants are HbS (expressed in red blood cells of
patients that suffer from sickle cell anemia) and HbC (causing a milder condition). In both
disorders, the mutation affects codon 6 in  globin mRNA: the glutamic acid (Glu) at position
6 in the  globin chain is replaced by valine (Val) or lysine (Lys) in HbS and HbC,
respectively (the formulae of these amino acids are shown in Figure 1.
H2N CH COOH H2N CH COOH H2 N CH COOH
CH2 CH CH2
2 5
1 CH2 CH2
CH 3 CH3
COOH CH2
3 4 CH2
NH2
Glu Val 6
Lys
Figure 1: The chemical formula of glutamic acid, valine and lysine.

2
Five-choice Completion
(This type of question consists of a question or incomplete statement followed by five suggested answers or completions .
Select the one best answer.)
1.____ After the incorporation of these amino acids into the globin chain, which of
these groups can be involved in peptide formation?
A. Group 1
B. Group 2
C. Group 3
D. Groups 1 & 2
E. All three groups
2.____ After the incorporation of these amino acids into the globin chain, which of these
groups can acquire a positive charge in aqueous solution?
A. Group 1
B. Group 2
C. Group 5
D. Group 6
E. Groups 1, 5 & 6
groups can acquire a negative charge in aqueous solution?
A. Group 1
B. Group 2
C. Group 3
D. Groups 2 & 3
E. Group 4
groups is most likely buried in the internal „core” of the hemoglobin molecule?
A. Group 1
B. Group 3
C. Group 4
D. Group 6
E. Groups 3 & 4
Quantitative Comparison
(In this type of question paired statements describe two entities that are to be compared in a quantutative sense. Select
A if A is greater than B;
B if B is greater than A;
C if the two are equal or very nearly equal.)
5.____ A. The isoelectric point of HbA

B. The isoelectric point of HbS
3
Red blood cell extracts from four individuals with different hemoglobin variants
(HbA, HbC and/or HbS) in their erythrocytes were analyzed by gel electrophoresis in the
experiment described in this test. After electrophoretic fractionation of the samples the gel
was stained with a protein dye (Figure 2).
+
I
II
III
Origin
-
1 2 3 4
Figure 2. Electrophoretic analysis of red blood cell extracts from four individuals (- and +
indicate the positions of the electrodes during electrophoresis). (After Fig. 6.9. in
Gelehrter, T.D., Collins, F.S., Principles of Medical Genetics, Williams & Wilkins,
Baltimore, 1990.)
Study the figures and solve the following multiple-choice questions. (Note that the
single amino acid differences do not affect significantly the molecular masses of HbA, HbS
and HbC: the size of these proteins is essentially the same.)
Figure Analysis
(The following statements are related to the information presented above. Based on the information given, select:
A if the statement is supported by the information given;
B if the statement is contradicted by the information given;
C if the statement is neither supported nor contradicted by the information given.)
6.____ Under the electrophoretic conditions used all the hemoglobin variants (I, II and III)
were negatively charged.
7.____ The pH of the electrophoretic buffer was the same as the isoelectric point of
hemoglobin variant III.
8.____ The isoelectric point of protein I is higher than that of protein III.
9.____ Individuals 1 and 4 are carriers of the HbC mutation.
4
10.____ Which of these individuals has/have the worst prognosis?

A. Individual 1
B. Individual 2
C. Individual 3
D. Individual 2 & 3
E. Individual 4
11.____ Which of these individuals has/have only mutant hemoglobin in the red blood cells?
A. Individual 1
B. Individual 2
C. Individual 1 & 2
D. Individual 3
E. Individual 4
12.____ Which of the following statements describes the hemoglobin status of red blood cells
in individual 2 best?
A. The structure, function and cellular level of hemoglobin is normal
B. Hemoglobin synthesis in the reticulocytes is normal, but degradation is
increased
C. Hemoglobin synthesis in the reticulocytes is reduced, but degradation is
normal
D. Hemoglobin is secreted to the plasma by these cells
E. The water solubility of hemoglobin is decreased
Correct anwers
1. D 7. B
2. D 8. B
3. C 9. B
4. C 10. B
5. B 11. C
6. A 12. E
This test was published in Biochemistry and Molecular Biology Education and is
presented here with the permission of the International Union of Biochemistry and Molecular
Biology.
Szeberényi J. (2004) Problem-solving test: Electrophoretic behavior of hemoglobin
variants. Biochem.Mol.Biol.Educ. 32, 350-351.
Supported by a grant from the European Union (TÁMOP-4.1.1.C-13/1/KONV-2014-

0001).
5
REAL-TIME POLYMERASE CHAIN REACTION
polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, HER2 gene,
genomic DNA, in vitro DNA synthesis, template, primer, Taq polymerase, 5’3’ elongation
activity, 5’3’ exonuclease activity, deoxyribonucleoside triphosphates, DNA structure,
proofreading, thermocycler, fluorescence
The experiment
In traditional polymerase chain reaction (PCR) the analysis of the amplified DNA
region takes place after 30-40 cycles the product is studied by electrophoresis and DNA
staining. The advantage of real time PCR is that the process can be monitored during the
reaction: the extent of DNA amplification can be determined after each PCR cycle. Several
different methods have been developed for this, the principle of one of the most popular
techniques (TaqMan reaction)1 is described in the following experiment.
Figure 1. The principle of TaqMan method (details in the text).
A tumor was removed from the breast of a patient. Genomic DNA was isolated from
the tumor and the surrounding normal tissue and PCR reaction was performed using identical
amounts of the two DNA samples. Reaction mixtures contained the following components.
DNA template (the genomic DNA samples); many copies of 2 primers specific for a
region of the HER2 gene (their binding to the template is shown in Fig. 1);
many copies of a TaqMan probe, an oligonucleotide binding to one of the template
strands in the region flanked by the two primers (a fluorescent reporter dye is
attached to the 5’-end and a quenching molecule to the 3’-end of the probe,
inhibiting the fluorescensce of the reporter);
Taq polymerase (heat-resistant DNA polymerase with 5’→3’ elongation and 5’→3’
exonuclease activities);
the four dexoyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP).
6
Using your knowledge of bacterial DNA replication solve the following multiple-
choice questions (MCQs).
Four-choice Association
(In this type of question a set of lettered headings is followed by a list of numbered words or phrases. Select
A if the word or phrase is associated with A only;
B if the word or phrase is associated with B only;
C if the word or phrase is associated with A and B;
D if the word or phrase is associated with neither A nor B.)
A: 5’→3’ elongation activity of Taq polymerase

B: 5’→3’ exonuclease activity of Taq polymerase
C: Both of them
D: Neither of them
1.____ Generates phosphodiester bonds.
2.____ Cleaves.
3.____ Cleaves hydrogen bonds.
4.____ Is primer-dependent.
5.____ Degrades the primers into mononucleotides in the mixture described above.
6.____ Degrades the TaqMan probe into mononucleotides in the mixture described above.
7.____ Has a proofreading function.
Reaction mixtures were incubated in a thermocycler capable of monitoring

fluorescence. Fig. 2 shows relative fluorescence values measured after each cycle using the
breast tumor (A) and normal (B) DNA sample.
7
1000
100
Sample A
10
F luorescence
1 Sample B
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Number of cycles
Figure 2. Real-time PCR performed with a breast cancer (A) and a normal (B) genomic
DNA sample from the same patient (details in the text).
Study the figure and solve the following MCQs.
(This type of question consists of a question or incomplete statement followed by five suggested answers or
completions. Select the one best answer.)
8.____ As PCR reactions proceed, at one point fluorescence increases in both mixtures.
What process can explain this?
A: TaqMan probe molecules are degraded into nucleotides
B: TaqMan probe molecules are degraded into smaller oligonucleotides
C: TaqMan probe molecules are released from the template as intact
oligonucleotides
D: TaqMan probe molecules serve as primers for Taq polymerase
E: TaqMan probe molecules are incorporated into the newly synthesized DNA strands
(In this type of question paired statements describe two entities that are to be compared in a quantitative sense.
Select
9.____ A: The number of free primer molecules in sample A after cycle 4

B: The number of free primer molecules in sample A after cycle 10
10.____ A: The number of free primer molecules in sample A after cycle 10

B: The number of free primer molecules in sample B after cycle 10
8
11.____ A: The number of free TaqMan probe molecules in sample A after cycle 10
B: The number of free TaqMan probe molecules in sample B after cycle 10
12.____ A: The number of free TaqMan probe molecules in sample A after cycle 18
B: The r number of free TaqMan probe molecules in sample B after cycle 18
13.____ A: The number of reporter dye/mononucleotide complexes in sample A after cycle 18

B: The number of reporter dye/mononucleotide complexes in sample B after cycle 18
14.____ A: The number of amplified HER2 fragments in sample A after cycle 20

B: The number of amplified HER2 fragments in sample B after cycle 20
15.____ A: The number of amplified HER2 fragments in sample A after cycle 30

B: The number of amplified HER2 fragments in sample B after cycle 30
16.____ Why there is no detectable fluorescence in the samples after the first few PCR
cycles?
A. Because Taq polymerase degrades the TaqMan probe molecules

B. Because Taq polymerase degrades the primers
C. Because there is no DNA synthesis
D. Because the quenchers block the fluorescence of all reporter dye molecules
E. Because the fluorescence detector is not sensitive enough
17.____ Why fluorescence does not increase after cycle 30 in either samples ?
A. Because all primer molecules have been used

B. Because all TaqMan probe molecules have been used
C. Because all template molecules have been used
D. A and B
E. A, B and C
18.____ What happened to the HER2 gene in the breast tumor cells?
A. Its copy number increased approximately 30-fold

B. Its copy number increased approximately 5-fold
C. Its copy number decreased approximately 30-fold
D. Its copy number decreased approximately 5-fold
E. Its expression decreased approximately 5-fold
9
Correct anwers
1. A 8. A 15. C
2. B 9. A 16. E
3. D 10. B 17. D
4. A 11. B 18. A
5. D 12. B
6. B 13. A
7. D 14. A
1
This test is based on a product description of Applied Biosystems (Foster City, California,
USA; www.appliedbiosystems.com).
Biology.
Szeberényi J. (2009) Problem-solving test: Real-time polymerase chain reaction.
Biochem.Mol.Biol.Educ. 37, 250-252.

0001).
10
TARGETED GENE DISRUPTION
mutation, vector, plasmid, origin of replication, promoter, introns/exons, open reading frame,
transfection, circular and linear DNA, DNA integration, homologous recombination, DNA
replication, gene expression, heterozygote, homozygote.
The experiment
Mutational inactivation of a specific gene is the most powerful technique to analyze

the biological function of the gene. This approach has been used for a long time in viruses,
bacteria, yeast, fruit fly, but looked quite hopeless in more complex organisms. Targeted
inactivation of specific genes (also known as knock-out mutation) in mice is arguably one of
the most significant achievements in modern biology. The following test describes the
procedure developed in the laboratory of one of the Nobel laureates of 2007, Mario Capecchi
[1] and the reader is expected to interpret the principle of K.O. mutation by solving the
multiple-choice questions (MCQs).
In this procedure part of the gene to be targeted (designated gene X) is inserted into a
knock-out vector (Fig. 1). This vector is a plasmid that does not contain a mammalian origin
of replication, but carries two selectable markers. A neoR gene that will make the cells
resistant to the highly toxic protein synthesis inhibitor Geneticin is inserted into one of the
protein coding exons of the targeted gene. The neoR gene is supplied with a strong mammalian
promoter.
1.____ What can be the consequence of inserting neoR gene into the gene X exon?
A. The replication of the targeting vector is stimulated in mammalian cells
B. The expression of protein X is stimulated
C. The reading frame of mRNA X is disrupted
D. A and B
E. A and C
2.____ What is the significance of linking a mammalian promoter to neoR ?

A. To make neoR expressed in the cells used for gene targeting
B. To inhibit the expression of gene X
C. To stimulate the expression of gene X
D. A and B
E. A and C
11
R
neo
HSV
tk
gene X region
targeting vector
linearized
with a restriction enzyme
R
neo
HSV
tk
gene X region
Figure 1. The structure of circular (above) and linearized (below) targeting vector (black
bars, exons; light gray blocks, introns of gene X).
The second selection marker in the targeting vector is tkHSV, a thymidine kinase gene
of herpes simplex virus that, if expressed in mammalian cells, causes cell death when treated
with the antiviral drug Gancyclovir.
Mouse cells in a culture are then treated with many linearized copies of the targeting
vector under conditions that help the transfer of DNA into the cells. (Interactions between the
foreign DNA and the host cell genome described below are more efficient with linear than
with circular plasmid DNA.) Many cells do not take up DNA or if they do the foreign nucleic
acid is quickly degraded in them. In some cells, however, the targeting vector interacts with
the genome of the cells. Two genetic events can take place (Fig. 2). In most cases the linear
DNA is randomly integrated into double-stranded breaks of the genome (Fig. 2A). Much less
frequently sequences of gene X in the K.O. vector find their genomic counterparts, get into
physical contact with them and homologous recombination takes place between the two
sequences (Fig. 2B). If recombination happens on both sides of the neoR gene, this region is
transferred into the genomic gene X, while the corresponding normal sequences will become
part of the plasmid. A key step in targeted gene disruption is to distinguish between these two
genetic events.
12
Figure 2. Random integration of the targeting vector (A) and homologous recombination
between the targeting vector and gene X (B).
13
A. Cells with random integration (as shown in Fig. 2A)

B. Cells with homologous recombination (as shown in Fig. 2B)
C. Both of them
D. Neither of them
3.____ Replicate the neoR gene during the S phase of the cell cycle.
4.____ Express the neoR gene.
5.____ Degrade and loose the tkHSV gene rapidly.
6.____ Express the tkHSV gene only transiently.
7.____ The function of gene X is inhibited in these cells.
A. Geneticin treatment
B. Gancyclovir treatment
C. Both of them
D. Neither of them
8.____ Kills cells that did not take up foreign DNA during transfection.
9.____ Kills cells in which foreign DNA is degraded in the cytoplasm.
10.____ Kills cells in which the targeting vector randomly integrated into the genome (as
shown in Fig. 2A).
11.____ Kills cells with homologous recombination between the targeting vector and gene X
(as shown in Fig. 2B).
12.____ What are the characteristic features of cells surviving double selection with
Geneticin and Gancyclovir?
A. They contain the targeting vector in integrated form only
B. They contain targeting vector sequences as a result of both integration and
homologous recombination (as shown in Fig. 2)
C. They are heterozygous gene X knock-outs (gene X+/-)
D. They are homozygous gene X knock-outs (gene X-/-)
E. B and D
14
Correct anwers
1. C 7. B
2. A 8. A
3. C 9. A
4. C 10. B
5. B 11. D
6. B 12. C
REFERENCE
[1] S. L. Mansour, K. R. Thomas, M. R. Capecchi (19889) Disruption of the proto-

oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting
mutations to non-selectable genes. Nature 336, 248-352.
Biology.
Szeberényi J. (2008) Problem-solving test: Targeted gene disruption.

0001).
15
THE EFFECT OF BISULFITE TREATMENT ON GENOMIC DNA
Polymerase chain reaction (PCR)  primer  promoter  restriction endonucleases 

agarose gel electrophoresis  ethidium bromide staining  DNA methylation * Taq
polymerase * single nucleotide polymorphisms (SNPs)* CpG islands * tumor suppressor
genes * protooncogenes * epigenetic regulation
The experiment
The assay described in this test was designed to detect an important regulatory DNA
modification. Genomic DNA samples from a normal tissue (samples 2 and 3 in Fig. 1) or a
tumor (samples 4 and 5) were divided into two aliquots: samples 3 and 5 were treated with
bisulfite (that converts unmethylated cytosines into uracils while methylated cytosines remain
unchanged), samples 2 and 4 were left untreated. Polymerase chain reaction (PCR) was
performed with the two DNA samples using a pair of primers specific for the promoter region
of a gene. The PCR products were digested with EcoRI restriction endonuclease, the samples
were fractionated by agarose gel electrophoresis and stained with ethidium bromide (Fig. 1).
(The recognition site of EcoRI is:
The arrows indicate the cleavage sites.)
Normal Tumor
Origin of D NA
tissue tissue
M
- + - + Bisulfite treatment
bp
1000
800 a
600
500
400 b
300 c
200
1 2 3 4 5
Figure 1: Agarose gel electrophoresis of the DNA samples (for details see the text; M, size
marker; bp, base pair).
16
Study the figure and solve the following multiple-choice questions (MCQs).
A: Band a
B: Band b
C: Both of them
D: Neither of them
1.____ Consists of fragments with one intact 5’-GAATTC-3’ sequence.

3’-CTTAAG-5’
5’-GAATTC-3’
2.____ Consists of fragments with several intact sequences.
3’-CTTAAG-5’
5’-GAATTC-3’
5’-GAATTT-3’
3.____ Consists of fragments with one intact ssesequence.
3’-CTTAAG-5’
3’-CTTAAA-5’
4.____ Consists of fragments with one intact 5’-AAATTT-3’ sessequence.

3’-TTTAAA-5’
5.____ Consists of fragments with two blunt ends.
6.____ Consists of fragments with one blunt and one sticky end.
7.____ Consists of fragments with two sticky ends.
Experiment Analysis
(The following statements are related to the information presented in the description of the experiment.
Based on the information given, select
A if the statement is supported by the information given

B if the statement is contradicted by the information given
8.____ Both strands of the EcoRI recognition site were methylated in the original normal
DNA molecule.
9.____ Both strands of the EcoRI recognition site were methylated in the original tumor
DNA molecule.
10.____ DNA strands containing uracil serve as templates for Taq polymerase.
17
11.____ Taq polymerase has DNA methyl transferase activity.
12.____ Bisulfite treatment of the template DNA inhibited the polymerase chain reaction.
13.____ The EcoRI site of the promoter region analyzed in this experiment is flanked by
C≡G pairs at both sides.
14.____ What can be the biomedical significance of this assay?

A. To detect single nucleotide polymorphisms (SNPs) in genomic DNA
B. To analyze the methylation state of the promoters of tumor suppressor
genes in cancer cells
C. To study the expression of protooncogenes in normal cells
D. B and C
E. A, B and C
Correct answers
1. D 8. D
2. D 9. A
3. A 10. A
4. D 11. C
5. A 12. B
6. B 13. A
7. D 14. B
The test describes a fictitious experiment based on: Zymo Research Catalog, 2006/2007, p42.
Biology.
Szeberényi J. (2008) Problem-solving test: The effect of in vitro bisulfite treatment on
genomic DNA. Biochem.Mol.Biol.Educ. 36, 66-67, 2008.

0001).
18
ANALYSIS OF THE CELL CYCLE BY FLOW CYTOMETRY
cell cycle * flow cytometry * cell culture * fluorescent DNA dye * diploid and tetraploid cells
* mitosis * phases of the cell cycle * growth factors * microtubules * apoptosis * DNA
synthesis * proteasome * [3H]thymidine * pulse labeling * histones * protein phosphorylation
* M-phase promoting factor (MPF) * cyclins * cyclin-dependent kinases (Cdks) *
synchronized culture
The experiment
Human tumor cell cultures were treated with a drug profoundly affecting the cell
cycle. Thereafter the drug was washed out from the cultures (at time zero), and the cells were
kept under conditions optimal for growth. At the indicated times cultures of identical cell
numbers were stained with a fluorescent DNA dye and then subjected to flow cytometry.
Figure 1 shows the flow cytometric curves.
Study the figure and solve the following multiple-choice questions!
Figure 1. Flow cytometric analysis of human tumor cell cultures (for experimental details
see the text). (Taken from D.O. Morgan: The Cell Cycle, with permission.)
19
1.____ Which of the following treatments could be used to produce the time-zero situation?
A. Treatment with a growth factor
B. Treatment with an inhibitor of microtubule assembly
C. Treatment with an apoptosis-inducing agent
D. Treatment with a DNA synthesis inhibitor
E. Treatment with a proteasome inhibitor
2.____ Which of the cultures would be most heavily labeled after a [3H]thymidine pulse?
A. The 0-hour culture
B. The 2-hour culture
C. The 8-hour culture
D. The 12-hour culture
E. The 24-hour culture
3.____ The cells of which culture contain the largest amount of histones?
4.____ The cells of which sample contain the highest number of phosphorylated H1 histone
molecules?
E. There is no difference between the cultures
5.____ The cells of which culture contain the highest levels of MPF activity?
20
6.____ The cells of which culture contain the lowest level of cyclin B?
7.____ At what time did the cells divide?

A. At time zero
B. Between 4 and 8 hours
C. Between 8 and 12 hours
D. At 24 hours
E. There was no cell division during the course of the experiment
8.____ Which of the cultures is the least synchronized?

9.____ The cells of which culture contain the highest amount of Cdk1, a component of
MPF?
E. All contain approximately the same amount of Cdk1
10.____ The cells of which culture contain the highest Cdk1 activity?
E. All contain approximately the same activity Cdk1
21
Correct Answers
1. D 6. D
2. B 7. C
3. C 8. E
4. C 9. E
5. C 10. B
The test is based on Figure 2-18 in David O. Morgan: The Cell Cycle. Principles of Control.
Oxford University Press, New Science Press Ltd., London, UK, 2007.
Biology.
Szeberényi J. (2007) Problem-solving test: Analysis of the cell cycle by flow
cytometry. Biochem.Mol.Biol.Educ. 35, 153-154.

0001).
22
α1-ANTITRYPSIN DEFICIENCY: AN EXAMPLE FOR A

PROTEIN FOLDING DISEASE
protein conformation * protein folding * proteases * protein synthesis * protein glycosylation

* glycoproteins * N-linked and O-linked oligosaccharides * endoplasmic reticulum * Golgi
complex * secretory pathway * microsomes * pulse/chase labeling * SDS-polyacrylamide gel
electrophoresis * immunoprecipitation * chaperones * protein translocation
The experiment
1-antitrypsin (1-AT) is an abundant protease inhibitor of human blood plasma

synthesized and secreted by liver cells. It is a glycoprotein containing 3 N-linked
oligosaccharide chains. A single amino acid substitution (Glu 342  Lys) results in the
autosomal recessive disorder 1-AT deficiency that is characterized by a 90% reduction of
1-AT in the blood. The main consequence of this is an increased activity of elastase enzyme
in the lungs leading to the destructive lung disease familial emphysema.
The following test describes a series of experiments designed to analyze the molecular
mechanisms involved in the pathogenesis of 1-AT deficiency. Study the description of
experiments and the results presented in the figures carefully and solve the multiple choice
questions (MCQs).
Before starting to study the experiments, answer this question:
Relationship Analysis
(This type of question consists of a sentence with two main parts: an assertion and a reason for that assertion.
Select
A if both assertion and reason are true statements and the reason is a correct explanation of the
assertion;
B if both assertion and reason are true statements but the reason is not a correct explanation of the
assertion;
C if the assertion is true but the reason is a false statement;
D if the assertion is false but the reason is a true statement;
E if both assertion and reason are false statements.)
1.____ The conformation of the 1-AT protein may be affected by this mutation,
BECAUSE this amino acid substitution causes an alteration of charged groups on
the surface of the protein.
Figure 1 shows the results of an experiment in which cells expressing wild-type or

mutant 1-AT were compared in a pulse/chase/immunoprecipitation experiment using
[35S]methionine for labeling (for details see the figure legend).
23
Cell extract Medium
Chase (min) 0 20 40 60 100 0 20 40 60 100

-
55 kd Wild-type 1-AT
52 kd
+
-
55 kd Muta nt 1-AT
52 kd
+
1 2 3 4 5 6 7 8 9 10
Figure 1. Analysis of the fate of wild-type and mutant 1-antitrypsin produced by cultured
human cells. Cell cultures expressing the normal (upper panel) and mutant forms
(lower panel) of 1-AT were pulse-labeled with [35S]methionine for 10 minutes
and then chase was performed for the intervals indicated in the figure. At the end
of each interval cell extracts (samples 1 to 5) and medium (samples 6 to 10)
corresponding to the same number of cells were immunoprecipitated using an
anti-1-AT antibody. The immunoprecipitates were resolved by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by
autoradiography. (The position of electrodes during electrophoresis are indicated
on the right side of the panels.)
2.____ What features of 1-AT can be studied in this experimental setting?

A. Its exact amount in the cells
B. Its rate of synthesis and turnover in the cell
C. Its rate of secretion
D. B and C
E. A, B and C
The experiment of Figure 2 was designed to characterize the oligosaccharide moieties

attached to 1-AT. To this end endoglycosidase H (endo H) treatment was used: endo H
cleaves N-linked oligosaccharides produced in the endoplasmic reticulum (see Fig. 4A), but
not O-linked and Golgi-processed N-linked oligosaccharides (experimental details are given
in the legend to Fig. 2).
24
Wild-type 1-AT Mutant 1-AT
Cell Medium Cell Medium

lysate lysate
Endo H - + - + - + - +
Chase (hours) 0 0 5 5 0 0 5 5
-
55 kd
52 kd
+
1 2 3 4 5 6 7 8
Figure 2. Effect of endoglycosidase H on wild-type and mutant 1-antitrypsin. Cell cultures

expressing wild-type (samples 1 to 4) or mutant 1-AT (samples 5 to 8) were
pulse/chase-labeled as described in the legend to Fig. 1. Cell lysates and medium
samples were subjected to mock digestion (-) or endo H digestion (+) as indicated
in the figure, and then to immunoprecipitation analysis with anti-1-AT.
A. The 52 kilodalton band

B. The 55 kilodalton band
C. Both of them
D. Neither of them
3.____ Corresponds to the free oligosaccharide moieties of 1-AT.
4.____ Contains glycosylated 1-AT.
5.____ Molecules of this band reach the Golgi complex within 10 minutes after termination
of translation.
6.____ Molecules of this band are secreted.
Figure 3 shows the results of a functional assay to compare wild-type and mutant 1-
AT (details in the legend).
25
Wild-type 1-AT Mutant 1-AT
Elastase 0 10 100 500 0 10 100 500

( ng) -
Y
+
1 2 3 4 5 6 7 8
Figure 3. Functional activity of wild-type and mutant 1-antitrypsin. Labeled wild-type

(samples 1 to 4) or mutant 1-AT (samples 5 to 8) were incubated with increasing
amounts of non-radioactive elastase enzyme as indicated and then the mixtures
were analyzed by SDS-PAGE and autoradiography.
7.____ What is the most likely explanation for the shift of electrophoretic mobility of 1-
AT (from band X to band Y) in this experiment?
A. Elastase cleaved 1-AT

B. Elastase caused complete degradation of 1-AT
C. Elastase formed a stable complex with 1-AT
D. Elastase removed the oligosaccharides from 1-AT
E. 1-AT induced aggregation of elastase molecules
In the second part of this test experiments are presented in which the effect of a plant
alkaloid, castanospermine (CST) was studied on the metabolism of 1-AT. CST inhibits
glucosidase I, the enzyme responsible for the removal of the distal glucose molecule of N-
linked oligosaccharide precursor (Fig. 4A). Fig. 4B shows the effect of CST on the mutant
form of 1-AT.
26
A B
G gluc osidase I
G
CST - +
G -
M M M
a
M M M b
M
M M +
N endoglucosidase H 1 2
N poly-
Asn peptide
chain
Figure 4. The effect of castanospermine on 1-antitrypsin. (A) The structure of N-linked

oligosaccharide precursor and the cleavage sites of glucosidase I and endo H (N,
N-acetylglucosamine; M, mannose; G, glucose). (B) The effect of CST. Cells
expressing the mutant 1-AT were labeled with [35S]methionine under conditions
favoring the labeling of the 55kd protein (see Figs. 1 and 2), in the absence
(sample 1) or presence of CST (sample 2). 1-AT was immunoprecipitated and
analyzed by SDS-PAGE and autoradiography.
8.____ What can be the consequence of CST treatment of cells on 1-AT?
A. The isoelectric point of 1-AT is altered

B. The protein becomes more hydrophilic
C. The molecular mass of 1-AT is altered
D. A and C
E. B and C
The effect of CST on 1-AT metabolism was studied in the experiment of Fig. 5
(details in the figure legend).
27
Control
S-Radioactivity
CST
35
30 60 90 120
Chase (min)
B
Cell extract Medium
Chase (hours) 0 1 2 4 6 0 1 2 4 6
-
a Control
b
+
-
a CST
b
+
1 2 3 4 5 6 7 8 9 10
Figure 5. The effect of castanospermine on the metabolism of wild-type (A) and mutant (B)
1-antitrypsin. Cells were pulse/chase-labeled with [35S]methionine in the
presence or absence of CST as indicated. 1-AT was analyzed in cell extracts (B,
left panel) or in the medium (A; B, right panel) as described in the legend to Fig.1.
(Note that Figs. A and B come from two separate experiments using different cell
lines and experimental protocols. Band a and b in panel B correspond to the
bands of Fig. 4B.)
Fig. 6 presents results from an in vitro microsomal translocation experiment designed

to study the role of the endoplasmic reticulum membrane chaperone calnexin in the traffic of
1-AT (for experimental details see the legend).
28
No immunoprecipitation anti-calnexin immunoprecipitation

Control CST Control CST
Chase (min) 0 15 30 45 90 0 15 30 45 90 0 15 30 45 90 0 15 30 45 90
-
a
b
+
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 6. Effect of castanospermine on the fate of mutant 1-antitrypsin in a cell-free

microsomal translocation system. Pulse/chase labeling was performed in cell-free
protein synthesizing systems containing mutant 1-AT mRNA, [35S]methionine,
microsomes and all components required for translation, in the presence or
absence of CST as indicated. Microsomes were collected by centrifugation and
were run directly (samples 1 to 10) or after immunoprecipitation with an anti-
calnexin antibody (samples 11 to 20) in an SDS-polyacrylamide gel. The figure
shows the autoradiogram of the gel.
9.____ What feature of calnexin was studied in this experiment?
A: Its effect on the synthesis of 1-AT

B: Its effect on the glycosylation of 1-AT
C: Its effect on the translocation of 1-AT across the endoplasmic reticulum
membrane
D: Its binding to 1-AT
E: All four
Summarize the conclusions of these experiments by solving the following MCQs.

29
A. Wild-type 1-AT
B. Mutant 1-AT
C. Both of them
D. Neither of them
10.____ Its gene is efficiently transcribed in the cells used in this experiment.
11.____ Its mRNA is efficiently translated.
12.____ It is able to translocate into the endoplasmic reticulum.
13.____ Its glycosylation starts in the endoplasmic reticulum.
14.____ It is mostly retained in the endoplasmic reticulum.
15.____ It is a substrate for glucosidase I.
16.____ It is able to bind to its target protein.
Experiment Analysis
(The following statements are related to the information presented in the description of the experiment. Based on
the information given, select:
17.____ Castanospermine inhibits the translocation of mutant 1-AT across the endoplasmic
reticulum membrane.
18.____ The distal glucose of the N-linked oligosaccharide is required for the secretion of
1-AT.
19.____ Glycosylation of 1-AT took place in the cell-free microsomal system.
20.____ Glucosidase I is present in the microsomes.
21.____ Calnexin selectively binds to mutant 1-AT molecules containing the untrimmed
oligosaccharide precursor (see Fig. 4A).
30
22.____ Castanospermine triggers the release of mutant 1-AT from its complex with
calnexin.
23.____ Castanospermine stimulates the secretion of mutant 1-AT.
24.____ Based on the results of this study what would you expect from the treatment of 1-
AT deficient patients with castanospermine?
A. Their plasma level of 1-AT would be increased
B. Their lung condition would be improved
C. The mutant 1-AT would disappear from their blood
D. A and B
E. B and C
Correct anwers
1. A 7. C 13. C 19. A
2. D 8. E 14. B 20. A
3. D 9. D 15. C 21. B
4. C 10. C 16. C 22. A
5. D 11. C 17. B 23. A
6. A 12. C 18. B 24. D
31
References
[1] V.W. Sasak, J.M. Ordovas, A.D. Elbein, R.W. Berninger (1985) Castanospermine
inhibits glucosidase I and glycoprotein secretion in human hepatoma cells. Biochem. J.
232, 759-766.
[2] J.A.J. Burrows, L.K. Willis, D.H. Perlmutter (2000) Chemical chaperones mediate
increased secretion of mutant 1-antitrypsin (1-AT) Z: A potential pharmacological
strategy for prevention of liver injury and emphysema in 1-AT deficiency. Proc. Natl.
Acad. Sci. USA 97, 1796-1801.
[3] N.Y. Marcus, D.H. Perlmutter (2000) Glucosidase and mannosidase inhibitors mediate
increased secretion of mutant 1-antitrypsin Z. J. Biol. Chem. 275, 1987-1992.
Biology.
Szeberényi J. (2007) Problem-solving test: 1-Antitrypsin deficiency: An example for
a protein folding disease. Biochem.Mol.Biol.Educ. 35, 300-304.

0001).
32
VECTORIAL TRANSPORT IN INTESTINAL

EPITHELIAL CELLS
polarized cells * vectorial transport * apical and basolateral membrane * tight junction *
occluding * transmembrane proteins * passive and active transport * glucose transporter *
Na+/glucose symporter * Na+/K+ ATPase * uniporter * antiporter * facilitated diffusion *
lateral diffusion
The Experiment
The main function of the small intestine is to absorb small molecules (sugars, amino
acids, nucleosides etc.) from the intestinal lumen and forward them to blood capillaries. The
molecular basis of this unidirectional, vectorial transport is based on the polarized nature of
the cell membrane: the apical and basolateral regions of the cell membrane contain different
transport proteins. The reader is expected to review textbook chapters describing details of the
membrane structure of epithelial cells lining the small intestine and then solve the multiple-
choice questions (MCQs) presented in the second part of the test. The first MCQs are
designed to refresh memories about vectorial transport.
Multiple Completion
(A question or incomplete statement is followed by four numbered completions, one or more of which are correct.
Select:
A: if 1, 2 and 3 are correct;
B: if 1 and 3 are correct;
C: if 2 and 4 are correct;
D: if only 4 is correct;
E: if all four are correct.)
1._____ Epithelial cells of the small intestine are held together by tight junctions. What are
the characteristic features of these structures?
1. Serve as barriers to prevent mixing of the components of the apical and

basolateral membranes
2. Their main components are transmembrane proteins called occludin
3. Seal the intestinal lumen from the extracellular space between epithelial
cells
4. These are the main communicative junctions between the epithelial cells
33
The most important membrane proteins involved in the vectorial transport of glucose
are: glucose transporter, Na+/glucose symporter and Na+/K+ ATPase. What are the main
features of these proteins?
Five-choice Association
(This type of question consists of a list of lettered headings followed by a list of numbered words or phrases. For
each numbered word or phrase, select the one heading which is most closely related to it.)
A: Glucose transporter
B: Na+/glucose symporter
C: Na+/K+ ATPase
D: A and C
E: All three proteins
2. _____ It is a transmembrane protein.
3. _____ It performs facilitated diffusion.
4. _____ Its only function is active transport.
5. _____ It performs both passive and active transport.
6. _____ It is an antiporter.
7. _____ It is localized to the apical membrane.
8._____ It is localized to the basolateral membrane.
9._____ It is a uniporter.
A fictitious experiment is described below in which the effect of a bacterial toxin

destroying tight junctions between intestinal cells is studied. Untreated control rats and
animals infected with the bacterium are used.
Considering the membrane structure of small intestinal cells, what differences do you
expect to see between the two groups of animals? Solve the following MCQs!
34
Select:
A: if both assertion and reason are true statements and the reason is a correct
explanation of the assertion;
B: if both assertion and reason are true statements but the reason is not a correct
explanation of the assertion;
C: if the assertion is true but the reason is a false statement;
D: if the assertion is false but the reason is a true statement;
E: if both assertion and reason are false statements.)
10._____The toxin reduces the concentration of glucose in the epithelial cells, BECAUSE the
Na+ concentration is lower than normal in the intestinal lumen of infected animals.
11._____ Blood sugar levels are higher than normal in the infected animals, BECAUSE the
Na+K+ pump is more active in their intestinal cells.
Select:
A: if A is greater than B;
B: if B is greater than A;
C: if the two are equal or very nearly equal.)
12._____ A: The density of Na+/glucose symporter molecules in the apical membrane of

intestinal cells in the infected animals
B.: The density of Na+/glucose symporter molecules in the basolateral membrane of
intestinal cells in the infected animals
13._____ A: The rate of glucose influx across the intestinal apical membrane in control
animals
B: The rate of glucose influx across the intestinal apical membrane in infected
animals
14._____ A: The rate of glucose outflux across the intestinal basolateral membrane in control
animals
B: The rate of glucose outflux across the intestinal basolateral membrane in infected
animals
15._____ A: The rate of glucose outflux across the intestinal apical membrane in control
animals
B: The rate of glucose outflux across the intestinal apical membrane in infected
animals
16._____ A: The rate of active Na+/K+ transport across the intestinal apical membrane in
control animals
B: The rate of active Na+/K+ transport across the intestinal apical membrane in
infected animals
35
Correct Answers
1. A 6. C 11. E 16. B
2. E 7. B 12. C
3. A 8. D 13. A
4. C 9. A 14. A
5. B 10. C 15 B
Biology.
Szeberényi J. (2006) Problem-solving test: Vectorial transport in intestinal epithelial

cells. Biochem. Mol. Biol. Educ. 34, 449-451.

0001).
36
EXPRESSION CLONING OF THE ERYTHROPOIETIN

RECEPTOR
cytokines * cytokine receptors * cDNA library * cDNA synthesis * poly(A)+ RNA * primer *
template * reverse transcriptase * restriction endonucleases * cohesive ends * expression
vector * promoter * Shine-Dalgarno sequence * poly(A) signal * DNA helicase * DNA ligase
* topoisomerases * [125I] labeling * transfection * mock transfection * SDS-polyacrylamide
gel electrophoresis * β-mercaptoethanol * autoradiography
The experiment
Erythropoietin (EPO) is a cytokine produced by certain kidney cells that stimulates the
production and differentiation of red blood cells. The following test describes experiments [1]
performed to clone the cDNA of murine erythropoietin receptor (EPO-R) and to express it in
suitable human host cells.
An expression cDNA library was constructed in this study using mouse

erythroleukemia cell total poly(A)+ RNA. The first step in library construction is cDNA
synthesis.
Multiple Completion
(A question or incomplete statement is followed by four numbered completions, one or more of which are
correct. Select
A if 1, 2 and 3 are correct;
B if 1 and 3 are correct;
C if 2 and 4 are correct;
D if only 4 is correct;
E if all four are correct.)
1.____ Besides total cytoplasmic poly(A)+ RNA, which of the following components are
required in the reaction mixture to synthesize a collection of cDNAs containing
copies of EPO-R cDNA?
1. Oligo(dT)
2. Reverse transcriptase
3. The four deoxyribonucleoside triphosphate
4. RNA polymerase II
cDNAs synthesized in this mixture were converted into double-stranded DNA

molecules supplied with Xho restriction endonuclease specific cohesive ends and ligated into
the Xho site of a mammalian expression plasmid.
37
2.____ What sequences should be present in this plasmid if it is to express inserted cDNAs
in human cells?
1. A mammalian promoter upstream of the Xho site

2. A Shine-Dalgarno sequence upstream of the Xho site
3. A poly(A) signal downstream of the Xho site
4. A Shine-Dalgarno sequence downstream of the Xho site
3.____ What enzyme should be added to the mixture of double-stranded cDNA and Xho-cut
plasmid to generate a collection of recombinant plasmids?
A. DNA helicase
B. DNA ligase
C. Topoisomerase I
D. B and C
E. A, B and C
The recombinant plasmids were transfected into human cells and a clone was selected
that bound radioactively labeled EPO under tissue culture conditions. Figure 1 shows the
results of an experiment in which [125I] EPO binding was compared using the original mouse
cell culture (A), human cells transfected with an empty plasmid (B) or the human cell clone
isolated from the experiment (C). [125I] EPO binding (Y axis) to the cells was measured at
different concentrations of the labeled hormone (see X axis), in the absence (●—●) or
presence of 100 nM unlabeled (“cold”) EPO (○—○).
A B C
[125I] radioactivity bound
1 2 1 2 1 2
125
[ I] erythropoietin (nM)
Figure 1. Binding of [125I] erythropoietin to mouse erythroleukemia cells (A), mock-

transfected human cells (B) and to human cells of the clone isolated as described
in the text (C).
38
4.____ What was the aim of using unlabeled EPO in this experiment?
A. To increase specific binding of [125 I] EPO
B. To decrease non-specific binding of [125 I] EPO
C. To distinguish specific and non-specific binding of [125 I] EPO
D. To stimulate EPO signaling
E. C and D
5.____ How can specific binding of [125 I] EPO be determined? (The symbols of Fig. 1 are
used in this question.) Specific binding equals to
A. Radioactivity corresponding to ●
B. Radioactivity corresponding to ○
C. Radioactivity corresponding to ● plus ○
D. Radioactivity corresponding to ● minus ○
E. Radioactivity measured in the medium in the presence of “cold” EPO
6.____ What can be determined by measuring specific [125 I] EPO binding?

A. Cell surface localization of EPO-R
B. EPO-R density
C. The rate of synthesis of EPO-R
D. A and B
E. A, B and C
Select
7.____ A. Specific EPO binding to mouse erythroleukemia cells

B. Non-specific EPO binding to mouse erythroleukemia cells
8.____ A. Specific EPO binding to mock-transfected human cells

B. Non-specific EPO binding to mock-transfected human cells
9.____ A. EPO-R density on mouse erythroleukemia cells

B. EPO-R density on the transfected human clone of Fig. 1C
39
In the second part of the experiment cultures of the cells in Fig.1C were incubated
with 1 nM [125 I] EPO in the presence (samples 2 and 4) or in the absence (samples 1 and 3) of
100 nM “cold” EPO (Fig. 2). After incubation the media were discarded and the cells were
washed with fresh medium that did not contain EPO. The cultures were subsequently
incubated in the presence (samples 1 and 2) or absence (samples 3 and 4) of the cross-linking
agent disuccinimidyl suberate. Cell membrane fractions were isolated and then subjected to
SDS-polyacrylamide gel electrophoresis in gels containing β-mercaptoethanol. The gels were
autoradiographed using an X ray film.
+ + - - cross-linker
- + - + cold EPO
200
a
b
97
68
43
c 34
1 2 3 4
Figure 2. Cross-linking of radiolabeled erythropoietin to its receptor expressed in human

cells (numbers on the right indicate molecular weights in kilodalton; for
experimental details see the text).
Figure Analysis
10.____ The gel regions corresponding to bands a, b and c all contain EPO.
11.____ The gel regions corresponding to bands a, b and c all contain EPO-R.
12.____ Most of the radiolabeled EPO molecules bound to specific receptor sites on the
cells.
13.____ Most of the radiolabeled EPO molecules were covalently cross-linked to EPO-R
molecules.
14.____ Native EPO-R molecules consist of several subunits.
15.____ Band a corresponds to EPO-EPO-R complexes held together by disulfide bonds.

40
Correct Answers
1. A 9. B
2. B 10. A
3. B 11. B
4. C 12. A
5. D 13. B
6. D 14. C
7. C 15. B
8. B
Reference
[1] A.D. D’Andrea, H.F. Lodish, G.G. Wong (1989) Expression cloning of the murine
erythropoietin receptor. Cell 57, 277-285.
Biology.
Szeberényi J. (2008) Problem-solving test: Expression cloning of the erythropoietin

receptor. Biochem.Mol.Biol.Educ. 36, 236-238.

0001).
41
THE YEAST TWO-HYBRID SYSTEM
transcription factors * enhancer * promoter * RNA polymerase * expression plasmid *

terminator * histidine * transfection * cell culture * fusion proteins * RNA splicing
The experiment
The following test describes the principle of a powerful technique of molecular

biology: the yeast two-hybrid system.
Yeast cells contain a transcription factor, GAL4, required for the expression of the -
galactosidase gene. It consists of a DNA-binding domain that recognizes the specific enhancer
sequence in the promoter region of the -galactosidase gene and an activator domain required
for the binding of RNA polymerase II (Fig 1).
Two mammalian proteins, designated X and Y, are studied in the following fictitious
experiment using the yeast two-hybrid system. Expression plasmids functioning in yeast cells
Figure 1. The role of GAL4 in the transcription of the -galactosidase gene (D, DNA binding
domain; A, activation domain; pol II, RNA polymerase II).
were used in the experiment (Fig. 2). They contained a promoter and a terminator region
specific for yeast cells. One plasmid (designated DX plasmid) contained a fusion gene
consisting of a cDNA coding for the DNA binding domain of GAL4 and a cDNA coding for
protein X. The other plasmid (AY plasmid) contained a hybrid gene constructed from cDNA
sequences coding for the activation domain of GAL4 and protein Y. A third, so-called
reporter plasmid was also used in the experiment: it contained the HIS3 gene as a reporter
gene hooked to the enhancer/promoter region of the -galactosidase gene. The three plasmids
were transfected into a culture of mutant yeast cells that contained neither GAL4, nor HIS3
(GAL4-, HIS3- mutant). The cells were spread on culture dishes with solid media lacking or
containing histidine as indicated in Table I.
42
Figure 2. Plasmid constructs used in the experiment. (P, promoter; T, terminator; E/P-gal,
the enhancer/promoter region of the -galactosidase gene; A and D, cDNAs
coding for the activation and DNA binding domain of GAL4, respectively; X and
Y, cDNAs coding for protein X and Y, respectively).
Table I. Transfection of GAL4-, HIS3- yeast cells with various plasmid constructs under
different culturing conditions. (The constructs are described in Fig. 2, experimental details
are given in the text.)
Transfection with Number of

Histidine in the colonies on the
DX plasmid AY plasmid reporter plasmid medium culture plates
- - - + many
- - - - none
- - + - none
+ - + - none
- + + - none
+ + + - several
Study the experimental strategy, evaluate the results and solve the following multiple-
choice questions.
Experiment Analysis
(The following statements are related to the information present in the description of the experiment. Based on the
information given, select:
1.____ Histidine is an essential amino acid in yeast.
2.____ The HIS3 gene codes for an enzyme required for histidine biosynthesis.
3.____ The HIS3 gene of the reporter plasmid is constitutively expressed in the
transfected mutant yeast cells.
43
4.____ The half-life of DX protein is shorter than that of GAL4.
5.____ Histidine can not be taken up by HIS3- cells.
6.____ Yeast cells are unable to splice pre-mRNAs of mammalian origine.
7.____ X and Y proteins act as transcription factors in their mammalian host cells.
8.____ The -galactosidase enhancer does not function in the HIS3- mutant cells.
9.____ The DX and AY fusion proteins can substitute the missing GAL4 activity in the
transfected cells.
10.____ The DX fusion protein is able to bind to the -galactosidase enhancer.
11.____ The AY fusion protein is able to activate RNA polymerase II.
12.____ What molecular event can be studied by the yeast two-hybrid system?
A. The regulation of -galactosidase gene expression

B. The regulation of HIS3 gene expression
C. The role of exogenous proteins (X and Y) in gene regulation
D. The role of exogenous proteins (X and Y) in RNA polymerase II
function
E: In vivo interaction between exogenous proteins (X and Y)
44
Correct answers
1. A 7. C
2. A 8. B
3. B 9. A
4. C 10. A
5. B 11. A
6. C 12. E
Reference
Spector, D.L., Goldman, R.D. and Leinwand, L.A. (editors): Cells – A Laboratory Manual.
Chapter 69: Two-hybrid System/Interaction Trap. Cold Spring Harbor Laboratory
Press, 1997.
Biology.
Szeberényi J. (2006) Problem-solving test: The yeast two-hybrid system.


0001).
45
ANALYSIS OF THE MECHANISM OF ACTION OF AN

APOPTOSIS INDUCING COMPOUND
apoptosis * Bcl-2 protein family * anti-apoptotic Bcl-2 proteins * pro-apoptotic multi domain
Bcl-2 proteins * BH3-only proteins * intrinsic (mitochondrial) pathway of apoptosis * cell
fractionation * targeted gene disruption * Western blotting * cytochrome c * loading control
* caspases * apoptosome
The Experiment
Most cancer cells loose their ability to undergo programmed cell death by apoptosis.
Proteins involved in the regulation and execution of cell death are therefore promising targets
of antitumor drug development. Among these members of the Bcl-2 protein family are
particularly important. They come in three forms: anti-apoptotic Bcl-2 members (e.g. Bcl-2,
Bcl-xL), pro-apoptotic multi domain Bcl-2 family proteins (e.g. Bax, Bak) and BH3-only
proteins (e.g. Bad, tBid; BH3 stands for Bcl-2 homology 3, this domain is common in all
proteins of the family). They regulate the permeability of the outer mitochondrial membrane,
thereby the intrinsic (mitochondrial) pathway of apoptosis.
A putative anticancer agent, designated compound 106, was tested in vitro on
isolated mitochondria in the study described in the following test [1]. In order to be able to
interpret the results of the following experiments, review the mitochondrial pathway of
apoptosis in an appropriate textbook (e.g. Ref. 2).
The experiments presented in this test were performed using mitochondria isolated
from murine embryonic fibroblasts.
1.____ Which of the following procedures is most suitable to isolate a pure mitochondrial
fraction from these cells?
A: Low-speed centrifugation of the homogenate

B: Medium-speed centrifugation of the homogenate
C: High-speed ultracentrifugation of the homogenate
D: Medium-speed centrifugation of the postnuclear supernatant
E: High-speed ultracentrifugation of the postnuclear supernatant
46
Experiment 1
The mitochondrial fraction was prepared from Bak-Bax- cells (in which these genes
had been knocked out by targeted gene disruption) and aliquots of the mitochondrial samples
were incubated without treatment (sample 1 in Fig. 1), with added Bax protein (samples 2 to
6), tBid protein (sample 3) and increasing amounts of compound 106 (samples 4 to 6) as
indicated in Figure 1. After incubation, mitochondria were pelletted by centrifugation,
proteins were solubilized in a suitable buffer and Western blot analysis was performed using
anti-Bax and anti-Tom 40 antibodies. (Tom 40 is a structural protein of the outer
mitochondrial membrane.)
Figure 1. Treatment of mitochondria of Bak-Bax- cells with Bax and tBid proteins and
compound 106 (for experimental details see the text.)
Experiment 2
Mitochondria isolated from Bak-Bax- cells were treated with the proteins Bax (samples
3 to 14), tBid (samples 5-6 and 11-12), Bcl-xL (samples 9 to 14) and compound 106 (samples
7-8 and 13-14) as indicated in Fig. 2 (samples 1-2 served as controls). Mitochondria were
sedimented by centrifugation and protein extracts of the pellets (P) and supernatants (S) were
subjected to Western blot analysis using anti-cytochrome c and anti-Tom 40 antibodies (Fig.
2).
47
Figure 2. Treatment of mitochondria of Bak-Bax- cells with Bax, tBid and Bcl-xL proteins and
compound 106 (P, pellet, S, supernatant; for experimental details see the text.)
Experiment 3
A similar experiment was performed using mitochondria from Bak +Bax- mouse
embryonic fibroblasts. Mitochondria were left untreated (samples 1-2 in Fig. 3) or incubated
with tBid protein (samples 3-4) or compound 106 (samples 5 to 10). Samples were processed
as in Experiment 2; Fig. 3 shows the results of Western blotting.
Figure 3. Treatment of mitochondria of Bak+Bax- cells with tBid and compound 10 (for
experimental details see the text.)
48
Study the experiments and solve the following multiple-choice questions!
2.____ What was the aim of using anti-Tom 40 antibody in these experiments?
A: It was used as a loading control

B: To check the integrity of mitochondrial structure
C: To check if mitochondrial fractions are free of cytosolic contaminations
D: A and B
E: A, B and C
Figure Analysis
(The following statements are related to the information presented above. Based on the information given.
Select: A: if the statement is supported by the information given;
B: if the statement is contradicted by the information given;
C: if the statement is neither supported nor contradicted by the information given.)
3.____ Murine embryonic fibroblasts overexpress Bcl-xL.
4.____ Compound 106 stimulates the translocation of Bax protein into the mitochondria.
5.____ The effect of compound 106 on mitochondria depends on the presence of tBid.
6.____ tBid enhances the action of compound 106.
7.____ Tumor cells expressing the Bax protein are expected to be sensitive to the apoptotic
effect of compound 106.
8.____ Tumor cells overexpressing the Bcl-xL protein are expected to be sensitive to the
apoptotic effect of compound 106.
9.____ Compound 106 destroys the outer mitochondrial membrane.
10.____ Compound 106 makes the outer mitochondrial membrane leaky.
11.____ Compound 106 stimulates the translocation of cytochrome c from the cytosol to the
mitochondria.
The following MCQs can be answered by extrapolating the results of the presented
experiments to the whole mitochondrial pathway of apoptosis.
49
(In this type of question a set of lettered headings is followed by a list of numbered words or phrases.
Select: A: if the word or phrase is associated with A only;
B: if the word or phrase is associated with B only;
C: if the word or phrase is associated with A and B;
D: if the word or phrase is associated with neither A nor B.)
A: Bak+Bax- cells
B: Bak-Bax- cells
C: Both of them
D: Neither of them
12.____ These cells are able to undergo apoptosis.
13.____ Expression of tBid in them leads to caspase 9 activation.
14.____ Treatment of these cells with compound 106 would lead to the formation of
apoptosomes.
15.____ Compound 106 triggers caspase 3 activation in these cells.
16.____ What protein is directly targeted by compound 106?
A: tBid
B: Bax
C: Bcl-xL
D: Bak
E: Cytochrome c
Correct Answers
1. D 9. B
2. D 10. A
3. C 11. B
4. A 12. C
5. B 13. C
6. C 14. D
7. A 15. D
8. B 16. B
50
Reference
[1] Zhao G., Y. Zhon, C.O. Eno, Y. Lin, L. DeLeenw, J.A. Burlison, J.B. Chaires, J.O.
Trent, C. Li (2014) Activation of the proapoptotic Bc-l2 protein Bax by a small
molecule induces tumor cell apoptosis. Mol. Cell. Biol. 34, 1198-1207.
[2] Cooper G.M., R.E. Hausman (2013) The Cell. A Molecular Appoarch. Sixth Edition.
Sinauer Associates, Inc., pp. 681-692.
Biology.
Szeberényi J. (2015) Analysis of the mechanism of action of an apoptosis-inducing

compound. Biochem.Mol.Biol.Educ. 43, .

0001).
51
THE MECHANISM OF ACTION OF A HUMAN PAPILLOMA VIRUS

ONCOPROTEIN
human papilloma virus * cervical cancer * oncoproteins * malignant transformation *

retinoblastoma protein * cell cycle * quiescent and cycling cells * cyclin/Cdk complexes *
E2F * S-phase genes * enhancer element * proto-oncogenes * tumor suppressor genes *
radioactive labeling * immunoprecipitation * SDS-polyacrylamide gel electrophoresis *
autoradiography * protein phosphorylation and dephosphorylation * gene induction *
agarose beads * centrifugation * Western blot analysis * phases of cell cycle * generation
time
The Experiment
One of the Nobel laureates in Physiology and Medicine of 2008, Harald Zur Hausen,
discovered that certain types of the human papilloma virus (HPV) are the causative agents of
cervical cancer [1]. HPV type 16 (HPV-16) is among the most common and best studied
human oncogenic viruses. Two oncoproteins (E6 and E7) encoded by the viral genome are
responsible for the transformation of normal epithelial cells into tumor cells. The experiment
presented in this test was designed to analyze the effects of E7 protein at the molecular level,
namely, its relationship to the retinoblastoma protein (RB) [2].
Since this research was not aimed at analyzing the behavior of RB protein, in order to
understand the mechanism of action of E7 oncoproteins the reader is expected to recall the
most important features of RB by solving the following multiple-choice questions (MCQs).
A. RB protein in quiescent cells

B. RB protein in cycling cells
C. Both of them
D. Neithet of them
1.____ Is present in the nuclei.
2.____ Is highly phosphorylated by cyclin/Cdk complexes.
3____ Is complexed to the transcription factor E2F.
4.____ Inhibits the transcription of S-phase genes.
5.____ Is bound to the enhancer elements of S-phase genes.

52
6.____ What are the main characteristics of the RB protein?
A. It is a proto-oncogenic protein
B. It is a tumor suppressor protein
C. It is a regulator of the cell cycle
D. A and C
E. B and C
Fig. 1 shows the results of a preliminary experiment. Cells of a human leukemia cell
line (HL-60) were cultured without (samples 1 and 3) or in the presence of phorbol ester
(samples 2 and 4), and labeled with [35S]methionine (samples 1 and 2) or [32P]phosphate
(samples 3 and 4). Cell extracts were immunoprecipitated with an antibody specific for the
retinoblastoma protein (anti-RB), SDS-polyacrylamide gel electrophoresis was performed
followed by autoradiography.
35 32
S P Labeling
- + - + Phorbol ester treatment

-
a
b
+
1 2 3 4 Samples
Figure 1. The effect of phorbol ester on the RB protein (for experimental details see the text).
What conclusions can be drawn from this experiment?
Figure Analysis
7.____ Band a is a degradation product of band b.
8.____ All RB protein molecules are phosphorylated in untreated HL-60 cells.
9.____ The RB protein is induced by phorbol ester in HL-60 cells.
10.____ The phorbol ester triggers dephosphorylation of the RB protein in HL-60 cells.
53
The results of the experiment carried out to analyze the function of the E7 protein are
shown in Fig 2. The human leukemia cells were cultured in the absence (samples 1 to 3) or
presence of phorbol ester (samples 4 to 6). Cell extracts were prepared and aliquots were
incubated with agarose beads to which E7 protein molecules had been covalently attached.
After incubation the beads were pelletted by centrifugation and Western blot analysis after
SDS-polyacrylamide gel electrophoresis was performed using the anti-RB antibody with total
cell extracts (samples 1 and 4), the supernatants (samples 2 and 5), and the sediments
(samples 3 and 6).
+ - - + - - Whole cell extract
- + - - + - Supernatant
- - + - - + Sediment
- - - + + + Phorbol ester treatment

-
a
b
+
1 2 3 4 5 6 Samples
Figure 2. Analysis of cell extracts using E7-agarose beads (experimental details are
described in the text).
11.____ What was the aim of using this E7-agarose bead protocol?
A. To study the expression of RB protein

B. To determine the rate of phosphorylation of RB protein
C. To analyze E7-RB interactions
D. A and B
E. A, B and C
To solve the following questions and describe the effect of E7 protein on the cell cycle
you need to combine your knowledge of the function of the RB protein (see MCQs 1 to 6) and
the conclusions drawn from the results of this study.
54
Select
A if both assertion and reason are true statements and the reason is a correct explanation of the
assertion;
B if both assertion and reason are true statements but the reason is not a correct explanation of
the assertion;
C if the assertion is true but the reason is a false statement;
D if the assertion is false but the reason is a true statement;
E if both assertion and reason are false statements.)
12.____ HL-60 cells were in the Go phase in the absence of phorbol esters, BECAUSE their
RB proteins were fully phosphorylated.
13.____ Phorbol ester treatment must have inhibited the proliferation of HL-60 cells,
BECAUSE it stimulated the binding of RB protein to the E7 oncoprotein.
A. Phorbol ester treatment of HL-60 cells

B. Expression of E7 oncoprotein in HL-60 cells
C. Both of them
D. Neither of them
14.____ Increases the amount of E2F transcription factor-bound RB protein.
15.____ Increases the expression of S-phase genes.
16.____ Increases the generation time of cells.
17.____ Increases the ratio of cells in the G1 phase of the cell cycle.
18.____ Let’s summarize the effects of E7 protein that can lead to malignant
transformation. E7 protein
A. binds and sequesters underphosphorylated RB protein
B. increases the amount of functionally active E2F transcription factor
C. pushes cells into the S-phase
D. increases the rate of cell proliferation
E. stimulates all the above processes
55
Correct Answers
1. C 8. A 15. B
2. B 9. B 16. A
3. A 10. A 17. A
4. A 11. C 18. E
5. D 12. D
6. E 13. B
7. B 14. A
References
[1] J. Cohen, M. Enserink (2008) Nobel prize in Physiology or Medicine. HIV, HPV
researchers honored, but one scientist is left out. Science, 322, 174-175.
[2] Y. Imai, Y. Matsushima, T. Sugimura, M. Terada (1991) Purification and

characterization of human papillomavirus type 16 E7 protein with preferential binding
to the underphosphorylated form of retinoblastoma gene product. J. Virol. 65, 4966-
4972.
Biology.
Szeberényi J. (2015) Problem-solving test: The mechanism of action of a human

papilloma virus oncoprotein. Biochem.Mol.Biol.Educ. 37, 118-120.

0001).
56
SIGNAL TRANSDUCTION IN PHILADELPHIA

CHROMOSOME POSITIVE LEUKEMIA CELLS
reciprocal translocation * proto-oncogene * gene expression * adaptor protein * SH2 and

SH3 domains * receptor proteins * tyrosine protein kinases * serine/threonine protein kinases
* steroid receptors * Src protein * malignant transformation * second messengers *
retroviral oncogenes and oncoproteins * transfection * immunoprecipitation * pre-immune
serum * [γ-32P]ATP * SDS-polyacrylamide gel electrophoresis * autoradiograph * Western
blot (immunoblot * co-immunoprecipitation * expression vector * cDNA * transient
transfection * promoter * Ras protein * transformed foci
The Experiment
The Philadelphia chromosome is the result of reciprocal translocation between

chromosome 9 and 22 in human cells. The unusually short chromosome 22 (Philadelphia or
Ph1 chromosome, named after the city were it was first described) contains a fusion gene
between the proximal region of the bcr gene and the distal part of the c-abl proto-oncogene
(originally carried by chromosome 9). Expression of this gene generates a Bcr/Abl fusion
protein that is biochemically different from the proto-oncogenic c-Abl protein. The aim of the
experiments [1] described in this test was to analyze the signaling mechanisms activated by
the Bcr/Abl protein, especially the role of the Grb2 adaptor protein characterized by an SH3-
SH2-SH3 domain structure. Before turning to the experiments, recall your knowledge
regarding adaptor proteins by solving the following multiple-choice questions (MCQs).
1.____ Which of the following statements best describes the role of adaptor proteins?
A. They connect cell surface receptors to intracellular target proteins

B. They connect tyrosine protein kinase receptors to intracellular target
proteins
C. They connect tyrosine protein kinase receptors to serine/threonine-specific
protein kinases
D. They connect tyrosine-phosphorylated proteins to their target proteins
E. They regulates tyrosine protein kinase activity
57
(In this type of question a set of lettered headings is followed by a list of numbered words or phrases.
Select: A: if the word or phrase is associated with A only;
B: if the word or phrase is associated with B only;
C: if the word or phrase is associated with A and B;
D: if the word or phrase is associated with neither A nor B.)
A. SH2 domain
B. SH3 domain
C. Both of them
D. Neither of them
2._____ This domain is the DNA binding region of steroid receptors.
3._____ The v-Src protein carries a similar region.
4._____ Only proteins carrying such domain are able to transform NIH3T3 fibroblast to
tumor cells.
5._____ It binds to protein regions phosphorylated on tyrosine side chains.
6._____ It recognizes proline-rich regions of target proteins.
7._____ It is present in second messengers.
8._____ It is present in all retroviral oncoproteins.
In the first part of the experiment (Fig. 1) the adaptor protein Grb2 was studied in
chromic myeloid leukemia (CML), acute lymphoid leukemia (ALL) cells and rat fibroblasts
transfected with the v-abl oncogene (Rat1/v-abl). Cell extracts were immunoprecipitated with
pre-immune serum (samples 1, 4 and 7 in Fig. 1), anti-Abl (samples 2, 5 and 8) or anti-Grb2
antibodies (samples 3, 6 and 9). The immunoprecipitates were incubated in vitro with [γ-
32
P]ATP, the proteins were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) and visualized by autoradiography.
9._____ What was the aim of using [γ-32P]ATP in this experimental setting?
A. To study Abl autophosphorylation

B. To study phosphorylation of proteins bound to Abl
C. To study the production of cAMP
D. A and B
E. A, B and C
58
CML ALL Rat1/v-abl Cell line
+ + + Pre-immune serum
+ + + Anti-Abl
+ + + Anti-Grb2
KDa
210
185
160
1 2 3 4 5 6 7 8 9 Samples
Figure 1. SDS-polyacrylamide gel electrophoresis of immunoprecipitates incubated with

[γ-32P]ATP (for details see the text).
In the next experiment (Fig. 2) CML cell extracts were incubated with pre-immune
serum (sample 1), anti-Abl (sample 2) or anti-Grb2 antibody (sample 3), the
immunoprecipitates were fractionated by SDS-PAGE and Western-blot analysis was
performed using the anti-Grb2 antibody.
Immunoprecipitating
+ Pre-immune serum
antibodies + Anti-Abl
+ Anti-Grb2
KDa
27
Western blotting
antibody Anti-Grb2
1 2 3 Samples
59
Figure 2. Immunoprecipitation/Western blotting experiment using an extract from CML

cells (for details see the text).
In the experiment of Fig. 3 a similar co-immunoprecipitation strategy was followed.

Cells transfected with expression vectors containing a bcr/abl fusion cDNA (samples 1 to 3)
or a c-abl proto-oncogenic cDNA (samples 4 to 6) were used. Cell extracts were
immunoprecipitated with control serum, anti-Abl or anti-Grb2 antibody as indicated in Fig. 3
and then immunoblotting was performed using the anti-Abl antibody.
bcr/abl c-abl Transfected cDNA
Immunoprecipitating
+ + Pre-immune serum
antibodies + + Anti-Abl
+ + Anti-Grb2
KDa
210
160
Western blotting
antibody Anti-Abl
1 2 3 4 5 6 Samples
Figure 3. Co-immunoprecipitation experiment using extracts from bcr/abl and c-abl-

transfected cells (for details see the text).
Finally, the significance of tyrosine at position 177 in the Bcr-region of the fusion
protein was analyzed. A mutant cDNA was generated that, at this position, coded for
phenylalanine (+
designated bcr/abl Y177F). Co-immunoprecipitation experiments were performed using
extracts from cells transfected with wild-type bcr/abl or bcr/abl Y177F cDNA, using the
antibodies shown in Fig. 4.
60
bcr/abl bcr/abl
wild-type Y177F
Transfected cDNA
Immunoprecipitating
+ + Pre-immune serum
antibodies + + Anti-Abl
+ + Anti-Grb2
KDa
210
160
Western blotting
antibody Anti-Abl
1 2 3 4 5 6 Samples
Figure 4. Co-immunoprecipitation experiment using extracts from wild-type bcr/abl and

bcr/abl Y177F-transfected cells (for details see the text).
The effect of bcr/abl fusion gene on gene expression was studied in a transient
transfection experiment (Fig. 5). Cells were transfected with a plasmid carrying the cDNA of
a reporter gene, the gene coding for chloramphenicol acetyltransferase (CAT), expressed from
a promoter containing a Ras-responsive element. Expression of the reporter gene can be easily
measured and quantitated. The reporter plasmid was co-transfected into rat fibroblasts with an
empty plasmid (sample 1 in Fig. 5) or with expression vectors containing cDNAs of wild-type
bcr/abl (sample 2), wt-bcr/abl together with an inhibitory mutant ras gene (sample 3) or the
mutant bcr/abl Y177F (sample 4). Figure 5 shows the CAT-activities in the transient by
transfected cell cultutes.
61
Co-transfecting Relative CAT activity

cDNAs
1 5 10
1. None
2. Wt-bcr/abl
3. Wt-bcr/abl
+ inhibitory ras
4. bcr/abl Y177F
Figure 5. CAT activities transfected with various expression plasmids (for details see the
text).
The biological activities of wt-bcr/abl and bcr/abl Y177F constructs were also tested
in transfection experiments: they produced 37 and 0 transformed foci, respectively, using rat
fibroblast cultures.
Study the results of the experiments and solve the following MCQs.
Select: A: if A is greater than B;
B: if B is greater than A;
C: if the two are equal or very nearly equal.)
10._____ A. The molecular mass of Bcr/Abl protein

B. The molecular mass of c-Abl protein
11._____ A. The molecular mass of Bcr/Abl protein

B. The molecular mass of Grb2 protein
12._____ A. RasGDP/RasGTP patio in Ph1-positive leukemia cells

B. RasGDP/RasGTP patio in normal leukocytes
13._____ A. ERK activity in Ph1-positive leukemia cells

B. ERK activity in normal leukocytes
62
Experiment Analysis
14._____ The Bcr/Abl fusion protein contains an SH1 domain.
15._____ Bcr/Abl phosphorylates Grb2 protein in an in vitro reaction.
16._____ Bcr/Abl and Grb2 form a complex in vivo.
17._____ Bcr/Abl and v-Abl proteins use the same signaling pathway to cause malignant
transformation.
18._____ Chromosomes break at different sites during the translocations cauring CML and
All.
19._____ The transforming potential of the Bcr/Abl fusion protein depends on its ability to
bind Grb2.
20._____ Gene activation by the Bcr/Abl protein is caused by the stimulation of the Ras
pathway.
Correct Answers
1. E 7. A
2. A 8. B
3. C 9. A
4. C 10. A
5. A 11. E
6. B 12. D
Reference
[1] A.M. Pendergast, L.A. Quilliam, L.D. Cripe, C.H. Bassing, Z. Dai, N. Li, A. batzer, K.M.
Rabun, C.J. Der, J. Schlessinger, M.L. Gishizky (1993): BCR-ABL-Induced Oncogenesis
Is Mediated by Direct Interaction with the SH2 Domain of the GRB-2 Adaptor Protein.
Cell, 75, 175-185.
Biology.
Szeberényi J. (2015) Problem-solving test: The mechanism of action of a human

papilloma virus oncoprotein. Biochem.Mol.Biol.Educ. 37, 118-120.

0001).

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PROBLEM-SOLVING TESTS IN

MOLECULAR CELL BIOLOGY

With the permission of the International Union of Biochemistry and

Supported by a grant from the European Union (TÁMOP-4.1.1.C-

Electrophoretic behavior of hemoglobin variants 1.

Real-time polymerase chain reaction 5.

Targeted gene disruption 10.

The effect of bisulfite treatment on genomic DNA 15.

Analysis of the cell cycle by flow cytometry 18.

α1-Antitrypsin deficiency: An example for a protein folding disease 22.

Vectorial transport in intestinal epithelial cells 32.

Expression cloning of the erythropoietin receptor 36.

The yeast two-hybrid system 41.

Analysis of the mechanism of action of an apoptosis inducing compound 45.

The mechanism of action of a human papilloma virus oncoprotein 51.

Signal transduction in Philadelphia chromosome positive leukemia cells 56.

ELECTROPHORETIC BEHAVIOR OF HEMOGLOBIN

Terms to be familiar with before you start to solve the test

Hemoglobins * erythrocytes * reticulocytes amino acids *  and  globin chains *

H2N CH COOH H2N CH COOH H2 N CH COOH

Figure 1: The chemical formula of glutamic acid, valine and lysine.

5.____ A. The isoelectric point of HbA

10.____ Which of these individuals has/have the worst prognosis?

Supported by a grant from the European Union (TÁMOP-4.1.1.C-13/1/KONV-2014-

REAL-TIME POLYMERASE CHAIN REACTION

Terms to be familiar with before you start to solve the test

Figure 1. The principle of TaqMan method (details in the text).

A: 5’→3’ elongation activity of Taq polymerase

1.____ Generates phosphodiester bonds.

3.____ Cleaves hydrogen bonds.

7.____ Has a proofreading function.

Reaction mixtures were incubated in a thermocycler capable of monitoring

9.____ A: The number of free primer molecules in sample A after cycle 4

10.____ A: The number of free primer molecules in sample A after cycle 10

13.____ A: The number of reporter dye/mononucleotide complexes in sample A after cycle 18

14.____ A: The number of amplified HER2 fragments in sample A after cycle 20

15.____ A: The number of amplified HER2 fragments in sample A after cycle 30

A. Because Taq polymerase degrades the TaqMan probe molecules

A. Because all primer molecules have been used

A. Its copy number increased approximately 30-fold

Supported by a grant from the European Union (TÁMOP-4.1.1.C-13/1/KONV-2014-

TARGETED GENE DISRUPTION

Terms to be familiar with before you start to solve the test

Mutational inactivation of a specific gene is the most powerful technique to analyze

2.____ What is the significance of linking a mammalian promoter to neoR ?

A. Cells with random integration (as shown in Fig. 2A)

4.____ Express the neoR gene.

5.____ Degrade and loose the tkHSV gene rapidly.

6.____ Express the tkHSV gene only transiently.

7.____ The function of gene X is inhibited in these cells.

9.____ Kills cells in which foreign DNA is degraded in the cytoplasm.

[1] S. L. Mansour, K. R. Thomas, M. R. Capecchi (19889) Disruption of the proto-

Supported by a grant from the European Union (TÁMOP-4.1.1.C-13/1/KONV-2014-

THE EFFECT OF BISULFITE TREATMENT ON GENOMIC DNA

Terms to be familiar with before you start to solve the test

Polymerase chain reaction (PCR)  primer  promoter  restriction endonucleases 

The arrows indicate the cleavage sites.)

1.____ Consists of fragments with one intact 5’-GAATTC-3’ sequence.

4.____ Consists of fragments with one intact 5’-AAATTT-3’ sessequence.