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 CHAPTER 10
ANALYZING GENES AND GENOMES
© 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
 A. Outline briefly the experiment you would do to determine which plasmid
is in which tube.
B. 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

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

B. Which individuals have a single defective gene and which have two
defective copies of the gene?
C. 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

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

B. 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

A. 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

B. 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 is false?
(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 cannot be 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.
A. You want to study the promoter of a gene A.
B. Gene A encodes a tRNA and you wish to isolate a piece of DNA
containing the full-length sequence of the tRNA.
C. You discover that gene A is alternatively spliced and you want to see
which predicted alternative splice products the cell actually produces.
D. You want to find both gene A and the genes located near gene A on the
chromosome.
E. 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.
A. What is the likely explanation for her failure?
B. 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

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

B. 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

A. 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.)
B. 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) is false?
(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.
B. 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.

B. The two parents have a single defective copy of the gene; the fetus has two
defective copies.
C. 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.

B. 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 as restriction 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 a vector 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.
B. 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.
B. 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.
C. 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.
D. 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.)
B. 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.)
C. cDNA library. (Because cDNAs are produced from mRNAs, isolating
cDNAs would tell you which splice variants are produced in a cell.)
 D. Genomic library. (A genomic DNA fragment can contain the genes next to
your gene of interest; a cDNA will not.)
E. 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.
B. 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 in DNA. 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′
B. 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.

B. 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. gravinius genome 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
Another random document with
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must get the remaining gasoline and provisions. Our only hope of
reaching Spitzbergen lay in salvaging this fuel from the N 24. We cut
out one of the empty tanks, filled it from one of the fresh ones,
loaded it in our canoe, put the canoe on the sledge and started back.
And now we found that a large lead had opened up behind us, over
which we were barely able to get across ourselves, so we had to
leave the tank and supplies on the further side over night. The next
day the lead had closed again and Dietrichson and Omdal
succeeded in getting the gasoline over. The light sledge got slightly
broken among the rough hummocks, which was an additional
catastrophe, in view of the probability of having to walk to Greenland.
We now had 245 liters additional fuel,—1,500 liters altogether,—
or a margin of 300 liters on which to make Spitzbergen, provided we
could get off immediately.
On May 31st an inventory of our provisions showed that we had
on hand:

285 half-pound cakes of pemmican,

300 cakes of chocolate,
3 ordinary cracker-tins of oatmeal biscuits,
3 20-lb. sacks of powdered milk,
3 sausages, 12 lbs. each,
42 condensed milk tins of Horlick’s Malted Milk Tablets,
liters of kerosene for our Primus stove (we later used
25 motor fuel for cooking).

Our observations for Latitude and Longitude this day showed our
position to be 87.32 N. and 7.30 W. It meant that the whole pack had
been steadily drifting southeast since our arrival. It was at least some
consolation to know that we were slowly but surely drifting south,
where we knew there was game. How we should have liked to have
had that seal we saw the first day! We had seen no life of any
description since, neither in the water nor in the air, not even a track
on the snow to show that there was another living thing in these
latitudes but ourselves. It is a land of misery and death.
 With a view to working the longest possible time in an attempt to
get the N 25 clear, and at the same time have sufficient provisions
left with which to reach Greenland, Captain Amundsen felt that it was
necessary to cut down our daily rations to 300 grams per man, or
just one half pound per man per day. This amounted to one-half the
ration that Peary fed his dogs a day on his journey to the Pole. By
thus reducing our rations, he figured that our provisions would last
for two months longer.
Captain Amundsen now set June 15th as the date upon which a
definite decision must be arrived at. On that date something must be
done; so a vote was taken, each man having the option of either
starting on foot for Greenland on that date, or else sticking by the
plane with the hope of open water coming while watching the food
dwindle. There was much divided opinion. It seemed absurd to
consider starting out on a long tramp when right by our side was 640
horsepower lying idle, which could take us back to civilization within
eight hours. Captain Amundsen was for staying by the plane. He
said that with the coming of summer the leads would open. Riiser-
Larsen said he would start walking on June 15th. Feucht said he
would not walk a foot and that he would stick by the motors. Omdal
said he would do what the majority did, and I said I would prefer to
wait until June 14th before making a decision.
My own mind was pretty well made up that if I ever succeeded in
traveling 100 miles towards Greenland on foot, I would be doing well.
Yet sitting down by the plane and watching the last of the food go
was a thing that ran counter to my every impulse. I agreed with
Captain Amundsen that I should much prefer to “finish it” on my feet.
I think that all really believed that in our worn-out condition, carrying
thirty pounds on our backs and dragging a canvas canoe along with
which to cross open leads, none of us would be able to reach the
Greenland coast.
Most of our doubt regarding the tramp to Greenland, of course,
came from our not knowing just how far the bad country that we
were in extended. Climb up as high as we could, we were never able
to see the end of it. Whether it extended to Greenland or not was the
question, and that was what made it so hard for us to decide what
course to take.
After our evening cup of chocolate Captain Amundsen and I
generally would put on our skis and take a few turns around the ice
floe we were on before turning into our sleeping-bags. I usually
asked him on these occasions what he thought of the situation. His
reply was that things looked pretty bad, but he was quick to add that
it had always been his experience in life that when things were
blackest, there was generally light ahead.
On May 31st there was eight inches of ice in the lead on the far
side of the floe we were on. We decided to try a take-off on this new
ice. From our ice-cake down into the lead there was a six-foot drop,
so that it was necessary to construct a slip upon which to get our
plane down into the lead. We built this slip in accordance with
standard road-making principles—first heavy blocks of ice, then
filling in on top with smaller pieces, and then tiny lumps and loose
snow, on top of which we spread a layer of loose snow which froze
into a smooth surface. It took us two days to build this slip and to
level off the ice ahead for 500 meters.
At this time we had established regular nightly patrols, each man
taking his turn at patrolling all night around and around the ice floe,
on his skis, looking for open water. The mental strain during this
period was terrific, for we never knew when the cake we were on
might break beneath us.
On June 2nd, at 5 p.m., we decided that our slip was worthy a
trial. We started up the motors and taxied across the floe and down
the slip, but we had built our slip too steep, and, therefore, not
having enough speed, the plane simply sagged through the ice and
for 1,000 meters we merely plowed through it. We shut off the
motors and prepared to spend the night in the lead.
At midnight I was awakened by Captain Amundsen yelling that
the plane was being crushed. I could plainly hear the pressure
against the metal sides. We lost no time in getting everything out
onto some solid ice near by, and by working the plane up and down
permitted the incoming ice to close in beneath her from both sides. It
was a narrow escape. We had expected the plane to be crushed like
an eggshell. Riiser-Larsen’s only comment after the screwing
stopped was, “Another chapter to be added to our book!” Before
morning our first heavy fog set in. The Arctic summer was upon us.
From then on the fog hung like a pall over us and for the remainder
of our stay in the Arctic we were never free from it, although we were
always able to see the rim of the sun through it and knew that above
it the sky was clear and the sun shining brightly, but we could not
rise into it. With the coming of the fogs the temperature rose to
freezing.
We were gradually working our way over towards where the
N 24 was lying. During the day we would level off a new course, but
there was not sufficient wind in which to rise, and as usual our
heavily loaded plane broke through the thin ice,—

“Trailing like a wounded duck, working out her soul.

Felt her lift and felt her sag, betted when she’d break;
Wondered every time she raced if she’d stand the shock.”

The N 25 started leaking so badly from the pressure she

received the other night that Captain Amundsen and I were obliged
to pitch our tent on the floe upon which the N 24 was resting. We
were wondering how much more she could stand. N 24 still lay with
her nose on the ice floe, as we left her, but she had now listed
sideways, so that the tip of one wing was firmly imbedded in the
freshly frozen ice around her. During the past few days the ice had
been freezing in from both sides, forming a long, narrow lane in front
of N 24, but parts of this lane have bent into a curve. It was a narrow,
crooked passage, but Riiser-Larsen felt that it offered one more
opportunity for a take-off. He taxied N 25 forward, narrowly escaping
an accident. As he slowed up to negotiate the curve, the nose broke
through the ice with the reduced speed. The plane suddenly stopped
and lifted its tail into the air. We jumped out and hacked away the ice
until the plane settled on an even keel. We dared not remain where
we were because the main body of the pack was fast closing in upon
us from both sides.
At two o’clock the next morning we commenced work on an
extension of our previous course and continued on throughout the
day and on into the following night. It was a tremendous task, as the
ice was covered with tightly frozen lumps, old pressure-ridges of
uptilted ice cakes. Hacking away with our short-handled pocket-ax
and ice anchor was such back-breaking work that we were
compelled to work on our knees most of the time. The sweat was
rolling down my face and blurred my snow-glasses, so that I was
compelled to take them off for a couple of hours. I paid the penalty
by becoming snow-blind in one eye. Dietrichson was not so
fortunate. He was badly attacked in both eyes, and had to lie in the
tent in his sleeping-bag for two days with his eyes bandaged and
suffering acutely from the intense inflammation.
We awoke on the morning of June 5th, tired and stiff, to look
upon the level track we had so frantically labored to prepare, but saw
in its place a jumbled mass of upturned ice blocks. With the
destruction of our fourth course our position was now desperate. But
we would hang on till the 15th, when the vital decision would have to
be made as to whether or not we should abandon N 25 and make for
the Greenland coast while there were yet sufficient provisions left.
But we had come here on wings, and I know we all felt only wings
could take us back to civilization. If we could only find a floe of
sufficient area from which to take off. That was our difficulty.
In the early morning of June 6th Riiser-Larsen and Omdal
started out into the heavy fog with the grim determination of men
who find themselves in desperate straits, to search for what seemed
to us all the unattainable. We saw no more of them till evening. Out
of the fog they came, and we knew by their faces, before they
uttered a word, that they had good news. Yes, they had found a floe!
They had been searching through the fog, stumbling through the
rough country. Suddenly the sun broke through and lit up one end of
a floe, as Riiser-Larsen puts it, which became our salvation. It was a
half mile off, and it would be necessary to build a slip to get out of
the lead and bridge two ice cakes before reaching the desired floe.
 The main body of the pack was now only ten yards away.
Immediately behind the N 25 a huge ice wall was advancing slowly,
inch by inch, and fifteen minutes after we started the motors the solid
ice closed in over the spot where our plane had lain. We were saved.
We worked our way slowly up to where we meant to build the
slip, using a saw to cut out the ice ahead where it was too heavy for
the plane to break through. After six hours of steady toil we had
constructed our slip and had the plane safe up on floe No. 1. That
night of June 6th we slept well, after the extra cup of chocolate that
was allowed us to celebrate our narrow escape.
The next morning began the most stupendous task we had yet
undertaken: cutting a passage through a huge pressure-ridge,—an
ice wall fifteen feet thick which separated floe No. 1 from floe No. 2,
—and then bridging between floe No. 1 and floe No. 2 two chasms
fifteen feet wide and ten feet deep, separating the two floes from one
another. In our weakened condition this was a hard task, but we
finished it by the end of the second day. Crossing the bridges
between the floes was exciting work. The sustaining capacity of such
ice blocks as we could manage to transport and lay in the water
could not be great. The heavier blocks which we used for a
foundation were floated into place in the sea and left to freeze—as
we hoped they would—into a solid mass during the night. When the
time came, we must cross at full speed, if we were not to sink into
the sea, and then instantly stop on the other side, because we had
taken no time to level ahead, so great was our fear that the ice floes
might drift apart during the operation of bridging. We made the
passages safely and were at last upon the big floe. In order to take
advantage of the south wind, which had continued to blow ever since
the day of our landing, we leveled a course across the shortest
diameter of this cake, which offered only 300 meters for a take-off.
But before we completed our work the wind died down. Nevertheless
we made a try, but merely bumped over it and stopped just short of
the open lead ahead. Our prospects did not look good. The southerly
winds had made the deep snow soft and soggy. But it was a relief to
know that we were out of the leads, with our plane safe from the
screwing of the pack-ice.
 It was June 9th, and now began the long grind of constructing a
course upon which our final hopes must rest. If we failed there was
nothing left. My diary shows the following entry for June 10th:—“The
days go by. For the first time I am beginning to wonder if we must
make the great sacrifice for our great adventure. The future looks so
hopeless. Summer is on. The snows are getting too soft to travel
over and the leads won’t open in this continually shifting ice.”
Riiser-Larsen looked the ground over and decided that we must
remove the two and a half feet of snow right down to the solid ice
and level a track twelve meters wide and four hundred meters long.
It was a heartbreaking task to remove this wet summer snow with
only our clumsy wooden shovels. It must be thrown clear an
additional six meters to either side, so as not to interfere with the
wing stretch. After but a few shovelfuls we stood weak and panting
gazing disheartened at the labor ahead.
One problem was how to taxi our plane through the wet snow
and get it headed in the right direction. We dug down to the blue ice,
and now we were confronted with a new difficulty. The moist fog,
which came over us immediately, melted the ice as soon as it was
exposed. We found that by working our skis underneath the plane
we were able finally to get her to turn, but after splitting a pair of skis
we decided to take no more chances that way. In desperation we
now tried stamping down the snow with our feet and found that it
served the purpose admirably. By the end of our first day of
shoveling down to the blue ice, we had succeeded in clearing a
distance of only forty meters, while with the new method we were
able to make one hundred meters per day. We adopted a regular
system in stamping down this snow. Each man marked out a square
of his own, and it was up to him to stamp down every inch in this
area. We figured that at this rate we would have completed our
course in five days.
During the first day’s work we saw our first sign of animal life
since the seal popped his head up out of the lead where we first
landed. Somebody looked up from his work of shoveling snow to see
a little auk flying through the fog overhead. It came out of the north
and was headed northwest. Next day two weary geese flopped down
beside the plane. They must have thought that dark object looming
up through the fog in all that expanse of desolate white looked
friendly. They seemed an easy mark for Dietrichson, but the rich
prize was too much for his nerves and he missed. The two geese ran
over the snow a long distance as if they did not seem anxious to take
wing again. They too came from the north and disappeared into the
northwest. We wondered if there could be land in that direction. It
was an interesting speculation.
On the 14th our course was finished. Then Riiser-Larsen paced
it again and was surprised to find that instead of four hundred meters
it was five hundred. When he informed Amundsen of this fact, the
Captain was quick to remark that one million dollars couldn’t buy that
extra hundred meters from him, and we all agreed that it was
priceless. And so it proved to be.

ELLSWORTH, AMUNDSEN, LARSEN AND FEUCHT WITH THE IMPLEMENTS

WITH WHICH THEY MOVED 300 TONS OF ICE
 On the evening of the 14th, after our chocolate, and with a
southerly wind still blowing—this was a tail-wind on this course and
of no help to us—we decided to make a try. But we only bumped
along and the plane made no effort to rise. What we needed to get
off with was a speed of 100 kilometers per hour. During all our
previous attempts to take off, forty kilometers had been the best we
could do. On this trial we got up to sixty, and Riiser-Larsen was
hopeful. It was characteristic of the man to turn in his seat as we
jumped out and remark to me: “I hope you are not disappointed,
Ellsworth. We’ll do better next time.” That calm, dispassionate man
was ever the embodiment of hope.
 LINCOLN ELLSWORTH AFTER THE TRIP
That night it was my watch all night. Around and around the ice-
cake I shuffled, with my feet thrust loosely into the ski straps and a
rifle slung over my shoulder, on the alert for open water. Then, too,
we were always afraid that the ice-cake might break beneath us. It
was badly crevassed in places. Many times during that night, on my
patrol, I watched Riiser-Larsen draw himself up out of the manhole in
the top of the plane to see how the wind was blowing. During the
night the wind had shifted from the south and in the morning a light
breeze was blowing from the north. This was the second time during
our twenty-five days in the ice that the wind had blown from the
north. We had landed with a north wind—but were we to get away
with a north wind? That was the question. The temperature during
the night was -1.5° c. and the snow surface was crisp and hard in the
morning. We now were forced to dump everything that we could
spare. We left one of our canvas canoes, rifles, cameras, field-
glasses; we even discarded sealskin parkas and heavy ski-boots,
replacing them with moccasins. All we dare retain was half of our
provisions, one canvas canoe, a shotgun and one hundred rounds of
ammunition.
Then we all climbed into the plane and Riiser-Larsen started up.
Dietrichson was to navigate. The plane began to move! After
bumping for four hundred meters the plane actually lifted in the last
hundred meters. When I could feel the plane lifting beneath me I was
happy, but we had had so many cruel disappointments during the
past twenty-five days that our minds were in a state where we could
feel neither great elation nor great suffering. Captain Amundsen had
taken his seat beside Riiser-Larsen, and I got into the tail.
For two hours we had to fly through the thick fog, being unable
either to get above or below it. During all this time we flew slowly,
with a magnetic compass, a thing heretofore considered to be an
impossibility in the Arctic. Dietrichson dropped down for drift
observations as frequently as possible. The fogs hung so low that we
were compelled to fly close to the ice, at one time skimming over it at
a height of but one hundred feet. Finally we were able to rise above
the fog and were again able to use our “Sun Compass.”
Southward we flew! Homeward we flew! One hour—two hours—
four, six hours. Then Feucht yelled back to me in the tail, “Land!” I
replied, “Spitzbergen?”—“No Spitzbergen, no Spitzbergen!” yells
back Feucht in his broken English. So I made up my mind that it
must be Franz-Josefs-Land. Anyway, it was land, and that meant
everything!
 Our rationing regulations were now off, and we all started to
munch chocolate and biscuits.
For an hour Riiser-Larsen had noticed that the stabilization
rudders were becoming more and more difficult to operate. Finally
they failed to work completely and we were forced down on the open
sea, just after having safely passed the edge of the Polar pack. We
landed in the sea, after flying just eight hours, with barely ninety liters
of gasoline in our tanks, one half hour’s fuel supply. The sea was
rough, and we were forced to go below and cover up the man-holes,
for the waves broke over the plane.
I had eaten seven cakes of chocolate when Feucht yelled, “Land
ahead!” But I was now desperately ill and cared little what land it was
so long as it was just land. After thirty-five minutes of taxi-ing through
the rough sea, we reached the coast.
In we came—“in the wash of the wind-whipped tide.”

“Overloaded, undermanned, meant to founder, we

Euchred God Almighty’s storm, bluffed the Eternal Sea!”

How good the solid land looked! We threw ourselves down on a

large rock, face upward to the sun, till we remembered that we had
better take an observation and know for sure where we were.
It seems remarkable, when I think about it now, how many
narrow escapes we really had. Again and again it looked like either
life or death, but something always just turned up to help us out.
Captain Amundsen’s answer was, “You can call it luck if you want,
but I don’t believe it.”
We got out our sextant and found that one of our position lines
cut through the latitude of Spitzbergen. While we were waiting to
take our second observation for an intersection, three hours later,
some one yelled, “A sail!”—and there, heading out to sea, was a little
sealer. We shouted after them and put up our flag, but they did not
see us, and so we jumped into our plane and with what fuel we had
left taxied out to them. They were after a wounded walrus that they
had shot seven times in the head, otherwise they would have been
gone long before. They were overjoyed to see us. We tried to tow
the plane, but there was too much headwind, so we beached her in
Brandy Bay, North Cape, North-East-Land, Spitzbergen, one
hundred miles east of our starting point at King’s Bay.
We slept continuously during the three days in the sealer, only
waking to devour the delicious seal meat steaks smothered in onions
and the eider-duck egg omelets prepared for us.
The homage that was accorded us upon our return to civilization
will ever remain the most cherished memory of our trip. We took
steamer from King’s Bay for Norway on June 25th, after putting our
plane on board, and nine days later arrived at Horten, the Norwegian
Naval Base, not far from Oslo.
On July 5th, with the stage all set, we flew N 25 into Oslo. It was
difficult to realize that we were in the same plane that had so
recently been battling in the midst of the Arctic ice. Good old N 25!
We dropped down into the Fjord amid a pandemonium of frantically
shrieking river craft and taxied on through the wildly waving and
cheering throngs, past thirteen fully manned British battleships, and
as I listened to the booming of the salute from the Fort and looked
ahead at the great silent expectant mass of humanity that waited to
greet us, I was overcome with emotion and the tears rolled down my
face. At that moment I felt paid in full for all that I had gone through.
 Part III
THE NAVIGATOR’S TASK

By Lieut. Hjalmar Riiser-Larsen

 THE NAVIGATOR’S TASK
“The Air Club has fixed up contracts with the publishers of
several countries for a book of at least seventy thousand words.
Therefore you must write several thousand. Come and stay with me so
that you can work in peace.” Such were Amundsen’s orders
immediately we stepped ashore in Oslo.
The manuscript of the entire 70,000 words should be delivered by
the 10th of August. In view of the big task of arranging charts and
pictorial matter, there would not be much time to spare, so we had to
get down to it as quickly as possible.
There were also many other things to be done in the meantime.
The expedition’s cinema film had to be cut and run off—run off again,
and recut, as the cinema owners wanted to “fit in” three shows daily at
5 p.m., 7 p.m. and 9 p.m. It would take fifteen minutes to clear the
theater, to ventilate it, and let the next audience get seated, therefore
the run of the film must not exceed one hour and three-quarters. At first
it took two and a half hours even without the caption lines. Berge
continued cutting, and the film got shorter daily. The worst task was to
arrange the sequence of the scenes. They were far from being in
chronological order, but after a time it began to present a better picture
of the expedition’s course—a picture which gave a calm
straightforward story—a calendar of daily episodes. The caption lines,
too, required writing, as they could not create themselves.
While we were busy with all this work we had also to attend to the
returning of the expedition’s unused stores to the suppliers. Much of
this had been bought conditionally so that we could return everything
we had not used. The ever-helpful Omdal, who never seemed to have
enough to do, took charge of this part of the work. The more I left to
him the better pleased he was. I asked him often in those days if he
would not like to be released to go home. “So long as I can be of use
to the expedition there is no hurry,” was his reply. At last on August 1st
he set off to his home in Kristiansand, which he had been longing for.
But I am sure he would have been quite happy about it if I, even then,
had said to him that he could not get off.
That’s the sort of man Omdal is!
In the meantime the post-bag was filled with requests for
information regarding the instruments and other equipment we had
used on the trip. Lantern slides for lectures had to be got ready and
advertising matter sent to our business managers.
Thus the days passed and the dreadful 10th of August got nearer,
so threateningly that at last to-day I had to take the bull by the horns
and go to Amundsen for further particulars.
Now I sit here experiencing the same feelings as in my
schooldays, when I used to put off writing Norwegian composition so
long that I had to do it during the games’ interval.
The first thing I shall render an account of is—

Why We Chose the Dornier-Wal Type

As the expense of using airships was prohibitive, we could only
consider the employment of flying-machines. The choice of type
depended upon the idea we could form of the landing conditions
among the ice. The highest authority in the “world of polar-exploration,”
and many others who had hunted and fished Greenland’s east coast
for many years, all contended that there would be many suitable
landing places on the numerous big flat ice-floes, and also that we
should find water-lanes where the seaplanes could land. Some voices
were raised against these contentions but as they were only “voices”
we didn’t lay much weight on their opinion, though, as was proved
later, these latter were right,—but that is a different matter. We
regarded it at that time as certain that we should find plenty of big-
enough landing places. Accordingly we based our plans, on making an
expedition which could land to carry out observations and which would
be of considerably more value than an exploration expedition which
would only fly over the ice. An expedition thus equipped would be
safer, as a forced landing might have to be made at any time. We
decided therefore to use two machines, which would allow the
expedition to continue with one plane if the other had to make a forced
landing on account of irreparable engine trouble. In a forced landing,
too, the machine might be damaged, as there would not be the same
opportunity to find a suitable landing-place, as in the case of a
voluntary landing. It is also certain that it would double the chances of
reaching the goal ahead to set off with two machines rather than with
only one,—always, of course, banking on the probability of good
opportunities for landing being found.
On the other hand, if such opportunities for landing did not offer,
the use of two machines would halve the chances of success, as the
risk of engine trouble where two are concerned is naturally double
what it would be if only one machine were employed. The
arrangements, therefore, were, that both seaplanes’ crews should
keep together.
When we made our forced landing on the ice we were convinced
that there were no suitable landing places to be found up there, and in
consequence we decided that we would only use one seaplane for the
homeward flight. We spent some days at first getting both machines
ready for a start, because starting conditions were so difficult, that it
was an advantage to hold one machine in reserve in case the other
should get damaged in attempting to get away; but we discovered that
it would take the six of us to tackle the work in each case, so we chose
the machine which was in the best condition and therefore safest for
the homeward flight.
The reason why I have gone into so many details regarding this
side of our plans and our conduct of the expedition, is that we have
been publicly criticized “because we flew with two machines over a
stretch of territory that offered no landing possibilities, and thus we
took a double risk of engine trouble.” This is putting a wrong
construction on it. The reason that we continued our northward flight
after we had reached 83°, and, being free of the fog, saw that there
were only bad chances of landing, was because we naturally had a
goal to reach and we thought conditions would improve further north.
 Back to the choice of type! In clear weather, especially in sunshine,
one can see from overhead unevennesses on a place, even when one
cannot be certain that all is clear, as the snow may have “covered-in”
some banks of drift-ice. If the weather is hazy, even a voluntary landing
is a matter of chance, for it is impossible to see even the biggest
undulations in the snow.
There are three kinds of under-carriages to choose from—skis,
floats, or flying boats. If one has chosen skis or floats, and should
strike against a projection with them, tearing off the under part, the
machine will turn over, and a continuation of the flight with the same
machine will be impossible.
A flying boat on the contrary has fewer sidewise projections (which
means that it would be less exposed to the danger of being damaged
by unevennesses) and, furthermore, it will not capsize so quickly. If
one has also ordered it of durable aluminium it will afford the uttermost
safety. Where a big strain would tear the bottom of a wooden boat
(making reparation impossible or at least very difficult in the conditions
prevailing up there) under the same strain durable aluminium would
only suffer some denting which could be straightened out again if it
proved sufficient to hinder progress. Aluminium does not break easily.
There were also other reasons that counted in making the choice
of a type of boat. Should one have the intention of rising from deep
snow, the burden (of the boat or the machine’s under-carriage) lying on
the snow must not be greater than a certain weight on the flat, namely,
600 kilograms per square meter.
As our machine would average a weight of six tons it was a simple
matter to calculate that we must lie on an area of at least ten square
yards, and even then it would be bearing the maximum weight. Thus, a
ski-attachment would be particularly heavy, and the floats would have
to be unnecessarily large if the bottom’s lines were to satisfy the
seamanlike desire “to rise from the water.”
After making these calculations we were never in doubt, but
decided that we should choose a flying boat built of durable aluminium.
With regard to ski-machines, we should gain a further advantage in
being able to land in, or rise from, possible water-lanes, while in a
wooden boat a collision with ice in the water-lanes presented a smaller
risk.
The point now was to find the right dur-aluminium boat as Dornier
was not the only builder of such boats. If one wishes to rise from loose
snow it is not only the flat-weight which counts, but it is distinctly
necessary that the bottom lines of the boat must be so designed that
no power shall be lost by the unnecessary pushing aside of snow
when gliding forward. There was thus only one type of boat which
satisfied our demands and that was Dornier-Wal.
Dornier-Wal has furthermore a distinct advantage which we first
became aware of up in the ice regions. It has not got wing-floats to
afford the necessary stability on the water, but for this purpose—as
shown in the illustration—has attached at each side of the propeller a
big “flyndre.” During our start from the water-lane the boat sank
through the new ice and a part of the weight fell on the “flyndres.” In
this way we were able to go to the assistance of N 25 in the capacity of
an ice-breaker and help it out of several critical situations. Had there
been floats on the wings, too great a weight would naturally have fallen
on these, and we should have been unable to avoid damage.
From the above it can be seen that there was nothing else for it but
to choose Dornier-Wal for our flight even though it might have been
handicapped by certain failings. I cannot at present mention one single
failing, but it had numerous advantages. The best of these in my
estimation is the fact that it is fitted with Rolls-Royce twin-engines
(Eagle IX). I should scarcely have agreed to undertake a flight of this
kind without a Rolls-Royce. It is not a matter of “chance” that made
Dornier fix Rolls-Royce engines to his Wal type: it would have been
bad policy to put anything but the very best engines in a flying-machine
of the “Wal’s” high standard.
 CAPTAIN ROALD AMUNDSEN, JUST BEFORE THE TAKE-OFF FROM
SPITZBERGEN
It will also be noticed from the illustrations that the “Wal” is fitted
with two engines and that these are placed immediately behind each
other—one pulls and one pushes—thus the aft propeller turns
contrariwise to the fore propeller, each rotating in its own way. The
wonderfully effective qualities which are thus attained, in conjunction
with the suitable lines and ingenious “wing-frontage,” make it possible
for a weight equal to that of the machine itself to be lifted. As we
started from King’s Bay we had a load of 3,100 kilograms, while the
“Wal” itself weighs 3,300 kilograms—yet the machine rose with such
ease from the ice that I am sure we could have taken an additional 200
kilograms on board. This very fact seemed most apparent during the
hardships we underwent in the ice regions, when we thought longingly
of how many boxes of biscuits or how much tobacco we might safely
have brought with us. We always closed these ruminations by a
unanimous agreement that it was a good thing we had carried no more