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JÓZSEF SZEBERÉNYI
EXPERIMENTS IN MOLECULAR
CELL BIOLOGY
A Problems Book With Multiple-choice Question-based Tests

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WWW . SCHENKBUCHVERLAG . DE

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JÓZSEF SZEBERÉNYI
EXPERIMENTS IN MOLECULAR
CELL BIOLOGY
A Problems Book With Multiple-choice
Question-based Tests

SCHENK VERLAG

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Reviewer
András Tigyi MD, PhD

Technical Assistance
Melinda Kiss
Mónika Vecsernyés

Bibliografische Information. Der Deutsche Bibliothek


Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliografie; detaillierte bibliografische Daten sind im Internet
über http.//dnb.ddb.de abrufbar.

ISBN 3-939337-13-7
ISBN 978-3-939337-13-3

© Schenk Verlag GmbH, Passau 2006

All rights reserved. No part of this publication may be reproduced, stored


in a retrieval system, or transmitted in any form or by any means,
electronic, mechanical, photocopying, recording, or otherwise, without the
prior written permission of the Publisher.

Printed in Hungary

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Contents

Preface 7
Types of multiple-choice questions 9
11. Restriction mapping of a plasmid DNA 11
12. Restriction mapping of a linear DNA fragment 17
13. Cutting and ligating λ phage DNA 23
14. Construction of a subtractive genomic library 29
15. Sequencing of DNA using the Maxam-Gilbert method 37
16. DNA fingerprinting with a minisatellite probe in a family 45
17. In vitro synthesis of infectious phage DNA 51
18. Replication of SV40 virus DNA 57
19. In vitro synthesis of Okazaki fragments 63
10. Proofreading by bacterial DNA polymerase I 69
11. Functional analysis of a DNA repair protein 75
12. The phases of cell cycle 83
13. Regulation of lamin function by protein kinases 89
14. In vitro transcription by a bacterial RNA polymerase 95
15. Isolation of RNA polymerases from frog cells 99
16. mRNA stability 105
17. Polysomes and protein synthesis 109
18. Analysis of protamine-synthesizing polysomes 113
19. Protein translocation across the endoplasmic reticulum membrane 119
20. The role of calnexin in the secretory pathway 125
21. Analysis of the lac-repressor 133
22. Lactose operon mutants 139
23. Regulation of gene expression in mitogen-activated lymphocytes 143
24. The role of the 3'-untranslated region in mRNA metabolism 149
25. Sucrosomes 155
26. Mitochondrial protein transport 161
27. Purification of tubulins 167

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6 CONTETS

28. SCG10, a cytoskeleton-associated protein 173


29. Consequences of the ΔF508 cystic fibrosis gene mutation 179
30. Signal transduction from the epidermal growth factor receptor 185
31. The regulation of a mitogen-actived protein kinase 191
32. The effect of nerve growth factor on an early-response gene 199
33. Targeted gene disruption 205
34. Catalytic activity of the SV40 T antigen 213
35. Identification of the v-src gene product 219
36. Analysis of the mutation that causes Burkitt lymphoma 225
37. The role of p53 protein in the cellular response to γ-irradiation 229
38. Analysis of a tumor by comparative genomic hybridization 235
39. Pedigree of hemophilia A 241
40. Colour blindness and Klinefelter syndrome 247
41. Inheritance of nail-patella syndrome and ABO blood groups 253

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Preface

“Medical practice is mainly problem-solving, so why not teach medicine through


problem-solving?” This slogan originates from E. Reerink of Maastricht Medical
School, Limburg University (The Netherlands), one of the leading universities in
experimenting in medical education. This philosophy of medical education can be
extended: it is true not only for clinical instruction, but for the teaching of basic medical
sciences as well. Molecular cell biology is one of the most rapidly developing fields
of natural sciences: its revolution has had a very strong impact on clinical disciplines
leading to the establishment of a new science, molecular medicine. The spectacular
advances in cell biology are mainly caused by the development of new, more and more
sophisticated techniques. Without understanding this methodology, it is impossible to
digest and understand the new findings, processes and principles of modern cell
biology. Teaching and learning molecular medicine thus requires a novel, problem-
oriented approach.
Application tests have been used for teaching and knowledge assessment of
medical students for more than two decades in the Molecular Cell Biology course
at the University Medical School of Pécs, Hungary. This type of test was designed
to enhance the problem-solving capabilities of medical students and to objectively
assess (by using multiple-choice questions) the higher domains of their intellectual
skills in cell biology. Applicaton tests are usually based on original scientific papers:
they raise an experimental problem, briefly describe the methods used in the study
and present the results in figures or tables. The student is expected to evaluate the
data and to draw conclusions by answering a set of multiple-choice questions. To
solve these questions, a thorough factual knowledge of the topic is required, but is
not sufficient: the student has to apply his knowledge creatively. According to the
principles of pedagogy, such a knowledge is more valuable and durable than
knowledge acquired by sheer memorization of facts.
I hope that solving these tests will not only help the reader to have an insight
into how science works, to acquire useful knowledge in cell biology, but maybe even
to have intellectual excitement and satisfaction.

January, 2006

József Szeberényi
MD, PhD, DSc

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Types of multiple-choice questions

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.

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.

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 Aand B;
D if the word or phrase is associated with neither A nor B.

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.

Quantitative comparison
In this type of question paired statements describe two entities that are to be
compared in a quantitative 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.

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10 TYPES OF MULTIPLE-CHOICE QUESTIONS

Variation relationship
This type of question consists of pairs of phrases which describe conditions or
quantities which may or may not vary in relation to each other. Select
A if increase in the first is accompanied by increase in the second or if
decrease in the first is accompanied by decrease in the second;
B if increase in the first is accompanied by decrease in the second or if
decrease in the first is accompanied by increase in the second;
C if changes in the second are independent of changes in the first.

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.

Text / Figure / Table 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;
C if the statement is neither supported nor contradicted by the information
given.

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P
1. RESTRICTION MAPPING
OF A PLASMID DNA

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Terms to be familiar with before you


start to solve the test

restriction endonucleases * plasmid * agarose gel electrophoresis * ethidium


bromide staining * circular DNA (see G.M. Cooper: The Cell. A Molecular
Approach. ASM Press/Sinauer Associates, Inc., 1997, pages 102-105, 108-109.)

The experiment

Restriction mapping of small DNA molecules is an important technique of modern


biology. An example of the mapping of a circular plasmid (pBR322) DNA is
described in this test. Purified samples of plasmid DNA were digested with the
restriction enzymes AccI, AvaI, BamHI, EcoRI or PvuII, or with various
combinations of these enzymes, and the reaction mixtures were fractionated by
agarose gel electrophoresis. Bands of DNA fragments were visualized by staining
with ethidium bromide. The image of the gel is displayed in Figure 1.1., and the
cleavage sites on the circular DNA are shown in Figure 1.2.
Using the information in Figure 1.1. determine the cleavage sites of the
enzymes. (An EcoRI site is already shown in Figure 1.2.)

Figure 1.1. Fractionation of the products of restriction endonuclease digestions by agarose gel
electrophoresis. (Undigested circular DNAs are usually present in the sample in three forms: closed,
superhelical circular DNA /I/, relaxed circular DNA /II/ or linear DNA /III/; see sample 1.
The arrow on the right side indicates the direction of electrophoresis.)

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14 RESTRICTION MAPPING OF A PLASMID DNA

Figure 1.2. The map of plasmid pBR322.


(The numbers indicate restriction sites to be identified on the basis of Figure 1.1.)

Five-choice association
A: AccI
B: AvaI
C: BamHI
D: PvuII
E: EcoRI

1.1._____ This enzyme cuts at site 1.


1.2._____ This enzyme cuts at site 2.
1.3._____ This enzyme cuts at site 3.
1.4._____ This enzyme cuts at site 4.
1.5._____ This enzyme cuts at site 5.

Correct answers
1.1. C 1.4. D
1.2. A 1.5. A
1.3. B

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RESTRICTION MAPPING OF A PLASMID DNA 15

Explanations

Agarose gel electrophoresis fractionates DNA fragments according to their size: the
smaller the molecule, the faster it runs. Restriction mapping can be done by
comparing fragment sizes.
Digesting pBR322 with AccI generates two fragments (sample 4 in Figure 1.1.),
therefore the plasmid must contain two cleavage sites. All the other enzymes have
a single recognition site in pBR322: alone they produce a single DNA fragment (e.g.
Ava I in sample 2) or in pairs they generate two fragments (samples 3 and 8). EcoRI
and PvuII give fragments of similar size (sample 8): the PvuII site is therefore either
site 4 or site 5. AccI gives two medium-sized fragments (sample 4): sites 1 or 2 and
sites 4 or 5 are the candidates for AccI cleavage. PvuII cuts into the small AccI
fragment (compare samples 4 and 7): site 4 is thus for PvuII (MCQ 1.4. D) and site
5 is for AccI (MCQ 1.5. A). BamHI generates a large and a very small fragment with
EcoRI (sample 3) indicating that the two enzymes cleave near to each other (MCQ
1.1. C). Site 2 is thus left for AccI (MCQ 1.2. A) and site 3 for AvaI (MCQ 1.3. B).
FURTHER READING

Bloch, K.D.: Mapping by multiple endonuclease digestions. In “Current Protocols in Molecular


Biology” (eds. Ausubel, F.M. et al.), John Wiley & Sons, Inc., 1994, Chapter 3.2.
Brooks, J.E.: Properties and uses of restriction endonucleases. In “Guide to Molecular Cloning
Techniques” (eds. S.L. Berger, A.R. Kimmel). Academic Press, Inc., 1987, pages 113-129.

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P
2. RESTRICTION MAPPING
OF A LINEAR DNA FRAGMENT

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Key terms to be familiar with before you


start to solve the test

restriction endonucleases * DNA polymerase I * in vitro DNA synthesis * chloroform


extraction * polyacrylamide gel electrophoresis * autoradiography * 5' → 3'
exonuclease * template * primer * DNA structure (see G.M. Cooper: The Cell. A
Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997, pages 46-47, 92-
93, 102-105, 175-184.)

The experiment

A DNA fragment generated by EcoRI (cleavage site: 5'-G↓AATTC-3') and BamHI


(cleavage site: 5'-G↓GATCC-3') digestion was analyzed in this experiment. (One
strand of the recognition site of the enzymes is shown only; the cleavage sites are
indicated by the arrows in the sequences.) The purified DNA fragment was incubated
in vitro with a DNA polymerase I lacking 5' → 3' exonuclease activity, dATP, dGTP,
dTTP, [α-32P]dCTP, ions and pH required for enzyme activity. After incubation the
DNA fragment was purified by chloroform extraction, divided into five aliquots and
treated with AluI restriction enzyme for various periods of time (0, 2, 5, 10 and 120
minutes). The products of the reaction were fractionated by polyacrylamide gel
electrophoresis and then analyzed by autoradiography. Figure 2.1. shows the auto-
radiogram.

Figure 2.1. Autoradiogram of AluI digests fractionated by gel electrophoresis.

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20 RESTRICTION MAPPING OF A LINEAR DNA FRAGMENT

Figure analysis
2.1._____ The DNA fragment was labelled by repair synthesis.
2.2._____ All BamHI-EcoRI fragments contain one labelled phosphate group.
2.3._____ The radioactive phosphate is present in the 3' end nucleotide of labelled
strands.
2.4._____ The BamHI-EcoRI fragment is 1000 base pairs (bp) long.
2.5._____ There are 100 bp fragments among the products of AluI digestion.
2.6._____ The AluI enzyme used in the experiment is contaminated by a 3' → 5'
exonuclease.
2.7._____ The sample digested for 2 minutes contains labelled DNA molecules
only.

Five-choice completion
2.8._____ Which map of Figure 2.2. is compatible with the experimental results?
A: Map A
B: Map B
C: Map C
D: Map D
E: None of them

Figure 2.2. Possible maps of the EcoRI-BamHI fragment. (E, EcoRI; A, AluI; B, BamHI)

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