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CHAPTER 10
ANALYZING GENES AND GENOMES

Ó 2009 Garland Science Publishing

10-1 Recombinant DNA technologies involve techniques that permit the

creation of custom-made DNA molecules that can be introduced back into
living organisms. These technologies were first developed in the ______.

(a) 1930s

(b) 1950s

(c) 1970s

(d) 1990s
Manipulating and Analyzing DNA Molecules

10-2 Which of the following statements about restriction nucleases is false?

(a) A reproducible set of DNA fragments will be produced every time a

restriction nuclease digests a known piece of DNA.

(b) Restriction nucleases recognize specific sequences on single-

stranded DNA.

(c) Some bacteria use restriction nucleases as protection from foreign

DNA.

(d) Some restriction nucleases cut in a staggered fashion, leaving short

single-stranded regions of DNA at the ends of the cut molecule.

10-3 You have purified DNA from your recently deceased goldfish. Which of
the following restriction nucleases would you use if you wanted to end up with
DNA fragments with an average size of 70 kilobase pairs (kb) after complete
digestion of the DNA? The recognition sequence for each enzyme is indicated
in the right-hand column.

(a) Sau3AI GATC

(b) BamHI GGATCC

(c) NotI GCGGCCGC

(d) XzaI GAAGGATCCTTC

10-4 You have a piece of circular DNA that can be cut by the restriction
nucleases XhoI and SmaI, as indicated in Figure Q10-4.

Figure Q10-4

If you were to cut this circular piece of DNA with both XhoI and SmaI, how
many fragments of DNA would you end up with?

(a) 1

(b) 2

(c) 3

(d) 4

10-5 You have a piece of circular DNA that can be cut by the restriction
nucleases EcoRI, HindIII, and NotI, as indicated in Figure Q10-5

Figure Q10-5

Which of the following statements is false?

(a) One piece of DNA will be obtained when this DNA is cut by NotI.
(b) A piece of DNA that cannot be cut by EcoRI will be obtained by cutting
this DNA with both NotI and HindIII.

(c) Two DNA fragments that cannot be cut by HindIII will be obtained
when this DNA is cut by EcoRI and NotI.

(d) Two DNA fragments of unequal size will be created when this DNA is
cut by both HindIII and EcoRI.

10-6 You have accidentally torn the labels off two tubes, each containing a
different plasmid, and now do not know which plasmid is in which tube.
Fortunately, you have restriction maps for both plasmids, shown in Figure
Q10-6. You have the opportunity to test just one sample from one of your
tubes. You have equipment for agarose-gel electrophoresis, a standard set of
DNA size markers, and the necessary restriction enzymes.

Figure Q10-6

1. Outline briefly the experiment you would do to determine which plasmid

is in which tube.
2. Which restriction enzyme or combination of restriction enzymes would
you use in this experiment?

10-7 Assume that defects in a hypothetical gene X have been linked to

antisocial behavior. Two copies of a defective gene X predispose a child to
bad behavior from childhood, whereas a single copy of the gene seems to
produce no symptoms until adulthood. Because early treatment can
counteract the effects of the gene, a program of voluntary genetic testing is
being performed with delinquent prospective parents. Charles S. and Caril
Ann F. have been arrested on charges of robbery and assault, and Caril Ann
is pregnant with Charles’s child. You obtain DNA samples from Charles, Caril
Ann, and the fetus. You digest these samples with NotI and use these
samples to perform two Southern blots, which you probe with two different
oligonucleotide probes, A and B, that hybridize to different parts of the normal
gene X, as shown in Figure Q10-7A. Your results are shown in Figure 10-7B.
Figure Q10-7

1. Which of the three individuals have defects in gene X?

2. Which individuals have a single defective gene and which have two
defective copies of the gene?
3. Indicate the nature (single-base-pair mutation or deletion) and location
of each individual’s defects on gene X.

10-8 Figure Q10-8 shows a restriction map of a piece of DNA containing

your favorite gene. The arrow indicates the position and orientation of the
gene in the DNA. In part (B) of the figure are enlargements showing the
portions of the DNA whose sequences have been used to make
oligonucleotide probes A, B, C, and D. Which of the oligonucleotides can be
used to detect the gene in each of the following?

Figure 10-8

1. A Southern blot of genomic DNA cut with HindIII.

2. A Northern blot.

10-9 During gel electrophoresis, DNA fragments

_______________________.

(a) travel through a matrix containing a microscopic network of pores

(b) migrate toward a negatively charged electrode

(c) can be visualized without stains or labels

(d) are separated on the basis of their sequence

10-10 A double-stranded DNA molecule can be separated into single strands

by heating it to 90°C because _______________________.

(a) heat disrupts the hydrogen bonds holding the sugar-phosphate

backbone together

(b) DNA is negatively charged

(c) heat disrupts hydrogen bonding between complementary nucleotides

(d) DNA is positively charged

10-11 You are interested in a single-stranded DNA molecule that contains

the following sequence:

5′- ….. GATTGCAT …. -3′

Which molecule can be used as a probe that will hybridize to your sequence
of interest?

(a) 5′-GATTGCAT-3′

(b) 5′-TACGTTAG-3′

(c) 5′-CTAACGTA-3′

(d) 5′-ATGCAATC-3′

DNA Cloning
10-12 For each of the following sentences, fill in the blanks with the best
word or phrase selected from the list below. Not all words or phrases will be
used; use each word or phrase only once.

A nuclease hydrolyzes the __________________ bonds in a nucleic acid.

Nucleases that cut DNA only at specific short sequences are known as
__________________. DNA composed of sequences from different sources
is known as __________________. __________________ can be used to
separate DNA fragments of different sizes. Millions of copies of a DNA
sequence can be made entirely in vitro by the __________________
technique.

DNA sequencing phosphodiester

endonucleases polymerase chain reaction

exonucleases recombinant DNA

gel electrophoresis restriction nucleases

hydrogen ribonucleases

nucleic acid hybridization

10-13 For each of the following sentences, fill in the blanks with the best
word or phrase selected from the list below. Not all words or phrases will be
used; use each word or phrase only once.

Two fragments of DNA can be joined together by __________________.

Restriction enzymes that cut DNA straight across the double helix produce
fragments of DNA with __________________. A fragment of DNA is inserted
into a __________________ in order to be cloned in bacteria. A
__________________ library contains a collection of DNA clones derived
from mRNAs. A __________________ library contains a collection of DNA
clones derived from chromosomal DNA.
blunt ends DNA polymerase RNA

cDNA genomic staggered ends

DNA ligase probe vector

10-14 Name three features that a cloning vector for use in bacteria must
contain. Explain your answers.

10-15 Figure Q10-15 shows the cleavage site of several restriction

nucleases.

Figure Q10-15

You cut a vector using the PclI restriction nuclease. Which of the following
restriction nucleases will generate a fragment that can be ligated into this cut
vector with the addition of only ligase and ATP?

(a) HindIII

(b) NcoI

(c) MmeI
(d) NspV

10-16 Figure Q10-16 depicts a strategy by which a DNA fragment produced

by cutting with the EcoRI restriction nuclease can be joined to a DNA
fragment produced by cutting DNA with the HaeIII restriction nuclease.

Figure Q10-16

Note that cutting DNA with EcoRI produces a staggered end, whereas cutting
DNA with HaeIII produces a blunt end. Why must polymerase be added in this
reaction?

(a) Polymerase will fill in the staggered end to create a blunt end.

(b) Polymerase is needed to seal nicks in the DNA backbone.

(c) Polymerase will add nucleotides to the end produced by the HaeIII
restriction nuclease.

(d) Without polymerase, there will not be enough energy for the reaction to
proceed.

10-17 DNA can be introduced into bacteria by a mechanism called

____________.

(a) transcription

(b) ligation

(c) replication

(d) transformation

10-18 Which of the following statements about gel-transfer hybridization (or

Southern blotting) is false?
(a) This technique involves the transfer of DNA molecules from gel onto
nitrocellulose paper or nylon paper.

(b) In this technique, single-stranded DNA is separated by

electrophoresis.

(c) A labeled DNA probe binds to the DNA by hybridization.

(d) The DNA that is separated on a gel is not labeled.

10-19 Figure Q10-19 shows the recognition sequences and sites of cleavage
for the restriction enzymes SalI, XhoI, PstI, and SmaI, and also a plasmid with
the sites of cleavage for these enzymes marked.

Figure Q10-19

1. After which of the five treatments listed below can the plasmid shown in
Figure Q10-19 re-form into a circle simply by treating it with DNA
ligase? Assume that after treatment any small pieces of DNA are
removed, and it is the larger portion of plasmid that will reassemble into
a circle.
After digestion with __________.

1. SalI alone
2. SalI and XhoI
3. SalI and PstI
4. SalI and SmaI
5. SmaI and PstI

1. In which of the treatments can the plasmid re-form into a circle by the
addition of DNA ligase after treating the cut DNA with DNA polymerase
in a mixture containing the four deoxyribonucleotides? Again assume
that you are trying to get the larger portion of plasmid to reassemble into
a circle.

10-20 Which of the following statements about genomic DNA libraries

isfalse?

(a) The larger the size of the fragments used to make the library, the
fewer colonies you will have to examine to find a clone that hybridizes to your
probe.

(b) The larger the size of the fragments used to make the library, the
more difficult it will be to find your gene of interest once you have identified a
clone that hybridizes to your probe.

(c) The larger the genome of the organism from which a library is derived,
the larger the fragments inserted into the vector will tend to be.

(d) The smaller the gene you are seeking, the more likely it is that the
gene will be found on a single clone.

10-21 A DNA library has been constructed by purifying chromosomal DNA

from mice, cutting the DNA with the restriction enzyme NotI, and inserting the
fragments into the NotI site of a plasmid vector. What information cannotbe
retrieved from this library?

(a) gene regulatory sequences

(b) intron sequences

(c) sequences of the telomeres (the ends of the chromosomes)

(d) amino acid sequences of proteins

10-22 You want to design a DNA probe used for hybridization to isolate a
clone from a cDNA library. Which of the following statements about DNA
probes is true?

(a) The shorter the DNA probe used to probe the library, the greater the
number of colonies to which the probe will hybridize.

(b) A DNA probe that contains sequences that span two exons is better
suited to the purpose than a DNA probe that only contains sequences from
one exon.

(c) A DNA probe that contains sequences immediately upstream of the

DNA that codes for the first methionine in the open reading frame will usually
not hybridize to clones in a cDNA library.

(d) Hybridization of a DNA probe to the plasmid of interest will permit the
detection of the clone of interest; labeling of the DNA probe is not necessary.

10-23 You have the amino acid sequence of a protein and wish to search for
the gene encoding this protein in a DNA library. Using this protein sequence,
you deduce a particular DNA sequence that can encode this protein. Why is it
unwise to use only this DNA sequence you have deduced as the probe for
isolating the gene encoding your protein of interest from the DNA library?

10-24 What is the main reason for using a cDNA library rather than a
genomic library to isolate a human gene from which you wish to make large
quantities of the human protein in bacteria?

10-25 Some clones from cDNA libraries can have defects because of the
way in which a cDNA library is constructed. For each dilemma (A to D) listed
below, indicate which of the outcomes (1 to 4) you might encounter, and
explain why. Outcomes may be used more than once.

10-26 You have an oligonucleotide probe that hybridizes to part of gene A

from a eucaryotic cell. Indicate whether a cDNA library or a genomic DNA
library will be more appropriate for use in the following applications.

1. You want to study the promoter of a gene A.

2. Gene A encodes a tRNA and you wish to isolate a piece of DNA
containing the full-length sequence of the tRNA.
3. You discover that gene A is alternatively spliced and you want to see
which predicted alternative splice products the cell actually produces.
4. You want to find both gene A and the genes located near gene A on the
chromosome.
5. You want to express gene A in bacteria to produce lots of protein A.

Note: The following codon table is to be used for Problems Q10-27, Q10-
28, Q10-40, Q10-43, Q10-44, and Q10-46.

10-27 Which of the restriction nucleases listed below can potentially cleave a
segment of cDNA that encodes the peptide KIGDACF?

10-28 Your biochemist friend has isolated a protein he thinks is responsible

for making you feel sleepy. Since he knows you’re taking Cell Biology, he
wants you to help him isolate the gene encoding this protein. Unfortunately,
because your friend could only isolate small amounts of protein, he was only
able to obtain three short stretches of amino acid sequence from the protein:
(a) H-C-W-K-M

(b) R-S-L-L-S

(c) D-A-Q-W-Y

For each of the three peptides above, you design a set of DNA oligonucleotide
probes that can be used to detect the gene in a library. Which of the three
sets of oligonucleotide probes would be preferable for screening a library?
Explain. (Refer to the codon table above Q10-27.)

10-29 Your friend has isolated a protein present in the cheek cells of all
straight-A seniors at your school. She says that this protein helps you
remember everything you read and therefore will help cut down on the
number of hours needed to study for exams. She sequences the protein,
which she calls ―geniuszyme‖, and designs a probe to isolate the gene that
encodes it. To make sure she designed the probe correctly, she consults with
the company that cloned Factor VIII. They have 100% confidence that her
probe will work. She also obtains a high-quality liver cDNA library from the
company and uses her probe to try to isolate the gene for geniuszyme.
Unfortunately, she is unable to isolate any clones.

1. What is the likely explanation for her failure?

2. Not to be discouraged, your friend has obtained some genomic DNA
isolated from the nuclei of liver cells and has made a genomic library
from that DNA. Do you expect she will succeed in isolating the
geniuszyme gene from this library? Why or why not?

10-30 Insulin is a small protein that regulates blood sugar level, and it is
given by injection to people who suffer from the disease diabetes. Diabetics
once used insulin purified from pig pancreas to control their diabetes. Give
two reasons why the drug companies that produce insulin wanted to clone the
human insulin gene to provide an alternative source of the hormone.
10-31 Which of the following statements about PCR is false?

(a) PCR uses a DNA polymerase from a thermophilic bacterium.

(b) PCR is particularly powerful because after each cycle of replication,

there is a linear increase in the amount of DNA available.

(c) For PCR, every round of replication is preceded by the denaturation of

the doubled-stranded DNA molecules.

(d) The PCR reaction will generate a pool of double-stranded DNA

molecules, most of which will have DNA from primers at the 5′ ends.

10-32 Why is a heat-stable DNA polymerase from a thermophilic bacterium

(the Taq polymerase) used in the polymerase chain reaction rather than a
DNA polymerase from E. coli or humans?

10-33 Which of the following limits the use of PCR to detect and isolate
genes?

(a) The sequence at the beginning and end of the DNA to be amplified
must be known.

(b) It also produces large numbers of copies of sequences beyond the 5′

or 3′ end of the desired sequence.

(c) It cannot be used to amplify cDNAs or mRNAs.

(d) It will amplify only sequences present in multiple copies in the DNA
sample.

10-34 You want to amplify the DNA between the two stretches of sequence
shown in Figure Q10-34. Of the listed primers, choose the pair that will allow
you to amplify the DNA by PCR.
Figure Q10-34

10-35 Your friend works at the Centers for Disease Control and has
discovered a brand-new virus that has recently been introduced into the
human population. She has just developed a new assay that allows her to
detect the virus by using PCR products made from the blood of infected
patients. The assay uses primers in the PCR reaction that hybridize to
sequences in the viral genome.

Your friend is distraught because of the result she obtained (see Figure Q10-
35) when she looked at PCR products made using the blood of three patients
suffering from the viral disease, using her own blood, and using a leaf from
her petunia plant.

You advise your friend not to panic, as you believe she is missing an
important control. Which one of the choices listed below is the best control for
clarifying the results of her assay? Explain your answer.

Figure Q10-35

(a) a PCR reaction using blood from a patient who is newly infected but
does not yet show symptoms

(b) a PCR reaction using blood from a dog

(c) a PCR reaction using blood from an uninfected person

(d) repeating the experiments she has already done with a new tube of
polymerase

Deciphering and Exploiting Genetic Information

10-36 For each of the following sentences, fill in the blanks with the best
word or phrase selected from the list below. Not all words or phrases will be
used; each word or phrase should be used only once.

The technique of __________________ hybridization can be used to detect a

specific RNA expression in a particular region of the brain. Northern blotting
detects a specific sequence in __________________. Southern blotting
detects a specific sequence in __________________. A short, single-
stranded DNA is a(n) __________________. A piece of DNA used to detect a
specific sequence in a nucleic acid by hybridization is known as a(n)
__________________.

DNA polymerase chain reaction

in situ probe

in vitro RNA

oligonucleotide vector

10-37 In situ hybridization can be used to determine the

_________________.

(a) sequence of a cloned gene

(b) distribution of proteins in tissues

(c) size of a gene

(d) distribution of a given type of mRNA in different tissues

10-38 Why are dideoxyribonucleoside triphosphates used during DNA

sequencing?

(a) They cannot be incorporated into DNA by DNA polymerase.

(b) They are incorporated into DNA particularly well by DNA polymerases
from thermophilic bacteria.

(c) Incorporation of a didoxyribonucleoside triphosphate leads to the

termination of replication for that strand.

(d) Dideoxyribonucleotide triphosphates are more stable than

deoxyribonucleoside triphosphates.

10-39 Which of the following describes a feature found in bacterial

expression vectors but not in cloning vectors?

(a) origin of replication

(b) cleavage sites for restriction nucleases

(c) promoter DNA sequences

(d) a polyadenylation signal

10-40 You have sequenced a short piece of DNA and produced the gel
shown in Figure Q10-40.

Figure Q10-40

1. What is the sequence of the DNA, starting from the 5′ end?

2. If you knew that this sequence is from the middle of a protein-coding
cDNA clone, what amino acid sequence can be deduced from this
sequence? (Refer to the codon table above Q10-27.)

10-41 You have sequenced a fragment of DNA and produced the gel shown
in Figure Q10-41. Near the top of the gel, there is a section where there are
bands in all four lanes (indicated by the arrow). Which of the following
mishaps would account for this phenomenon? Explain your answer.

Figure Q10-41

(a) You mistakenly added all four dideoxynucleotides to one of the

reactions.

(b) You forgot to add deoxynucleotides to the reactions.

(c) Your primer hybridizes to more than one area of the fragment of DNA
you are sequencing.

(d) A restriction nuclease cut a fraction of the DNA you are sequencing.

10-42 You are interested in understanding the gene regulation of a protein

called LKP1, which is normally produced in liver and kidney cells in mice.
Interestingly, you find that LKP1 is not expressed in heart cells. You replace
the coding sequence for LKP1 with the DNA encoding green fluorescent
protein (GFP) and examine the expression of GFP in mice. (GFP is your
reporter gene.) You find expression of GFP in liver and kidney cells but not
heart cells; this expression pattern is similar to how LKP1 is expressed
normally. Further experiments demonstrate that there are three regulatory
sequences in the promoter, labeled A, B, and C in Figure Q10-42, that are
important for this expression pattern. You want to determine the significance
of each regulatory sequence, so you create situations where only a subset of
regulatory regions are present upstream of the reporter gene, as diagrammed
in Figure Q10-42.

Figure Q10-42

Given the data, which of the statements below is false?

(a) Regulatory sequence A switches on LKP1 in the kidney.

(b) Regulatory sequence B switches on LKP1 in the liver.

(c) Neither regulatory sequence A nor regulatory sequence B is required

for LKP1 expression in the heart.

(d) Regulatory sequence C is not necessary for proper expression.

10-43 You are studying a protein, and a small fragment of its sequence is
shown below. You have decided that the glutamine in the protein segment has
an important role in its function. You decide to change this glutamine to a
lysine using DNA technology with the use of site-directed mutagenesis. You
have a plasmid that contains the full-length version of the gene that encodes
this protein and wish to create a new DNA molecule that has a change in only
one base and that substitutes a lysine for a glutamine. Given the partial
mRNA sequence that codes for this stretch of protein below, devise a 21-
nucleotide DNA oligonucleotide that can be used to make this mutation. Be
sure to label the 5′ and 3′ ends. (Refer to the codon table above Q10-27.)

F D P Q G S H

5′-uucgacccgcagggcagccac–3′

10-44 You are studying a protein that contains the peptide sequence
RDWKLVI. The part of the DNA encoding this peptide is included in the
sequence shown below.

5′-GGCGTGACTGGAAGCTAGTCATC-3′

3′-CCGCACTGACCTTCGATCAGTAG-5′

This sequence does not contain any HindIII restriction enzyme sites; the
target sequence for the HindIII restriction nuclease is shown in Figure Q10-44.
Figure Q10-44

Your goal is to create a HindIII site on this plasmid without changing the
coding sequence of the protein. Explain how you would do this. (Refer to the
codon table above Q10-27.)

10-45 You are interested in understanding how the brain works, and are
using the fruit fly Drosophila as a model system to study brain development.
You perform a microarray analysis to try to determine genes expressed in the
fly brain. For your microarray experiment, you first prepare cDNA from fly
brains and label it with a red fluorochrome. Then you isolate cDNA from whole
flies and label it with a green fluorochrome. Next you hybridize these cDNA
populations to a microarray containing the Drosophila genes. From this you
obtain a list of genes that are specifically enriched in the brain (those that
show up as a red spot on the microarray).

You are disappointed because your favorite fly gene, tubby, does not appear
on this list, even though you have repeated the microarray experiment 10
times and did not encounter any technical difficulties. The reason you
thought tubby would appear on this list is that you believe tubby is important
for brain development, because flies mutant in tubby have no brains. Not to be
discouraged, you perform in situ analysis using the tubby DNA as a probe,
and see that it is expressed at high levels in the fly brain of normal flies but not
expressed in animals lacking the tubby gene.

Why do you think tubby did not show up as a gene specifically enriched in the
brain in your microarray experiment?
10-46 You have created a piece of recombinant DNA by placing a cDNA
from a gene you believe is important for the differentiation of liver cells (called
LC1) onto an expression plasmid that contains all the sequences necessary
for propagation of this DNA in bacteria and for the production of the LC1
protein in bacteria. A picture of this plasmid is shown in Figure Q10-46A, with
the segment of the DNA containing the PK1 gene depicted as a grey
rectangle; the promoter sequence is depicted as a white rectangle. The LC1
protein is phosphorylated on serine 54; the nucleotide sequence of the portion
of the DNA that encodes this region is shown below. All HindIII and SalI
restriction sites have also been mapped on the plasmid; the recognition
sequences for these restriction nucleases are shown in Figure Q10-46B.

Figure Q10-46

54. Given the information above, write out the amino acids 52 to 57,
encoded by the nucleotide sequence shown above. Be sure to number
the amino acids appropriately. (Hint: remember, serine is amino acid
number 54.) (Refer to the codon table above Q10-27.)
55. You want to create a mutant version of the PK1 gene that
replaces serine 54 found on this peptide with a glycine. You want to do
this by changing only one nucleotide, and you also want to destroy the
HindIII recognition sequence with this change. Write out a 21-nucleotide
DNA primer that can be used for site directed mutagenesis to
accomplish this task. Be sure to (i) write out the DNA and label the 5′
and 3′ ends, (ii) underline the mutated HindIII recognition site, and (iii)
circle any change made to the original sequence.

10-47 Which of the following statements about RNA interference (or RNAi)
isfalse?

(a) RNAi is a natural mechanism used to regulate genes.

(b) During the process of RNAi, hybridization of a small RNA molecule

with the mRNA degrades the mRNA.

(c) Because RNAi depends on the introduction of a double-stranded RNA

into a cell or an organism, it is not a process that can cause heritable changes
in gene expression.
(d) In C. elegans, RNAi can be introduced into the animals by feeding
them with bacteria that produce the inhibitory RNA molecules.

10-48 You have been hired to create a cat that will not cause allergic
reactions in cat-lovers. Your co-workers have cloned the gene encoding a
protein found in cat saliva, expressed the protein in bacteria, and shown that it
causes violent allergic reactions in people. But you soon realize that even if
you succeed in making a knockout cat lacking this gene, anyone who buys
one will easily be able to make more hypoallergenic cats just by breeding
them. Which of the following will ensure that people will always have to buy
their hypoallergenic cats from you?

(a) Inject the modified ES cells into embryos that have a genetic defect to
prevent the mature adult from reproducing.

(b) Implant the injected embryos into a female cat that is sterile as a result
of a genetic defect.

(c) Sell only the offspring from the first litter of the female cat implanted
with the injected embryos.

(d) Surgically remove the sexual organs of all the knockouts before you
sell them.

How We Know: Sequencing the Human Genome

10-49 You have been asked to consult for a biotech company that is seeking
to understand why some fungi can live in very extreme environments, such as
the high temperatures inside naturally occurring hot springs. The company
has isolated two different fungal species, F. cattoriae and W. gravinius, both of
which can grow at temperatures exceeding 95°C. The company has
determined the following things about these fungal species:

By sequencing and examining their genomes, the biotech company hopes to

understand why these species can live in extreme environments. However,
the company only has the resources to sequence one genome, and would like
your input as to which species should be sequenced and whether you believe
a shotgun strategy will work in this case. (Be sure to explain your answer.)

10-50 Figure Q10-50A depicts the restriction map of one segment of the
human genome for four restriction nucleases W, X, Y, and Z. Figure Q10-50B
depicts the restriction maps of four individual BAC clones that contain
segments of human DNA from the region depicted in Figure Q10-50A.

Figure Q10-50

From this information, how would you order these BAC clones, from left to
right?

(a) 1, 2, 3, 4

(b) 2, 1, 4, 3

(c) 3, 4, 2, 1

(d) 4, 1, 3, 2

Answers

10-1 (c)

10-2 (b). Restriction nucleases cleave double-stranded DNA.

10-3 (c). A restriction nuclease that has a 4-base-pair recognition sequence
cuts on average once every 44 or 256 base pairs; one that has a 6-base-pair
recognition sequence cuts once every 46 or 4096 base pairs; one that has an
8-base-pair recognition sequence cuts once every 48 or 65,536 base pairs;
one that has a 12- base-pair recognition sequence cuts once every 412 or 16
million base pairs. Thus, to obtain fragments of about 70 kb in size, you would
cut with a nuclease that recognizes an 8-base-pair site.

10-4 (b)

10-5 (d)

10-6 A. You would first digest your sample with a combination of

restriction enzymes selected so that they give a set of fragment sizes that
could have come from only one of the plasmids. Then you would run the
resulting mixture of DNA fragments on a gel alongside a set of size markers
and determine the size of each fragment. By looking at the restriction maps,
you should then be able to match your results to one of the plasmids.

1. Digestion with any of the following combinations will enable you to

distinguish which plasmid you have: HindIII + BglII; EcoRI + BglII; EcoRI
+ BglII + HindIII. The plasmids are the same size, so you cannot
distinguish between them simply by making a single cut (with HindIII)
and determining the size of the complete DNA by gel electrophoresis.
Nor can you distinguish them by cutting with all four restriction
nucleases, Because the set of fragment sizes produced from both
plasmids will be the same. Cutting with BamHI or EcoRI on their own is
not sufficient because you will get bands of the same size from both
plasmid A and plasmid B. The only difference between the two plasmids
is in the location of the BglII site relative to the two BamHI sites, so if
you cut with an enzyme that cuts outside the BamHI fragment and with
BglII, you will get different-sized fragments from the two plasmids.

10-7 A. All three are affected.

 1. The two parents have a single defective copy of the gene; the fetus has
two defective copies.
2. As seen in the two blots in Figure Q10-7B, Caril Ann and Charles each
have one full-length copy of gene X (the bands at the top of the gel),
which hybridizes with both probe A and probe B. The fetus does not.
The blot with probe A shows that Caril Ann and the fetus have a short
fragment of gene X that hybridizes with probe A only, indicating that this
copy of gene X has a deletion somewhere other than in the region
recognized by probe A. The second blot (with probe B) shows that
Charles and the fetus have a short fragment that hybridizes with probe
B, indicating that this copy of gene X has a deletion somewhere other
than in the region recognized by probe B. Because the shortened gene
found in Charles does not show up on the probe A blot, this deletion
must be in the region of A; similarly, because the shortened gene found
in Caril Ann does not show up on the probe B blot, her deletion must be
in the region of B. The fetus has inherited two defective copies of gene
X, one from each parent.

10-8 A. All four oligonucleotide probes.

1. Oligonucleotide probe B and oligonucleotide probe D. Both the upper

and lower strands of DNA are present in genomic Southern blots, so all
four oligonucleotides will hybridize to either Southern blot.
(Oligonucleotides A and B will still be able to hybridize to genomic DNA
cut with BglII, because they can still base-pair with the individual
fragments that result from the digest.) Northern blots contain only RNA,
which has the sequence of the upper strand of the DNA. Hence, only
oligonucleotide probe B and oligonucleotide probe D will hybridize to a
Northern blot.

10-9 (a)

10-10 (c)

10-11 (d)
10-12 A nuclease hydrolyzes the phosphodiester bonds in a nucleic acid.
Nucleases that cut DNA only at specific short sequences are known
asrestriction nucleases. DNA composed of sequences from different
sources is known as recombinant DNA. Gel electrophoresis can be used to
separate DNA fragments of different sizes. Millions of copies of a DNA
sequence can be made entirely in vitro by the polymerase chain
reaction technique.

10-13 Two fragments of DNA can be joined together by DNA ligase.

Restriction enzymes that cut DNA straight across the double helix produce
fragments of DNA with blunt ends. A fragment of DNA is inserted into
avector in order to be cloned in bacteria. A cDNA library contains a collection
of DNA clones derived from mRNAs. A genomic library contains a collection
of DNA clones derived from chromosomal DNA.

10-14 A cloning vector for use in bacteria must contain the following:

1. a bacterial replication origin (to allow the plasmid to be replicated)

2. at least one unique restriction site (to allow easy insertion of foreign
DNA)
3. an antibiotic-resistance gene or some other selectable marker gene (to
allow selection for bacteria that have taken up the recombinant
plasmids)

10-15 (b). However, you will not be able to excise the fragment after ligation,
because you will destroy both the PclI site and the NcoI site, creating a new
site with the sequence

5′-ACATGG-3′

3′-TGTACC-5′
10-16 (a)

10-17 (d)

10-18 (b). During Southern blotting, double-stranded DNA is loaded onto the
agarose gel. The DNA becomes denatured (and thus single stranded) as it
gets transferred by the alkali solution from the gel to the nitrocellulose or nylon
sheet.

10-19 A. 1 and 2. When SalI and XhoI cut DNA, the staggered ends left
behind will match up by base pairing and can therefore be joined by ligase
alone.

4. 1, 2, and 4. SmaI cuts and leaves a blunt end. Addition of DNA

polymerase and the four deoxynucleotides will fill in the 5′ overhangs
generated by digestion with SalI and XhoI, leaving blunt ends. DNA
ligase joins the blunt ends. However, 3′ overhangs (that is, those
generated by PstI) will not be filled in, because DNA polymerase moves
in a 5′ to 3′ direction. DNA ligase will not join 3′ overhangs to blunt-
ended DNA, which are the situations presented in treatments 3 and 5.

10-20 (c). The sizes of the fragments left after a restriction digest do not
depend on the total size of the genome; they depend on the sequence of the
genome and the frequency with which the restriction enzyme recognition site
is found in the genome. Choices (a) and (b) are true: as a limiting case, think
of what would happen if a fragment the size of the entire genome were
inserted into the bacterial vector. You would have to screen only one colony to
find the clone that hybridized to your probe, but it would be very difficult to find
out where on the insert your gene of interest lay. Choice (d) is true: the larger
the gene you are seeking, the more likely it is that there will be a restriction
fragment in the gene (or that the gene will be broken if the DNA was
fragmented by random shearing), and hence the less likely it is that the entire
gene will be found in one clone.
10-21 (c). The very ends of all of the chromosomes are unlikely to be NotI
sites, meaning that the fragments containing the ends of the chromosomes
will not be able to insert into the bacterial vector (because they have not been
cut by NotI at both ends) and will be lost from the library. All sequences
present in genomic DNA (which includes regulatory sequences and introns)
should be present in a genomic library. The coding sequence of the gene (and
hence the amino acid sequence of the encoded protein) is also present in a
genomic clone, although it is interrupted by intron sequences and is therefore
somewhat difficult (but not impossible) to determine.

10-22 (a). The shorter the DNA probe, the more likely it is that that particular
sequence will show up in the genome at random. cDNA libraries contain
sequences represented by exons, so it does not really matter whether or not
the probe spans two exons (choice (b)). mRNAs usually have 5′ untranslated
regions that should be represented in a cDNA library, so choice (c) is not true.
DNA probes are usually labeled (for example, with radioactivity) for
visualization (choice (d)).

10-23 Because most amino acids can be encoded by more than one codon,
a given sequence of amino acids could be encoded by several different
nucleotide sequences. Probes corresponding to all these possible sequences
have to be synthesized, to be sure of including the one that corresponds to
the actual nucleotide sequence of the gene and thus will hybridize with it.

10-24 The gene isolated from a genomic library would still contain introns,
and bacteria do not contain the biochemical machinery for removing introns by
RNA splicing. The same gene isolated from the cDNA library will already have
had its introns removed.

10-25 A. Outcome 1 would occur. If the mRNA is degraded from the 5′

end, it will still be reverse transcribed and will end up in the library as a clone
lacking its 5′ end.

1. Outcome 4 would occur. If the mRNA is degraded from the 3′ end, it will
lack its 3′ poly A tail. In the construction of a cDNA library, only
 molecules that still have their poly A tail will be reverse transcribed, so
mRNAs lacking their 3′ end will not be represented in the library.
2. Outcome 1 would occur. If the 5′ end hybridizes to sequences in the
middle of the gene, the ―hairpin‖ formed when the single-stranded DNA
loops back on itself to form the primer for DNA polymerase will be very
large. After this loop has been digested, the remaining double-stranded
DNA fragment will lack the 5′ end of the gene.
3. Outcome 2 would occur. If the gene has a long stretch of internal A’s,
the poly T primer used in the reverse transcription step can hybridize to
the internal poly A stretch rather than to the poly A tail, and the resulting
cDNA will have lost its 3′ end.

10-26 A. Genomic library. (cDNAs are produced from mRNAs; therefore,

the promoters will not be included in a cDNA library.)

1. Genomic library. (cDNAs are usually constructed by using an oligo dT

primer; tRNAs do not have poly A tails. If the cDNA library were made
using random primers, it would be unlikely to contain the full-length
version of the tRNA.)
2. cDNA library. (Because cDNAs are produced from mRNAs, isolating
cDNAs would tell you which splice variants are produced in a cell.)
3. Genomic library. (A genomic DNA fragment can contain the genes next
to your gene of interest; a cDNA will not.)
4. cDNA library. (Bacteria do not have the ability to remove introns, which
may exist in DNA isolated from a genomic library.)

10-27 The nucleotide sequences that can encode the peptide KIGDACF are
shown below.

The enzyme NsiI cleaves at ATGCAT.

10-28 (a) H-C-W-K-M. There is the least amount of degeneracy in the

nucleotides that could code for this peptide; see below.
10-29 A. Geniuszyme is not expressed in the liver. Because cDNA is
made from mRNA, a cDNA library reflects the genes expressed in a particular
tissue.

1. Yes, she should be able to isolate the gene, because genomic DNA is
essentially the same in all tissues.

10-30 Any two of the following would be acceptable.

1. Cloning the gene allows human insulin to be produced in large

quantities from bacteria or other cells carrying the cloned DNA
sequence.
2. It is easier and less costly to extract the same amount of insulin from a
bacterial culture than from pig pancreas.
3. Insulin made in a bacterial culture and then purified will be free of any
possible contaminating viruses that pigs (and any other whole animal)
tend to harbor.
4. The pig protein has slight differences from the human protein, which can
lead to side effects on prolonged use. Whenever possible, a human
protein would be preferred for clinical treatment of this sort.

10-31 (b)

10-32 The PCR technique involves heating the reaction at the beginning of
each cycle to separate the newly synthesized DNA into single strands so that
they can act as templates for the next round of DNA synthesis. Using a heat-
stable polymerase avoids having to add it afresh for each round of DNA
replication.

10-33 (a). To construct primers that will bracket the desired gene, you have
to know the sequence at the beginning and end of the DNA to be copied.
10-34 The appropriate PCR primers are primer 1 (5′-GACCTGTGGAAGC-3′)
and primer 8 (5′-TCAATCCCGTATG-3′). The first primer will hybridize to the
bottom strand and prime synthesis in the rightward direction. The second
primer will hybridize to the top strand and prime synthesis in the leftward
direction. (Remember that strands pair antiparallel.)

The middle two primers in each list (primers 2, 3, 6, and 7) would not hybridize
to either strand. The remaining pair of primers (4 and 5) would hybridize, but
would prime synthesis in the wrong direction—that is, outward, away from the
central segment of DNA. Each wrong choice has been made at one time or
another in most laboratories that use PCR. In most cases the confusion arises
because the conventions for writing nucleotide sequences have been ignored.
By convention, nucleotide sequences are written 5′ to 3′, with the 5′ end on the
left. For double-stranded DNA, the 5′ end of the top strand is on the left.

10-35 (c). Primers can sometimes hybridize to unintended sequences and

produce unintended products. The appropriate control for your friend’s
experiment would be DNA from an uninfected person; in that way she would
be able to determine whether the bands present in the PCR from her blood
truly correspond to product generated from viral DNA rather than cross-
hybridization to DNA sequences in the human genome, because the bands
would be absent from a person uninfected by the virus in the former case
only. Doing PCR from an infected but asymptomatic person would not be
useful (choice (a)), because it would not allow your friend to distinguish
whether she is infected. Although doing PCR from dog blood (choice (b))
should not give any viral bands, any nonspecific products from a dog would
probably be different from those in your friend. The absence of PCR
fragments in the petunia lane suggests that there is no viral contaminant in
any of your friend’s reagents, so using a new tube of polymerase is not the
solution (choice (d)).

10-36 The technique of in situ hybridization can be used to detect a specific

RNA expression in a particular region of the brain. Northern blotting detects a
specific sequence in RNA. Southern blotting detects a specific sequence
inDNA. A short, single-stranded DNA is called a(n) oligonucleotide. A piece
of DNA used to detect a specific sequence in a nucleic acid by hybridization is
known as a(n) probe.
10-37 (d)

10-38 (c)

10-39 (c). Origins of replication (choice (a)) and cleavage sites for restriction
nucleases (choice (b)) are found in both cloning vectors and expression
vectors. Bacterial mRNAs do not undergo polyadenylation (choice (d)).

10-40 A. 5′-TAGACTGACCTG-3′

1. Arg-Leu-Thr (Only the second reading frame can be used, because

reading frame 1 contains a stop codon (TAG), as does reading frame 3
(TGA).)

10-41 (d). If some of the DNA templates you are sequencing are cut at one
specific site (as would be the case if a restriction enzyme cut the DNA), the
polymerase will stop when it comes to the end of the DNA, giving rise to at
least some product of one particular size in all the reaction mixtures. If this
were the case, all four lanes will have a band of this particular size. In
addition, you would get a normal sequence from the full-length templates, and
a normal sequence from those templates in which the polymerase
incorporated a dideoxynucleotide before encountering the end. The other
options are incorrect: if you added all four dideoxynucleotides to one of the
reactions (choice (a)), that lane would have a band at every position because
the polymerase would stop at A’s, C’s, G’s, and T’s instead of at only one type
of nucleotide. If you forgot to add deoxynucleotides to the reactions (choice
(b)), you would not get any polymerization, and all of your lanes would be
blank. If your primer hybridized to more than one part of the fragment of DNA
you were sequencing (choice (c)), your gel would look as though two different
sequences had been superimposed on each other.
10-42 (d). Regulatory sequence C is needed to turn off LKP1 in the heart.
Without regulatory sequence C (see experiments 2 and 6), inappropriate
expression of LKP1 in the heart is seen.

10-43 Either 5′-ttcgacccgaagggcagccac–3′ or 5′-gtggctgcccttcgggtcgaaa-3′

would work. The changed nucleotide is indicated in gray in the sequences
above. The codon for glutamine (CAG) has been changed to AAG, which
codes for lysine.

10-44 The new DNA sequence is shown in Figure A10-44.

Figure A10-44

The change in the sequence is indicated by the rectangle; the HindIII

recognition sequence has been underlined. The peptide encoded by this piece
of DNA is indicated above the DNA sequence. Note that this DNA will still
encode leucine, despite the change from a CUA codon to a CUU codon.

10-45 The tubby gene is expressed in all tissues, including the brain. A red
spot on a microarray is indicative of a difference in gene expression between
the two RNA samples being compared. You may expect in this experiment
that the tubby gene would be a yellow spot (a gene expressed at equal levels
in both samples). An in situ experiment looks at the RNA level directly in the
tissue of interest, which is why in this case you see ample levels
of tubby RNA.

10-46 A. 52 alanine, 53 valine, 54 serine, 55 leucine, 56 tryptophan, 57

cysteine. See Figure A10-46 for how this answer was derived.
 1. Either of the two nucleotide sequences shown in Figure A10-46B is
correct. The peptide encoded is shown above the top sequence, for
reference. The bottom nucleotide sequence is the reverse complement
of the top sequence.

Figure A10-46

10-47 (c)

10-48 (d). Neutering all the knockout animals that you sell is the only option
of the four listed that will prevent happy pet owners from becoming happy pet
breeders. The situation described in choice (a) will not allow you to make any
knockout cats because the first litter (which will at best have a few mosaics in
which one copy of the gene has been knocked out in the germ cells) will be
sterile and you will not be able to mate them. The genotype of the female cat
in which you implant the embryos has no effect on the genotype of the
embryos, which is why choice (b) is incorrect. Choice (c) is incorrect because
the first litter will yield mosaic cats that still have one copy of the allergen-
producing gene in their cells and are therefore not hypoallergenic.

10-49 Even though the genome of F. cattoriae is smaller, the W.

graviniusgenome is more attractive to sequence because it contains less
repetitive DNA. Repetitive DNA makes the assembly of sequenced fragments
difficult. Shotgun sequencing would not be an unrealistic approach for W.
gravinius, because the genome of W. gravinius contains little repetitive DNA
and is relatively small. The genome of H. influenzae is 1.83 megabases long
and was successfully sequenced with the shotgun approach. (For
comparison, the genome of S. cerevisiae is 14 megabases long.)

10-50 (b). The order of the BAC clones relative to the segment of human
DNA is shown in Figure A10-50.

Figure A10-50
Essential Cell Biology: 3rd Edition Test Bank – Alberts