Because learning changes everything.

Chapter 13
DNA Replication and
Recombination
Lecture Outline

Essentials of the Living World

Seventh Edition

George Johnson, Joel Bergh

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 13.1 The Human Genome 1

The publication of the sequence of the entire human genome

occurred on June 26, 2000.
• The human genome contains more than 3 billion base
pairs.
• It is estimated that there are 20,000 to 25,000 protein-
encoding genes, only 1% of the genome.

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 Figure 13.1: The human genome

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 13.1 The Human Genome 2

The number of genes is much less than expected based on

the number of unique mRNAs found in human cells.
• In eukaryotic DNA, protein-encoding segments called
exons are interspersed among noncoding introns.
• Alternative splicing of exons accounts for the fact that
about 25,000 genes can encode four times as many
proteins.
Some chromosomes are packed with genes; others contain
relatively few genes.

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 13.1 The Human Genome 3

There are four major types of noncoding DNA, which account

for 99% of the DNA in humans.
• Noncoding DNA within genes:
• These are introns.
• Structural DNA:
• Tightly coiled DNA that remains untranscribed.
• Repeated and duplicated sequences:
• Simple sequences of a few nucleotides repeated thousands of
times.
• Transposable elements:
• Bits of DNA that jump from one location on a chromosome to
another.

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 Table 13.1: Types of DNA Sequences Found in the
Human Genome

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 13.2 A Scientific Revolution 1

Genetic engineering is moving genes from one organism to

another.
• The first stage in a genetic engineering experiment is to
chop up the source DNA and obtain a copy of the gene
you want to transfer.
• Restriction enzymes, or restriction endonucleases,
bind to specific short sequences on the DNA and make a
specific cut.
• The sequence is symmetrical.
• The cut generates DNA fragments that are “sticky” because the
incision made by the restriction enzyme is usually made to the side
of the sequence, not in the center of it.

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 13.2 A Scientific Revolution 2

DNA from another source that is cut with the same restriction
enzyme will have the same sticky ends.
• These ends can be joined together by the enzyme ligase.
Restriction enzymes are the basic tools of genetic
engineering.

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 Figure 13.4: How restriction enzymes produce DNA
fragments with sticky ends

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 13.2 A Scientific Revolution 3

The need for complementary DNA (cDNA).

• Recall that eukaryotic genes contain both exons and
introns.
• Before the gene is translated and expressed, the introns are cut
out from the primary RNA transcript.

• Enzymes stitch together the remaining exon fragments to form

mRNA, which is eventually translated in the cytoplasm.

• Bacteria lack the enzymes to remove introns.

• It is desirable for genetic engineers to transfer an
intron-free copy of the gene into bacteria.

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 13.2 A Scientific Revolution 4

Forming cDNA:
• To obtain DNA without introns, genetic engineers isolate
first the processed mRNA corresponding to a particular
gene.
copier

• The enzyme reverse transcriptase can then produce a

DNA version of this mRNA, called complementary DNA
(cDNA).

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 Figure 13.5: cDNA: Producing an intron-free version of a
eukaryotic gene for genetic engineering

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 13.2 A Scientific Revolution 5

DNA fingerprinting is a revolutionary technique used in

forensic evidence.
• The process uses probes on DNA samples that have been
cut with the same restriction endonucleases.
• The probes are complementary to unique DNA sequences
found in noncoding regions of human DNA that are highly
variable among individuals.
• The resulting DNA fingerprints consist of autoradiographs,
rows of parallel bands on X-ray film.
• Each band represents the position of a DNA fragment that
complementarily binds to a probe.

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 13.2 A Scientific Revolution 6

DNA fingerprints have been used in courts of law since 1987.

• While an individual DNA fingerprint is not 100% accurate,
it is as reliable as traditional fingerprinting used in
evidence when multiple probes are used.
• Any source of DNA (that is, a hair, a speck of blood, or
semen) can be used in DNA fingerprinting to convict or to
clear a suspect.
• However, laboratory analyses of DNA must be carried out
properly to ensure accuracy.

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 Figure 13.6: DNA fingerprints that led to conviction

Hologic

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 13.2 A Scientific Revolution 7

Polymerase chain reaction (PCR) is a method which

produces millions of copies of DNA from a single copy.

• A double-stranded DNA fragment is heated to separate the

strands.

• Each of the strands is copied by DNA polymerase.

• The cycle is repeated many times, each time doubling the

number of DNA copies.

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 13.3 Genetic Engineering and Medicine 1

Much of the promise of genetic engineering lies in treating

illness.
Many genetic defects occur because our cells fail to make
critical proteins.
• For example, diabetics cannot control their blood sugar
levels because a critical protein, insulin, is not made.
• Through genetic engineering, the genes encoding insulin have
been introduced in bacteria, which can cheaply produce large
quantities of protein.

• Other proteins, such as factor VIII to promote clotting, are

now safely produced by bacteria, which eliminates the
risks associated with donated blood.

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 13.3 Genetic Engineering and Medicine 2

Genetic engineering is also used to create subunit vaccines

against viruses, such as herpes and hepatitis.
• Engineers splice genes from the coat of the virus into a
fragment of cowpox (vaccinia) virus genome, which is
used as a vector to carry the viral coat genes.
• The recombinant virus is injected into humans and causes
the production of antibodies against the virus, which is
essentially harmless to humans.
• This method of using one virus as a vector to introduce
fragments of the DNA of a disease-causing virus produces
what are called piggyback vaccines.

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 Figure 13.8: Constructing a subunit, or piggyback,
vaccine for the herpes simplex virus

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 13.4 Genetic Engineering and Agriculture 1

Engineering crops to be resistant to insect pests reduces the

need to add insecticides to the environment.
• For example, genes from the soil bacterium Bacillus
thuringiensis (Bt), which produces a protein that is toxic
when eaten by crop pests, have been inserted into the
chromosomes of tomatoes.
• Because the plants can synthesize Bt protein, they are toxic to
pests, such as the tomato hornworm.

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 13.4 Genetic Engineering and Agriculture 2

Herbicide resistance has also been genetically engineered.

• Glyphosate is a powerful herbicide that kills most actively
growing plants and is used to control weeds.
• Using a gene gun, engineers inserted into crop plants an
isolated gene from a bacterium that is resistant to
glyphosate.
• The glyphosate can now be widely applied to fields and orchards
where it retards weed growth but not crop growth.

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 Figure 13.9: Shooting genes into cells

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 Figure 13.10: Genetically engineered herbicide
resistance

Courtesy Monsanto Company

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 13.4 Genetic Engineering and Agriculture 3

The real promise of genetically modified (GM) plants is to

produce crops with desirable traits that directly benefit the
consumer.
• For example, to combat iron and vitamin A micronutrient
deficiencies among the world’s rice eaters, genetic
engineers created GM “golden” rice.
• This transgenic rice contains genes from a bean, a fungus, wild
rice, and a daffodil to increase its nutritional value.

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 Figure 13.11: Transgenic “golden” rice

Courtesy Golden Rice Humanitarian Board: www.goldenrice.org

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 13.4 Genetic Engineering and Agriculture 4

Genetically modified (GM) crops are commonly cultivated in

the United States.
• Some of the benefits of GM crops include increased soil
preservation and reduced pesticide usage.
Is eating GM food dangerous?
• Does adding genes introduce novel proteins that may be
potentially harmful when consumed?
• Could introduced proteins become allergens?
• All introduced proteins are extensively tested and the risk
of eating GM crops seems to be slight.

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 Table 13.2: Genetically Modified Crops 1

Genetically Modified Crops

Rice Genes have been added to commercial

rice from daffodils for vitamin A, and
from beans, fungi, and wild rice to
An illustration of rice. supply dietary iron; transgenic strains
that are cold-tolerant are under
development.

Wheat New strains of wheat, resistant to the

herbicide glyphosate, greatly reduce
the need for tilling and so reduce loss
An illustration of wheat. of topsoil.

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 Table 13.2: Genetically Modified Crops 2

Genetically Modified Crops

Soybean A major animal feed crop, soybeans tolerant of the

herbicide glyphosate were used in over 94% of U.S.
soybean acreage in 2014. Varieties are being
An illustration developed that contain the Bt gene to protect the
of soybean.
crop from insect pests without chemical pesticides.

Corn Corn varieties resistant to insect pests (Bt corn) are

widely planted (81% of U.S. acreage in 2016; fully
89% of corn acreage was planted with Roundup-
An illustration of corn. resistant seeds in 2017). Varieties that are drought-
resistant are being developed, as well as
nutritionally improved lines.

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 Table 13.2: Genetically Modified Crops 3
Genetically Modified Crops

Cotton Cotton crops are attacked by cotton bollworm,

budworm, and other lepidopteran insects; more
than 40% of all chemical pesticide tonnage
An illustration
worldwide is applied to cotton. A form of the Bt
of a cotton
plant. gene toxic to all lepidopterans but harmless to other
insects has transformed cotton to a crop that
requires few chemical pesticides. Over 85% of U.S.
acreage is Bt cotton.
Peanut The lesser cornstalk borer causes serious damage
to peanut crops. An insect-resistant variety is under
An illustration of a peanut. development by gene engineers to control this pest.

Potato Verticillium wilt (a fungal disease) infects the water-

conducting tissues of potatoes, reducing crop yields
An illustration of a potato. 40%. An antifungal gene from alfalfa reduces
infections sixfold.

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 13.5 Reproductive Cloning 1

• Hans Spemann proposed in 1938 that cloning might be

possible by removing the nucleus from an egg cell and
replacing it with a nucleus from another cell.
• Attempts at cloning were made many years later by
several researchers.
• Some success was obtained but only with a donor nucleus
from an early embryo, not an adult nucleus.

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 13.5 Reproductive Cloning 2

Keith Campbell, a geneticist, proposed that, in order for a

successful nuclear transplant to take place, both the egg and
the donated nucleus need to be in the same stage of the cell
cycle.
• By first starving the cells so that they paused at the G1
checkpoint, researchers succeeded in cloning farm
animals from advanced embryos.

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 13.5 Reproductive Cloning 3

Campbell’s colleague Wilmut attempted to transfer a nucleus

from an adult cell into an enucleated egg.
• Wilmut used an adult sheep’s mammary gland as the
nuclear donor.
• Both the donor mammary cells and the enucleated eggs
were first starved and a brief electrical shock allowed the
contents to fuse together.
• The resulting embryo developed into a blastula and was
implanted into a surrogate mother.
• “Dolly” the cloned lamb was born on July 5, 1996.

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 Figure 13.13: Wilmut’s animal cloning experiment

AFP PHOTO/FILES/ALESSANDRO ABBONIZIO/COLIN MCPHERSON/Getty Images

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 13.5 Reproductive Cloning 4

• A wide variety of farm animals have been cloned since

“Dolly” the sheep.
• Cloning procedures have become increasingly efficient.
• However, most cloned animals do not live a normal
lifespan.

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 13.5 Reproductive Cloning 5

The problem with cloned embryos may be that as

mammalian eggs and sperm mature, their DNA is
conditioned by the parent through a type of reprogramming
called epigenetics.
• Chemical changes are made to the DNA that alter when
particular genes are expressed.
• Genes can be locked in the “off” position by methylation, in which
a –CH3 group is added to some of the cytosine nucleotides.

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 13.6 Stem Cell Therapy 1

Embryonic stem cells are special cells that form early in

development.
• Embryonic stems are called totipotent, meaning that each
cell has the ability to form any body tissue, even an adult
animal.
Adult stem cells are pluripotent because they can
differentiate into many different cell types, but not all.

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 Figure 13.15: Human embryonic stem cells (×20)

Mary Martin/Science Source

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 13.6 Stem Cell Therapy 2

As development proceeds, some of the embryonic stem cells

become committed to forming a certain type of tissue only.
• Every major tissue is formed from its own tissue-specific
adult stem cell.
Because they can develop into any tissue, embryonic stem
cells offer the exciting possibility of restoring damaged
tissues.

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 Figure 13.17: Using embryonic stem cells to restore
damaged tissue

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 13.7 Gene Therapy

Gene transfer therapy involves introducing “healthy” genes

into cells that lack them.
• Early work with a cold virus vector, called adenovirus, was
unsuccessful in humans because of immune attack.
• A smaller vector, adeno-associated virus (AAV), does
not elicit a strong immune response and seems promising.

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 Figure 13.19: Using gene therapy to cure a retinal
degenerative disease in dogs

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