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4: A Number of Pathways Repair Changes in DNA
Transposable genetic elements
1 Pairing by looping and
crossing over between
in the same direction…
2 …leads to deletion.
3 Pairing by bending and
crossing over between
two transposable elements
oriented in opposite directions…
4 …leads to an inversion.
5 Misalignment and unequal
exchange between transposable elements located
on sister chromatids…
6 …leads to one chromosome with a deletion…
remains remarkably low, thanks to the efficiency with which
DNA is repaired. Less than one in a thousand DNA lesions is
estimated to become a mutation; all the others are corrected.
There are a number of complex pathways for repairing
DNA, but several general statements can be made about
DNA repair. First, most DNA-repair mechanisms require
two nucleotide strands of DNA because most replace whole
nucleotides, and a template strand is needed to specify the
A second general feature of DNA repair is redundancy,
meaning that many types of DNA damage can be corrected
by more than one pathway of repair. This redundancy testifies to the extreme importance of DNA repair to the survival
of the cell: it ensures that almost all mistakes are corrected.
If a mistake escapes one repair system, it’s likely to be
repaired by another system.
One type of DNA repair is mismatch repair, which corrects incorrectly inserted nucleotides that arise in the course
of replication (Figure 13.27). Incorrectly paired bases distort
the three-dimensional structure of DNA, and mismatchrepair enzymes detect these distortions. A complex of mismatch-repair enzymes cuts out the distorted section of the
newly synthesized strand and fills the gap with new
nucleotides, by using the original DNA strand as a template.
The template strand is recognized by the presence of methyl
groups on special sequences of the old strand.
Another type of DNA-repair mechanism is direct repair,
which does not replace altered nucleotides but, instead, changes
them back into their original (correct) structures. For example,
direct repair corrects O6-methylguanine, an alkylation product
of guanine that pairs with adenine, producing G·C S T·A
transversions. An enzyme called O6-methylguanine-DNA
methyltransferase removes the methyl group from O6-methylguanine, restoring the base to guanine (Figure 13.28).
In base-excision repair, a modified base is first excised
and then the entire nucleotide is replaced. The excision of
modified bases is catalyzed by a set of enzymes called DNA
glycosylases, each of which recognizes and removes a specific
type of modified base. Uracil glycosylase, for example, recognizes and removes uracil produced by the deamination of
cytosine. Other glycosylases recognize hypoxanthine, 3methyladenine, 7-methylguanine, and other modified bases.
A final repair pathway that we’ll consider is nucleotideexcision repair, which removes bulky DNA lesions (such as
pyrimidine dimers) that distort the double helix. Nucleotideexcision repair is quite versatile and can repair many different
types of DNA damage. It is found in cells of all organisms from
bacteria to humans and is among the most important of all
7 …and one chromosome
with a duplication.
13.26 Chromosomal rearrangements can be generated by
A number of pathways exist for the repair of DNA. Most require
two nucleotide strands because a template strand is needed to
specify the correct base sequence.
Gene Mutations, Transposable Elements, and DNA Repair
1 In DNA replication, a
mismatched base was
added to the new strand.
2 Methylation at GATC sequences allows old and newly synthesized nucleotide strands
to be differentiated: a lag in methylation means that, immediately after replication,
the old strand will be methylated but the new strand will not.
Old (template) DNA
13.27 Many incorrectly inserted nucleotides that escape
proofreading are corrected by mismatch repair.
3 The mismatch-repair complex brings the
mismatched bases close to the methylated
GATC sequence, and the new strand is identified.
(may mispair with adenine)
13.28 Direct repair changes nucleotides back into their
4 Exonucleases remove nucleotides
on the new strand between the
GATC sequence and the mismatch.
Genetic Diseases and Faulty
Several human diseases are connected to defects in DNA
repair. These diseases are often associated with high
incidences of specific cancers, because defects in DNA
repair lead to increased rates of mutation. This concept
is discussed further in Chapter 15.
Among the best studied of the human DNA-repair
diseases is xeroderma pigmentosum (Figure 13.29), a
5 DNA polymerase then replaces the nucleotides,
correcting the mismatch, and DNA ligase seals
the nick in the sugar–phosphate backbone.
13.29 Xeroderma pigmentosum results from defects in
DNA repair. The disease is characterized by frecklelike spots on the
skin (shown here) and predisposition to skin cancer. [Ken Greer/Visuals
rare autosomal recessive condition that includes abnormal
skin pigmentation and acute sensitivity to sunlight. Persons
who have this disease also have a strong predisposition to
skin cancer, with an incidence ranging from 1000 to 2000
times that found in unaffected people.
Sunlight includes a strong UV component; so exposure
to sunlight produces pyrimidine dimers in the DNA of skin
cells. Most pyrimidine dimers in humans can be corrected by
nucleotide-excision repair. However, the cells of most people
with xeroderma pigmentosum are defective in nucleotideexcision repair, and many of their pyrimidine dimers remain
uncorrected and may lead to cancer.
Genetic diseases associated with defects in DNA-repair systems
Frecklelike spots on skin, sensitivity to sunlight,
predisposition to skin cancer
Defects in nucleotide-excision repair
Dwarfism, sensitivity to sunlight, premature
aging, deafness, mental retardation
Defects in nucleotide-excision repair
Brittle hair, skin abnormalities, short stature, immature
sexual development, characteristic facial features
Defects in nucleotide-excision repair
Predisposition to colon cancer
Defects in mismatch repair
Increased skin pigmentation, abnormalities of skeleton,
heart, and kidneys, predisposition to leukemia
Possibly defects in the repair
of interstrand cross-links
Defective muscle coordination, dilation of blood
vessels in skin and eyes, immune deficiencies,
sensitivity to ionizing radiation, predisposition to cancer
Defects in DNA-damage detection
Predisposition to cancer in many different tissues
Defects in DNA-damage response
Another genetic disease caused by faulty DNA repair is
an inherited form of colon cancer called hereditary nonpolyposis colon cancer (HNPCC). This cancer is one of the
most common hereditary cancers, accounting for about
15% of colon cancers. Research findings indicate that
HNPCC arises from mutations in the proteins that carry
out mismatch repair. Some additional genetic diseases
associated with defective DNA repair are summarized in
Defects in DNA repair are the underlying cause of several genetic
diseases. Many of these diseases are characterized by a predisposition to cancer.
✔ Concept Check 7
Why are defects in DNA repair often associated with increases in
• Mutations are heritable changes in genetic information.
sequence but does not change the functioning of the protein.
A suppressor mutation reverses the effect of a mutation at a
different site and may be intragenic (within the same gene as
the original mutation) or intergenic (within a different gene).
Somatic mutations occur in somatic cells; germ-line
mutations occur in cells that give rise to gametes.
• The simplest type of mutation is a base substitution, a change
in a single base pair of DNA. Transitions are base substitutions
in which purines are replaced by purines or pyrimidines are
replaced by pyrimidines. Transversions are base substitutions
in which a purine replaces a pyrimidine or a pyrimidine
replaces a purine.
Insertions are the addition of nucleotides, and deletions are
the removal of nucleotides; these mutations often change the
reading frame of the gene.
Expanding trinucleotide repeats are mutations in which the
number of copies of a trinucleotide increases through time;
they are responsible for several human genetic diseases.
A missense mutation alters the coding sequence so that one
amino acid substitutes for another. A nonsense mutation
changes a codon that specifies an amino acid into a
termination codon. A silent mutation produces a synonymous
codon that specifies the same amino acid as does the original
sequence, whereas a neutral mutation alters the amino acid
• Mutation rate is the frequency with which a particular
mutation arises in a population. Mutation rates are influenced
by both genetic and environmental factors.
Some mutations occur spontaneously. These mutations
include the mispairing of bases in replication and spontaneous
depurination and deamination.
Insertions and deletions can arise from strand slippage in
replication or from unequal crossing over.
Base analogs can become incorporated into DNA in the course
of replication and pair with the wrong base in subsequent
replication events. Alkylating agents and hydroxylamine
modify the chemical structure of bases and lead to mutations.
Intercalating agents insert into the DNA molecule and cause
single-nucleotide additions and deletions.
Ionizing radiation is mutagenic, altering base structures and
breaking phosphodiester bonds. Ultraviolet light produces
pyrimidine dimers, which block replication.
Gene Mutations, Transposable Elements, and DNA Repair
• The Ames tests uses bacteria to assess the mutagenic potential
of chemical substances.
Transposable elements are mobile DNA sequences that insert
into many locations within a genome and often cause mutations
and DNA rearrangements.
Most transposable elements have two common characteristics:
terminal inverted repeats and the generation of short direct
repeats in DNA at the point of insertion.
Transposition may take place through a DNA molecule or
through the production of an RNA molecule that is then reverse
transcribed into DNA. Transposition may be replicative, in
which the transposable element is copied and the copy
moves to a new site, or nonreplicative, in which the
transposable element excises from the old site and moves to a
Transposons are mutagenic and have played an important role
in genome evolution.
Damage to DNA is often corrected by DNA-repair mechanisms.
Most repair pathways require two strands of DNA and exhibit
some overlap in the types of damage repaired.
Defects in DNA repair are the underlying cause of several
mutation (p. 322)
somatic mutation (p. 322)
germ-line mutation (p. 322)
gene mutation (p. 323)
base substitution (p. 323)
transition (p. 323)
transversion (p. 323)
insertion (p. 323)
deletion (p. 323)
frameshift mutation (p. 323)
in-frame insertion (p. 324)
in-frame deletion (p. 324)
expanding trinucleotide repeat (p. 324)
forward mutation (p. 325)
reverse mutation (reversion) (p. 325)
missense mutation (p. 326)
nonsense mutation (p. 326)
silent mutation (p. 326)
neutral mutation (p. 326)
loss-of-function mutation (p. 326)
gain-of-function mutation (p. 326)
conditional mutation (p. 326)
lethal mutation (p. 326)
suppressor mutation (p. 326)
intragenic suppressor mutation (p. 327)
intergenic suppressor mutation (p. 327)
mutation rate (p. 328)
spontaneous mutation (p. 329)
induced mutation (p. 329)
incorporated error (p. 330)
replicated error (p. 330)
strand slippage (p. 330)
unequal crossing over (p. 331)
depurination (p. 332)
deamination (p. 332)
mutagen (p. 333)
base analog (p. 333)
intercalating agent (p. 334)
pyrimidine dimer (p. 336)
SOS system (p. 336)
Ames test (p. 336)
flanking direct repeat (p. 337)
terminal inverted repeat (p. 337)
transposition (p. 338)
DNA transposon (p. 338)
retrotransposon (p. 338)
replicative transposition (p. 338)
nonreplicative transposition (p. 338)
mismatch repair (p. 340)
direct repair (p. 340)
base-excision repair (p. 340)
nucleotide-excision repair (p. 340)
Answers to Concept Checks
1. Studying mutations that disrupt normal processes often leads
to the identification of genes that normally play a role in the
process and can help in understanding the molecular details of a
3. A reverse mutation restores the original phenotype by
changing the DNA sequence back to the wild type. A suppressor
mutation restores the phenotype by causing an additional
change in the DNA at a site that is different from that of the
4. An incorporated error is due to a change that takes place in
DNA. This change may be corrected by a DNA-repair pathway.
However, if the error has been replicated, it is permanent and
cannot be detected by repair pathways.
6. In transposition, staggered cuts are made in DNA and the
transposable element inserts into the cut. Later, replication of the
single-stranded pieces of DNA creates short flanking repeats on
either side of the inserted transposable element.
7. Changes in DNA structure do not undergo repair in people
with defects in DNA-repair mechanisms. Consequently, increased
numbers of mutations occur at all genes, including those that
predispose to cancer. This observation indicates that cancer arises
from mutations in DNA.
1. A codon that specifies the amino acid Asp undergoes a singlebase substitution that yields a codon that specifies Ala. Refer to
the genetic code in Figure 11.5 and give all possible DNA
sequences for the original and the mutated codon. Is the mutation
a transition or a transversion?
There are two possible RNA codons for Asp: GAU and GAC. The
DNA sequences that encode these codons will be complementary
to the RNA codons: CTA and CTG. There are four possible RNA
codons for Ala: GCU, GCC, GCA, and GCG, which correspond to
DNA sequences CGA, CGG, CGT, and CGC. If we organize the
original and the mutated sequences as shown in the following
table, the types of mutations that may have occurred can be
Possible original sequence
Possible mutated sequence
If the mutation is confined to a single-base substitution, then the
only mutations possible are that CTA mutated to CGA or that
CTG mutated to CGG. In both, there is a T S G transversion in
the middle nucleotide of the codon.
*7. How do base analogs lead to mutations?
*1. What is the difference between a transition and a transversion?
Which type of base substitution is usually more common?
8. What types of mutations are produced by ionizing and UV
*2. Briefly describe expanding trinucleotide repeats.
9. What is the purpose of the Ames test? How are his– bacteria
used in this test?
3. What is the difference between a missense mutation and a
nonsense mutation? A silent mutation and a neutral
4. Briefly describe two different ways that intragenic
suppressors may reverse the effects of mutations.
*5. What causes errors in DNA replication?
6. How do insertions and deletions arise?
*10. What general characteristics are found in many transposable
elements? Describe the differences between replicative and
*11. What is a retrotransposon and how does it move?
*12. List at least three different types of DNA repair and briefly
explain how each is carried out.
Application Questions and Problems
*13. A codon that specifies the amino acid Gly undergoes a
single-base substitution to become a nonsense mutation.
In accord with the genetic code given in Figure 11.5, is this
mutation a transition or a transversion? At which position
of the codon does the mutation occur?
*14. a. If a single transition occurs in a codon that specifies Phe,
what amino acids can be specified by the mutated
b. If a single transversion occurs in a codon that specifies
Phe, what amino acids can be specified by the mutated
c. If a single transition occurs in a codon that specifies
Leu, what amino acids can be specified by the mutated
d. If a single transversion occurs in a codon that specifies
Leu, what amino acids can be specified by the mutated
15. Hemoglobin is a complex protein that contains four
polypeptide chains. The normal hemoglobin found in
adults—called adult hemoglobin—consists of two alpha and
two beta polypeptide chains, which are encoded by different
loci. Sickle-cell hemoglobin, which causes sickle-cell anemia,
arises from a mutation in the beta chain of adult hemoglobin.
Adult hemoglobin and sickle-cell hemoglobin differ in a
single amino acid: the sixth amino acid from one end in
adult hemoglobin is glutamic acid, whereas sickle-cell
hemoglobin has valine at this position. After consulting the
genetic code provided in Figure 11.5, indicate the type and
location of the mutation that gave rise to sickle-cell anemia.
*16. The following nucleotide sequence is found on the template strand of DNA. First, determine the amino acids of
the protein encoded by this sequence by using the genetic
code provided in Figure 11.5. Then, give the altered amino
acid sequence of the protein that will be found in each of
the following mutations:
3Ј–TAC TGG CCG TTA GTT GAT ATA ACT–5Ј
a. Mutant 1:
b. Mutant 2:
c. Mutant 3:
A transition at nucleotide 11
A transition at nucleotide 13
A one-nucleotide deletion at nucleotide 7
Gene Mutations, Transposable Elements, and DNA Repair
d. Mutant 4:
e. Mutant 5:
f. Mutant 6:
A T S A transversion at nucleotide 15
An addition of TGG after nucleotide 6
A transition at nucleotide 9
17. A polypeptide has the following amino acid sequence:
The amino acid sequence of this polypeptide was determined in a series of mutants listed in parts a through e. For
each mutant, indicate the type of mutation that occurred in
the DNA (single-base substitution, insertion, deletion) and
the phenotypic effect of the mutation (nonsense mutation,
missense mutation, frameshift, etc.).
a. Mutant 1:
b. Mutant 2:
c. Mutant 3:
d. Mutant 4:
e. Mutant 5:
*18. A gene encodes a protein with the following amino acid
A mutation occurs in the gene. The mutant protein has the
following amino acid sequence:
An intragenic suppressor restores the amino acid sequence
to that of the original protein:
Give at least one example of base changes that could
produce the original mutation and the intragenic
suppressor. (Consult the genetic code in Figure 11.5.)
19. XG syndrome is a rare genetic disease that is due to an
autosomal dominant gene. A complete census of a small
European country reveals that 77,536 babies were born in
2004, of whom 3 had XG syndrome. In the same year, this
country had a population of 5,964,321 people, and there
were 35 living persons with XG syndrome. What is the
mutation rate of XG syndrome in this country?
*20. The following nucleotide sequence is found in a short
stretch of DNA:
If this sequence is treated with hydroxylamine, what
sequences will result after replication?
21. The following nucleotide sequence is found in a short
stretch of DNA:
a. Give all the mutant sequences that may result from
spontaneous depurination in this stretch of DNA.
b. Give all the mutant sequences that may result from
spontaneous deamination in this stretch of DNA.
22. Mary Alexander studied the effects of radiation on mutation
rates in the sperm of Drosophila melanogaster. She irradiated
Drosophila larvae with either 3000 roentgens (r) or 3975 r,
collected the adult males that developed from irradiated
larvae, mated them with unirradiated females, and then
counted the number of mutant F1 flies produced by each
male. All mutant flies that appeared were used in subsequent
crosses to determine if their mutant phenotypes were
genetic. She obtained the following results (M. L. Alexander.
1954. Genetics 39:409–428):
a genetic mutation
Control (0 r)
Irradiated (3000 r)
Irradiated (3975 r)
a. Calculate the mutation rates of the control group and
the two groups of irradiated flies.
b. On the basis of these data, do you think radiation has
any effect on mutation? Explain your answer.
*23. A particular transposable element generates flanking direct
repeats that are 4 bp long. Give the sequence that will be
found on both sides of the transposable element if this
transposable element inserts at the position indicated on
each of the following sequences.
*24. What factor do you think determines the length of the
flanking direct repeats that are produced in transposition?
25. Zidovudine (AZT) is a drug used to treat patients with
AIDS. AZT works by blocking the reverse-transcriptase
enzyme used by the human immunodeficiency virus (HIV),
the causative agent of AIDS. Do you expect that AZT would
have any effect on transposable elements? If so, what type of
transposable elements would be affected and what would be
the most likely effect?
26. A transposable element is found to encode a reverse-transcriptase enzyme. On the basis of this information, what
conclusions can you make about the likely method of
transposition of this element?
*27. A plant breeder wants to isolate mutants in tomatoes that
are defective in DNA repair. However, this breeder does not
have the expertise or equipment to study enzymes in DNArepair systems. How can the breeder identify tomato plants
that are deficient in DNA repair? What are the traits to
28. Robert Bost and Richard Cribbs studied a strain of E. coli
DATA (araB14) that possessed a nonsense mutation in the
structural gene that encodes L-ribulokinase, an enzyme that
allows the bacteria to metabolize the sugar arabinose (R.
Bost and R. Cribbs. 1969. Genetics 62:1–8). From the araB14
strain, they isolated some bacteria that possessed mutations
that caused them to revert back to the wild type. Genetic
analysis of these revertants showed that they possessed two
different suppressor mutations. One suppressor mutation
(R1) was linked to the original mutation in the L-ribulokinase
and probably occurred at the same locus. By itself, this
mutation allowed the production of L-ribulokinase, but the
enzyme was not as effective in metabolizing arabinose as the
enzyme encoded by the wild-type allele. The second
suppressor mutation (SuB) was not linked to the original
mutation. In conjunction with the R1 mutation, SuB
allowed the production of L-ribulokinase, but SuB by itself
was not able to suppress the original mutation.
a. On the basis of this information, are the R1 and SuB
mutations intragenic suppressors or intergenic
suppressors? Explain your reasoning.
b. Propose an explanation for how R1 and SuB restore the
ability of araB14 to metabolize arabinose and why SuB is
able to more fully restore the ability to metabolize
29. Achondroplasia is an autosomal dominant disorder
characterized by disproportionate short stature—the legs
and arms are short compared with the head and trunk. The
disorder is due to a base substitution in the gene, located on
the short arm of chromosome 4, for fibroblast-growthfactor receptor 3 (FGFR3).
Although achondroplasia is clearly inherited as an
autosomal dominant trait, more than 80% of the people who
have achondroplasia are born to parents with normal stature.
This high percentage indicates that most cases are caused by
newly arising mutations; these cases (not inherited from an
affected parent) are referred to as sporadic. Findings from
molecular studies have demonstrated that sporadic cases of
achondroplasia are almost always caused by mutations
inherited from the father (paternal mutations). In addition,
the occurrence of achondroplasia is higher among older
fathers; indeed, approximately 50% of children with
achondroplasia are born to fathers older than 35 years of age.
There is no association with maternal age. The mutation rate
for achondroplasia (about 4 ϫ 10–5 mutations per gamete) is
high compared with those for other genetic disorders.
Explain why most spontaneous mutations for
achondroplasia are paternal in origin and why the occurrence of achondroplasia is higher among older fathers.
30. Mutations ochre and amber are two types of nonsense
mutations. Before the genetic code was worked out,
Sydney Brenner, Anthony O. Stretton, and Samuel Kaplan
applied different types of mutagens to bacteriophages in
an attempt to determine the bases present in the codons
responsible for amber and ochre mutations. They knew
that ochre and amber mutants were suppressed by
different types of mutations, demonstrating that each is a
different termination codon. They obtained the following
1. A single-base substitution could convert an ochre
mutation into an amber mutation.
2. Hydroxylamine induced both ochre and amber
mutations in wild-type phages.
3. 2-Aminopurine caused ochre to mutate to amber.
4. Hydroxylamine did not cause ochre to mutate to amber.
These data do not allow the complete nucleotide sequence
of the amber and ochre codons to be worked out, but they
do provide some information about the bases found in the
a. What conclusions about the bases found in the codons
of amber and ochre mutations can be made from these
b. Of the three nonsense codons (UAA, UAG, UGA), which
represents the ochre mutation?
A family of three who have
achondroplasia. [Gail Burton/AP.]
31. To determine whether radiation associated with the atomic
bombings of Hiroshima and Nagasaki produced recessive
germ-line mutations, scientists examined the sex ratio of the
children of the survivors of the blasts. Can you explain why
an increase in germ-line mutations might be expected to
alter the sex ratio?
Feeding the Future
Population of the World
n the year 2000, the world’s population reached 6 billion.
Because the human population has exhibited exponential
growth, the pace of increase in the number of people is ever
quickening: more than 100,000 years were required for
humans to reach 1 billion in number (in 1830); only
another 100 years were required for the population to double to 2 billion (in 1930); and only 45 years were required
for it to double again (in 1975) to 4 billion. The United
Nations projects that the world population will reach somewhere between 7.3 billion and 10.7 billion by the year 2050
and, because the tendency is for people to have smaller families, will eventually level off or even drop in the last part of
the twenty-first century.
How will we feed the additional billions of people that
will populate the planet Earth 40 years from now? So far, we
have been able to sustain the tremendous increase in human
numbers because advances in agriculture have greatly
increased worldwide food production. Much of this
increase was between 1950 and 1980 through the Green
Revolution, which utilized traditional techniques of plant
breeding and genetics to develop new varieties of corn,
wheat, and rice. For example, worldwide grain production
increased 260% between 1950 and 1990; worldwide cereal
production increased from 275 kg/person in the 1950s to
370 kg/person in the 1980s, during a time in which human
population almost doubled. Thus, even though human
Genetic engineering is being used to modify rice and other crops
numbers have increased tremendously in the past 60 years,
to grow in environments that are currently unable to support
the world’s farmers today produce more food per person
agriculture. [Friedrich Stark/Peter Arnold.]
than they did in 1950.
What about the next 40 years, when there will be
between 1 billion and 5 billion more people to feed? Most of the world’s cultivatable land
is already in use, and increases in crop yield achievable through traditional breeding and
genetics have leveled off. Many experts propose that feeding the future population of the
world can be achieved only through the application of genetic engineering to bring about
a “second” Green Revolution. Already, genetic engineering has been used to produce crops
that are resistant to pests, disease, and herbicides. Genetically engineered (often called
genetically modified) crops are today cultivated on more than 125 million hectares
(1 hectare ϭ 2.471 acres) of land worldwide; in 2008, 80% of corn, 92% of soybeans, and
86% of cotton grown in the United States was genetically engineered.
The potential of genetic engineering to help feed the future world population must be
weighed against concerns about the widespread use of genetically modified crops. Although
recent scientific reviews contain little evidence of risk to human health from eating genetically modified foods, many consumers remain wary of eating them. Findings from recent
studies in the United Kingdom demonstrated that genetically modified beets and oilseed
rape reduce the biodiversity of native plants and insects in agricultural fields, and there are
concerns that genetically modified plants may hybridize with native plants and cause ecological disruption.
his chapter introduces some of the techniques being
used to create genetically engineered crops and other
organisms. We begin by considering molecular genetic technology and some of its effects. We examine a number of
methods used to isolate, study, alter, and recombine DNA
sequences and place them back into cells. We then explore
some of the applications of molecular genetic analysis. The
last part of the chapter deals with genomics, the study of
whole genomes. We consider genetic and physical maps,
methods for sequencing entire genomes, and functional
genomics—how genes are identified in genomic sequences
and how their functions are defined. Finally, we compare the
genomes of different organisms and consider methods for
studying the proteins of a cell (proteomics).
14.1 Molecular Techniques
Are Used to Isolate,
Recombine, and Amplify
Recombinant DNA technology is a set of molecular techniques for locating, isolating, altering, and studying DNA
segments. The term recombinant is used because, frequently,
the goal is to combine DNA from two distinct sources. Genes
from two different bacteria might be joined, for example, or
a human gene might be inserted into a viral chromosome.
Commonly called genetic engineering, recombinant DNA
technology now encompasses many molecular techniques
that can be used to analyze, alter, and recombine virtually
any DNA sequences from any number of sources.
The Molecular Genetics Revolution
The techniques of recombinant DNA technology are just a
part of a vast array of molecular methods that are now available for the study of genetics. These molecular techniques have
drastically altered the way that genes are studied. Previously,
information about the structure and organization of genes
was gained by examining their phenotypic effects, but molecular genetic analysis allows the nucleotide sequences themselves to be read. Methods in molecular genetics have provided
new information about the structure and function of genes
and has altered many fundamental concepts of genetics. Our
detailed understanding of genetic processes such as replication, transcription, translation, RNA processing, and gene regulation has been learned through the use of molecular genetic
techniques. These techniques are used in many other fields as
well, including biochemistry, microbiology, developmental
biology, neurobiology, evolution, and ecology.
Recombinant DNA technology and other molecular
techniques are also being used to create a number of commercial products, including drugs, hormones, enzymes, and
crops (Figure 14.1). A complete industry—biotechnology—
has grown up around the use of these techniques to develop
new products. In medicine, molecular genetics is being used
to probe the nature of cancer, diagnose genetic and infectious diseases, produce drugs, and treat hereditary disorders.
Molecular genetics and recombinant DNA technology are used to
locate, analyze, alter, study, and recombine DNA sequences. These
techniques are used to probe the structure and function of genes,
address questions in many areas of biology, create commercial
products, and diagnose and treat diseases.
Working at the Molecular Level
The manipulation of genes at the molecular level presents a
serious challenge, often requiring strategies that may not, at
first, seem obvious. The basic problem is that genes are minute
and every cell contains thousands of them. Individual
nucleotides cannot be seen, and no physical features mark
the beginning or the end of a gene.
Let’s consider a typical situation faced by a molecular
geneticist. Suppose we wanted to use bacteria to produce large
quantities of a human protein. The first and most formidable
problem is to find the gene that encodes the desired protein. A
haploid human genome consists of 3.2 billion base pairs of
DNA. Let’s assume that the gene that we want to isolate is 3000
bp long. Our target gene occupies only one-millionth of the
genome; so searching for our gene in the huge expanse of
genomic DNA is more difficult than looking for a needle in the
proverbial haystack. But, even if we are able to locate the gene,
how are we to separate it from the rest of the DNA?
Molecular Genetic Analysis, Biotechnology, and Genomics
Cutting and Joining DNA Fragments
✔ Concept Check 1
Briefly outline the steps required to genetically engineer bacteria
that will produce a protein encoded by a human gene.
HindIII cuts the sugar–phosphate backbone of each strand
at the point indicated by the arrow, generating fragments
with short, single-stranded overhanging ends:
Such ends are called cohesive ends or sticky ends, because
they are complementary to each other and can
spontaneously pair to connect the fragments. Thus, DNA
fragments can be “glued” together: any two fragments
cleaved by the same enzyme will have complementary ends
and will pair (Figure 14.2 on p. 351). When their cohesive
ends have paired, two DNA fragments can be joined together
permanently by DNA ligase, which seals nicks between the
sugar–phosphate groups of the fragments.
Not all restriction enzymes produce staggered cuts and
sticky ends. PvuII cuts in the middle of its recognition site,
producing blunt-ended fragments:
Molecular genetic analyses require special methods because individual genes make up a tiny fraction of the cellular DNA and they
cannot be seen.
If we did succeed in locating and isolating the desired
gene, we would next need to insert it into a bacterial cell.
Linear fragments of DNA are quickly degraded by bacteria;
so the gene must be inserted in a stable form. It must also be
able to successfully replicate or it will not be passed on when
the cell divides. If we succeed in transferring our gene to bacteria in a stable form, we must still ensure that the gene is
properly transcribed and translated.
Finally, the methods used to isolate and transfer genes
are inefficient and, of a million cells that are subjected to
these procedures, only one cell might successfully take up
and express the human gene. So we must search through
many bacterial cells to find the one containing the
recombinant DNA. We are back to the problem of the needle in the haystack.
Although these problems might seem insurmountable,
molecular techniques have been developed to overcome all
of them, and human genes are routinely transferred to bacterial cells, where the genes are expressed.
genetically modified crops. Genetically engineered corn, which
produces a toxin that kills insect pests, now constitutes 57% of all
corn grown in the United States. [Chris Knapton/Photo Researchers.]
14.1 Recombinant DNA technology has been used to create
A first step in the molecular analysis of a DNA segment or
gene is to isolate it from the remainder of the DNA. A key discovery in the development of molecular genetic methods was
the discovery in the late 1960s of restriction enzymes (also
called restriction endonucleases) that recognize and make
double-stranded cuts in DNA at specific nucleotide sequences.
These enzymes are produced naturally by bacteria, where they
are used in defense against viruses. A bacterium protects its
own DNA from a restriction enzyme by modifying the recognition sequence, usually by adding methyl groups to its DNA.
More than 800 different restriction enzymes that recognize and cut DNA at more than 100 different sequences have
been isolated from bacteria. Many of these enzymes are commercially available; examples of some commonly used
restriction enzymes are given in Table 14.1. The name of
each restriction enzyme begins with an abbreviation that signifies its bacterial origin.
The sequences recognized by restriction enzymes are usually from 4 to 8 bp long; most enzymes recognize a sequence
of 4 or 6 bp. Most recognition sequences are palindromic—
sequences that read the same forward and backward.
Some of the enzymes make staggered cuts in the DNA.
For example, HindIII recognizes the following sequence:
Fragments that have blunt ends must be joined together
in other ways. One option is to use the enzyme DNA ligase,