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CAMPBELL
BIOLOGY
Reece • Urry • Cain • Wasserman • Minorsky • Jackson
19
Viruses
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
© 2014 Pearson Education, Inc.
TENTH
EDITION
A Borrowed Life
• A virus is an infectious particle consisting of genes packaged in a
protein coat
• Viruses are much simpler in structure than even prokaryotic cells
• Viruses cannot reproduce or carry out metabolism outside of a host
cell
Figure 19.1
Figure 19.1a
Concept 19.1: A virus consists of a nucleic
acid surrounded by a protein coat
• Viruses were detected indirectly long before they were actually seen
The Discovery of Viruses: Scientific Inquiry
• Tobacco mosaic disease stunts growth of tobacco plants and gives
their leaves a mosaic coloration
• In the late 1800s, some researchers hypothesized that a particle
smaller than bacteria caused the disease
• In 1935, Wendell Stanley confirmed this hypothesis by crystallizing
the infectious particle, now known as tobacco mosaic virus (TMV)
Experiment
Figure 19.2
1 Extracted sap
from tobacco
plant with
tobacco mosaic
disease
2 Passed sap
3 Rubbed filtered
through a
porcelain filter
known to trap
bacteria
4 Healthy plants
became infected
sap on healthy
tobacco plants
Figure 19.2a
Figure 19.2b
Figure 19.2c
Structure of Viruses
• Viruses are not cells
• A virus is a very small infectious particle consisting of nucleic acid
enclosed in a protein coat and, in some cases, a membranous
envelope
Viral Genomes
• Viral genomes may consist of either
• Double- or single-stranded DNA, or
• Double- or single-stranded RNA
• Depending on its type of nucleic acid, a virus is called a
DNA virus or an RNA virus
• The genome is either a single linear or circular molecule
of the nucleic acid
• Viruses have between three and several thousand
genes in their genome
Capsids and Envelopes
• A capsid is the protein shell that encloses the viral genome
• Capsids are built from protein subunits called capsomeres
• A capsid can have a variety of structures
RNA
Figure 19.3
Capsomere
DNA
Membranous RNA
Head
envelope
Capsid
Capsomere
of capsid
DNA
Tail
sheath
Tail
fiber
Glycoprotein
18  250 nm
Glycoproteins
70–90 nm (diameter) 80–200 nm (diameter)
20 nm
50 nm
(a) Tobacco mosaic (b) Adenoviruses
virus
80  225 nm
50 nm
50 nm
(c) Influenza viruses (d) Bacteriophage T4
RNA
Figure 19.3a
Capsomere
DNA
Capsomere
of capsid
Glycoprotein
18  250 nm
20 nm
(a) Tobacco mosaic
virus
70–90 nm (diameter)
50 nm
(b) Adenoviruses
Figure 19.3aa
20 nm
(a) Tobacco mosaic virus
Figure 19.3ab
50 nm
(b) Adenoviruses
Membranous RNA
envelope
Capsid
Figure 19.3b
Head
DNA
Tail
sheath
Tail
fiber
Glycoproteins
80–200 nm (diameter)
50 nm
(c) Influenza viruses
80  225 nm
50 nm
(d) Bacteriophage T4
Figure 19.3ba
50 nm
(c) Influenza viruses
Figure 19.3bb
50 nm
(d) Bacteriophage T4
• Some viruses have accessory structures that help them infect hosts
• Viral envelopes (derived from membranes of host cells) surround the
capsids of influenza viruses and many other viruses found in animals
• Viral envelopes contain a combination of viral and host cell molecules
• Bacteriophages, also called phages, are viruses that infect bacteria
• They have the most complex capsids found among viruses
• Phages have an elongated capsid head that encloses their DNA
• A protein tail piece attaches the phage to the host and injects the
phage DNA inside
Concept 19.2: Viruses replicate only in host
cells
• Viruses are obligate intracellular parasites, which means they can
replicate only within a host cell
• Each virus has a host range, a limited number of host cells that it can
infect
General Features of Viral Replicative Cycles
• Once a viral genome has entered a cell, the cell begins to
manufacture viral proteins
• The virus makes use of host enzymes, ribosomes, tRNAs, amino acids,
ATP, and other molecules
• Viral nucleic acid molecules and capsomeres spontaneously selfassemble into new viruses
VIRUS
DNA
Entry and
Figure 19.4
uncoating
1
2 Replication
3 Transcription and
manufacture of
capsid proteins
Capsid
HOST
CELL
Viral DNA
mRNA
Viral DNA
Capsid
proteins
4 Self-assembly of
new virus particles
and their exit from
the cell
Animation: Simplified Viral Reproductive
Cycle
Replicative Cycles of Phages
• Phages are the best understood of all viruses
• Phages have two alternative reproductive mechanisms: the lytic cycle
and the lysogenic cycle
The Lytic Cycle
• The lytic cycle is a phage replicative cycle that culminates in the death
of the host cell
• The lytic cycle produces new phages and lyses (breaks open) the
host’s cell wall, releasing the progeny viruses
• A phage that reproduces only by the lytic cycle is called a virulent
phage
• Bacteria have defenses against phages, including restriction enzymes
that recognize and cut up certain phage DNA
1 Attachment
Figure 19.5-1
1 Attachment
Figure 19.5-2
2 Entry of phage
DNA and
degradation
of host DNA
1 Attachment
Figure 19.5-3
2 Entry of phage
DNA and
degradation
of host DNA
3 Synthesis of viral
genomes and
proteins
1 Attachment
Figure 19.5-4
2 Entry of phage
DNA and
degradation
of host DNA
Phage assembly
Head
Tail
Tail
fibers
4 Self-assembly
3 Synthesis of viral
genomes and
proteins
1 Attachment
Figure 19.5-5
5 Release
2 Entry of phage
DNA and
degradation
of host DNA
Phage assembly
Head
Tail
Tail
fibers
4 Self-assembly
3 Synthesis of viral
genomes and
proteins
Animation: Phage T4 Lytic Cycle
The Lysogenic Cycle
• The lysogenic cycle replicates the phage genome without destroying
the host
• The viral DNA molecule is incorporated into the host cell’s
chromosome
• This integrated viral DNA is known as a prophage
• Every time the host divides, it copies the phage DNA and passes the
copies to daughter cells
Phage
DNA
Figure 19.6
The phage injects its DNA.
Daughter cell
with prophage
Many cell
divisions
create many
infected
bacteria.
Phage DNA
circularizes.
Tail fiber
Phage
Bacterial
chromosome
Lytic
cycle
The cell lyses,
releasing
phages.
Prophage exits
chromosome.
Lysogenic
cycle
Prophage
Phage DNA and proteins
are synthesized and
assembled.
Prophage is copied
with bacterial
chromosome.
Phage DNA integrates
into bacterial
chromosome.
Phage
DNA
Figure 19.6a
The phage injects its DNA.
Phage DNA
circularizes.
Tail fiber
Phage
Bacterial
chromosome
Lytic
cycle
The cell lyses,
releasing
phages.
Phage DNA and proteins
are synthesized and
assembled.
Daughter cell
with prophage
Many cell
divisions
create many
infected
bacteria.
Figure 19.6b
Prophage exits
chromosome.
Lysogenic
cycle
Prophage
Prophage is copied
with bacterial
chromosome.
Phage DNA integrates
into bacterial
chromosome.
Animation: Phage Lambda Lysogenic and Lytic
Cycles
• An environmental signal can trigger the virus genome to exit the
bacterial chromosome and switch to the lytic mode
• Phages that use both the lytic and lysogenic cycles are called
temperate phages
Replicative Cycles of Animal Viruses
• There are two key variables used to classify viruses that infect animals
• An RNA or DNA genome
• A single-stranded or double-stranded genome
• Whereas few bacteriophages have an envelope or an RNA genome,
many animal viruses have both
Table 19.1
Table 19.1a
Table 19.1b
Viral Envelopes
• Many viruses that infect animals have a membranous envelope
• Viral glycoproteins on the envelope bind to specific receptor
molecules on the surface of a host cell
• Some viral envelopes are derived from the host cell’s plasma
membrane as the viral capsids exit
• Other viral membranes form from the host’s nuclear envelope and
are then replaced by an envelope made from Golgi apparatus
membrane
Capsid
FigureRNA19.7
HOST CELL
Envelope (with
glycoproteins)
Template
Viral genome
(RNA)
mRNA
Capsid
proteins
ER
Glycoproteins
Copy of
genome
(RNA)
New virus
RNA as Viral Genetic Material
• The broadest variety of RNA genomes is found in viruses that infect
animals
• Retroviruses use reverse transcriptase to copy their RNA genome
into DNA
• HIV (human immunodeficiency virus) is the retrovirus that causes
AIDS (acquired immunodeficiency syndrome)
• The viral DNA that is integrated into the host genome is called a
provirus
• Unlike a prophage, a provirus remains a permanent resident of the
host cell
• RNA polymerase transcribes the proviral DNA into RNA molecules
• The RNA molecules function both as mRNA for synthesis of viral
proteins and as genomes for new virus particles released from the cell
Glycoprotein
Viral envelope
Figure 19.8
HIV
Reverse
transcriptase
HIV
Capsid
RNA (two
identical
strands)
Membrane of
white blood cell
HOST
CELL
Viral RNA
Reverse
transcriptase
RNA-DNA
hybrid
0.25 µm
HIV entering a cell
DNA
NUCLEUS
Provirus
Chromosomal
DNA
RNA genome
for the progeny
mRNA
viruses
New virus
New HIV leaving
a cell
Figure 19.8a
Glycoprotein
Viral envelope
Capsid
RNA (two
identical
strands)
HIV
Reverse
transcriptase
HOST
CELL
Viral RNA
RNA-DNA
hybrid
DNA
Reverse
transcriptase
Figure 19.8b
NUCLEUS
Provirus
Chromosomal
DNA
RNA genome
for the progeny
mRNA
viruses
New virus
Figure 19.8c
HIV
Membrane of
white blood cell
0.25 µm
HIV entering a cell
New HIV leaving
a cell
Figure 19.8ca
HIV
Membrane of
white blood cell
0.25 µm
HIV entering a cell
Figure 19.8cb
0.25 µm
HIV entering a cell
Figure 19.8cc
0.25 µm
New HIV leaving
a cell
Figure 19.8cd
0.25 µm
New HIV leaving
a cell
Figure 19.8ce
0.25 µm
New HIV leaving
a cell
Animation: HIV Reproductive Cycle
Evolution of Viruses
• Viruses do not fit our definition of living organisms
• Since viruses can replicate only within cells, they probably evolved as
bits of cellular nucleic acid
• Candidates for the source of viral genomes include plasmids and
transposons
• Plasmids, transposons, and viruses are all mobile genetic elements
• The largest virus yet discovered is the size of a small bacterium, and
its genome encodes proteins involved in translation, DNA repair,
protein folding, and polysaccharide synthesis
• There is controversy about whether this virus evolved before or after
cells
CAMPBELL
BIOLOGY
Reece • Urry • Cain • Wasserman • Minorsky • Jackson
20
DNA Tools and
Biotechnology
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
© 2014 Pearson Education, Inc.
TENTH
EDITION
The DNA Toolbox
• Recently the genome sequences of two extinct species—
Neanderthals and wooly mammoths—have been completed
• Advances in sequencing techniques make genome sequencing
increasingly faster and
less expensive
Figure 20.1
Figure 20.1a
• Biotechnology is the manipulation of organisms or their components
to make useful products
• The applications of DNA technology affect everything from
agriculture, to criminal law,
to medical research
Concept 20.1: DNA sequencing and DNA cloning
are valuable tools for genetic engineering and
biological inquiry
• The complementarity of the two DNA strands is the
basis for nucleic acid hybridization, the base pairing of
one strand of nucleic acid to the complementary
sequence on another strand
• Genetic engineering is the direct manipulation of genes
for practical purposes
DNA Sequencing
• Researchers can exploit the principle of complementary base pairing
to determine a gene’s complete nucleotide sequence, called DNA
sequencing
• The first automated procedure was based on a technique called
dideoxy or chain termination sequencing, developed by Sanger
Figure 20.2
(a) Standard sequencing machine
(b) Next-generation sequencing machines
Figure 20.2a
(a) Standard sequencing machine
Figure 20.2b
(b) Next-generation sequencing machines
Figure 20.3
Technique DNA
(template strand)
5′ C
T
G
A
C
T
T
C
G
A
C
A
3′ A
5′ C
T
G
A
C
T
T
C
G
A
C
A
3′ A
Primer
3′
T
G
T
T 5′
DNA
polymerase
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P
T
G
T
T 5′
P
dd A
G
C
T
G
T
T
T
G
T
T
dd A
A
G
C
T
G
T
T
dd G
A
A
G
C
T
G
T
T
dd T
G
A
A
G
C
T
G
T
T
Shortest
Direction
of movement
of strands
Longest labeled strand
Laser
Shortest labeled strand
Results
Last nucleotide
of shortest
labeled strand
dd C
T
G
A
A
G
C
T
G
T
T
dd A
C
T
G
A
A
G
C
T
G
T
T
G
dd G 3′
A
C
T
G
A
A
G
C
T
G
T
T 5′
Longest
Detector
Last nucleotide
of longest
labeled strand
P P P
G
Labeled strands
DNA (template
strand)
dd G
dd C 3′ C
Dideoxyribonucleotides
(fluorescently tagged)
Deoxyribonucleotides
G
A
C
T
G
A
A
G
C
Figure 20.3a
Technique
DNA
(template strand)
5′ C
T
G
A
C
T
T
C
G
A
C
A
3′ A
Primer
T
G
T
T
3′
Deoxyribonucleotides
5′
DNA
polymerase
P
Dideoxyribonucleotides
(fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P
P
G
P
P
P
G
Figure 20.3b
Technique
DNA (template
5′ C
strand)
T
G
A
C
T
T
C
G
A
C
A
3′ A
dd C 3′
dd G
T
G
T
T 5′
Shortest
C
T
G
T
T
Labeled strands
dd A
G
C
T
G
T
T
dd A
A
G
C
T
G
T
T
dd G
A
A
G
C
T
G
T
T
dd T
G
A
A
G
C
T
G
T
T
dd G 3′
dd C
T
G
A
A
G
C
T
G
T
T
dd A
C
T
G
A
A
G
C
T
G
T
T
A
C
T
G
A
A
G
C
T
G
T
T 5′
Longest
Figure 20.3c
Technique
Direction
of movement
of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
Results
Last nucleotide
of longest
labeled strand
Last nucleotide
of shortest
labeled strand
G
A
C
T
G
A
A
G
C
• “Next-generation sequencing” techniques use a single template
strand that is immobilized and amplified to produce an enormous
number of identical fragments
• Thousands or hundreds of thousands of fragments (400–1,000
nucleotides long) are sequenced in parallel
• This is a type of “high-throughput” technology
Figure 20.4
Technique
1 Genomic DNA is fragmented.
Results
4-mer
2 Each fragment is isolated with
3-mer
a bead.
A
T
G
C
2-mer
3 Using PCR, 106 copies of each
fragment are made, each attached
to the bead by 5′ end.
1-mer
4 The bead is placed into a well with
DNA polymerases and primers.
Template strand
of DNA
5′
3′
5′
3′
Primer
A T GC
5
A TGC
DNA
polymerase
Template
C
strand
C
of DNA
A
A
dATP
T
G
TA
PPi
GC
GC
AG
Primer
TA
6 If a nucleotide is joined to
a growing strand, PPi is
released, causing a flash
of light that is recorded.
A solution of each of the four nucleotides
is added to all wells and then washed off.
The entire process is then repeated.
A T GC
C
C
A dTTP
A
T
G
TA
GC
GC
AG
TA
7 If a nucleotide is not
complementary to the
next template base,
no PPi is released, and
no flash of light is recorded.
A TGC
C
C
A
dGTP
A
T
G
TA
GC
GC
AG
TA
A T GC
C
C
A
A
T
GC
TA
GC
GC
AG
TA
dCTP
PPi
8 The process is repeated until every
fragment has a complete complementary
strand. The pattern of flashes reveals the
sequence.
Figure 20.4a
Technique
1 Genomic DNA is fragmented.
2 Each fragment is isolated with
a bead.
3 Using PCR, 106 copies of each
fragment are made, each attached
to the bead by 5′ end.
4 The bead is placed into a well with
DNA polymerases and primers.
Template strand
of DNA
5′
3′
5′
3′
Primer
A T GC
5 A solution of each of the four nucleotides
is added to all wells and then washed off.
The entire process is then repeated.
Figure 20.4b
Technique
A T GC
DNA
polymerase
Template
C
strand
C
of DNA
A
A
dATP
T
G
TA
PPi
GC
GC
AG
Primer
TA
6 If a nucleotide is joined to
a growing strand, PPi is
released, causing a flash
of light that is recorded.
A T GC
C
C
A
dTTP
A
T
G
TA
GC
GC
AG
TA
7 If a nucleotide is not
complementary to the
next template base,
no PPi is released, and
no flash of light is recorded.
Figure 20.4c
Technique
A T GC
C
C
A
dGTP
A
T
G
TA
GC
GC
AG
TA
A T GC
C
C
A
A
T
GC
TA
GC
GC
AG
TA
dCTP
PPi
8 The process is repeated until every
fragment has a complete complementary
strand. The pattern of flashes reveals the
sequence.
Figure 20.4d
Results
4-mer
3-mer
2-mer
1-mer
A
T
G
C
• In “third-generation sequencing,” the techniques used are even faster
and less expensive than
the previous
Making Multiple Copies of a Gene or Other
DNA Segment
• To work directly with specific genes, scientists prepare well-defined
DNA segments in multiple identical copies by a process called DNA
cloning
• Plasmids are small circular DNA molecules that replicate separately
from the bacterial chromosome
• Researchers can insert DNA into plasmids to produce recombinant
DNA, a molecule with
DNA from two different sources
• Reproduction of a recombinant plasmid in a bacterial cell results in
cloning of the plasmid including the foreign DNA
• This results in the production of multiple copies of a single gene
• The production of multiple copies of a single gene is a type of DNA
cloning called gene cloning
Figure 20.5
Bacterium
Cell containing gene
of interest
1 Gene inserted
Bacterial
chromosome
into plasmid
Plasmid
Gene of
interest
Recombinant
DNA (plasmid)
DNA of
chromosome
(“foreign” DNA)
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest
Copies of gene
Gene for pest resistance
inserted into plants
Gene used to alter bacteria
for cleaning up toxic waste
Protein harvested
4 Basic research
and various
applications
Human growth hormone
treats stunted growth
Protein dissolves blood clots
in heart attack therapy
Figure 20.5a
Bacterium
Cell containing gene
of interest
1 Gene inserted
Bacterial
chromosome
into plasmid
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
DNA of
chromosome
(“foreign” DNA)
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture to form a
clone of cells containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
from gene of interest
Figure 20.5b
Gene of
interest
Protein expressed
from gene of interest
Copies of gene
Protein harvested
4 Basic research
and various
applications
Gene for pest resistance
inserted into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Human growth hormone
treats stunted growth
Protein dissolves
blood clots in heart
attack therapy
• A plasmid used to clone a foreign gene is called a cloning vector
• Bacterial plasmids are widely used as cloning vectors because they
are readily obtained, easily manipulated, easily introduced into
bacterial cells, and once in the bacteria they multiply rapidly
• Gene cloning is useful for amplifying genes to produce a protein
product for research, medical, or other purposes
Using Restriction Enzymes to Make a
Recombinant DNA Plasmid
• Bacterial restriction enzymes cut DNA molecules at specific DNA
sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction
fragments
• The most useful restriction enzymes cut DNA
in a staggered way, producing fragments with “sticky ends”
• Sticky ends can bond with complementary sticky ends of other
fragments
• DNA ligase is an enzyme that seals the bonds between restriction
fragments
Figure 20.6
Bacterial
plasmid
Restriction site
5′
3′
G AAT T C
C T T AAG
DNA
3′
5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
5′
3′
5′
3′
5′
3′
3′
Sticky end
5′
5′
3′
2 Base pairing of sticky
ends produces various
combinations.
5′
3′
3 DNA ligase
3′ 5′
G AAT T C
C T TA A G
5′ 3′
3′
5′
Fragment from different DNA molecule
cut by the same restriction enzyme
3′ 5′
G AAT T C
C T TAA G
5′ 3′
3′
5′
One possible combination
seals the strands.
5′
3′
3′
Recombinant DNA molecule
Recombinant
plasmid
5′
Figure 20.6a
Bacterial
plasmid
Restriction site
5′
3′
G A AT T C
C T TA A G
DNA
3′
5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
5′
5′
3′
3′
5′
3′
3′
Sticky end
5′
Figure 20.6b
5′
5′
3′
3′
5′
3′
3′
Sticky end
5′
5′
3′
2 Base pairing of sticky
ends produces various
combinations.
5′
3′
3′ 5′
G AATT C
C TTAA G
5′ 3′
3′
5′
Fragment from different
DNA molecule cut by the
same restriction enzyme
3′ 5′
G AATT C
C TTAA G
5′ 3′
One possible combination
3′
5′
Figure 20.6c
5′
3′
3 DNA ligase
3′ 5′
G AATT C
C TTAA G
5′ 3′
3′ 5′
G AATT C
C TTAA G
5′ 3′
3′
5′
One possible combination
seals the strands
3′
5′
3′
Recombinant DNA molecule
Recombinant
plasmid
5′
Animation: Restriction Enzymes
• To check the recombinant plasmid, researchers might cut the
products again using the same restriction enzyme
• To separate and visualize the fragments produced, gel electrophoresis
would be carried out
• This technique uses a gel made of a polymer to separate a mixture of
nucleic acids or proteins based on size, charge, or other physical
properties
Figure 20.7
Mixture of
DNA molecules of
different
sizes
Power
source
Cathode
Anode
Wells
Gel
(a) Negatively charged DNA molecules move
toward the positive electrode.
Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less than
longer ones, so they move faster through the gel.
Figure 20.7a
Mixture of
DNA molecules of
different
sizes
Power
source
Cathode
Anode
Wells
Gel
(a) Negatively charged DNA molecules move
toward the positive electrode.
Figure 20.7b
Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less
than longer ones, so they move faster
through the gel.
Video: Biotechnology Lab
Amplifying DNA: The Polymerase Chain
Reaction (PCR) and Its Use in DNA Cloning
• The polymerase chain reaction, PCR, can produce many copies of a
specific target segment of DNA
• A three-step cycle—heating, cooling, and replication—brings about a
chain reaction that produces an exponentially growing population of
identical DNA molecules
• The key to PCR is an unusual, heat-stable DNA polymerase called Taq
polymerase
• PCR uses a pair of primers specific for the sequence to be amplified
• PCR amplification occasionally incorporates errors into the amplified
strands and so cannot substitute for gene cloning in cells
Figure 20.8
Technique
5′
3′
Target
sequence
Genomic DNA
3′
5′
1 Denaturation 5′
3′
3′
5′
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Figure 20.8a
Technique
5′
3′
Target
sequence
Genomic DNA
3′
5′
Figure 20.8b-1
Technique
1 Denaturation
Cycle 1
yields
2
molecules
5′
3′
3′
5′
Figure 20.8b-2
Technique
1 Denaturation
5′
3′
3′
5′
2 Annealing
Cycle 1
yields
2
molecules
Primers
Figure 20.8b-3
Technique
1 Denaturation
5′
3′
3′
5′
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Figure 20.8c
Technique
Cycle 2
yields
4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Results After 30 more cycles, over 1 billion (109) molecules match
the target sequence.
• PCR primers can be designed to include restriction sites that allow the
product to be cloned into plasmid vectors
• The resulting clones are sequenced and
error-free inserts selected
Figure 20.9
DNA fragments obtained
by PCR with restriction
sites matching those in
the cloning vector
Cut with same restriction
enzyme used on cloning
vector
A gene that makes bacterial
cells resistant to an antibiotic
is present on the plasmid.
Cloning vector
(bacterial plasmid)
Mix and ligate
Recombinant
DNA plasmid
Only cells that take up
a plasmid will survive
Animation: Cloning a Gene
Expressing Cloned Eukaryotic Genes
• After a gene has been cloned, its protein product can be produced in
larger amounts for research
• Cloned genes can be expressed as protein in either bacterial or
eukaryotic cells
Bacterial Expression Systems
• Several technical difficulties hinder expression of cloned eukaryotic
genes in bacterial host cells
• To overcome differences in promoters and other DNA control
sequences, scientists usually employ an expression vector, a cloning
vector that contains a highly active bacterial promoter
• Another difficulty with eukaryotic gene expression in bacteria is the
presence of introns in most eukaryotic genes
• Researchers can avoid this problem by using cDNA, complementary to
the mRNA, which contains only exons
Eukaryotic DNA Cloning and Expression
Systems
• Molecular biologists can avoid eukaryote-bacterial incompatibility
issues by using eukaryotic cells, such as yeasts, as hosts for cloning
and expressing genes
• Even yeasts may not possess the proteins required to modify
expressed mammalian
proteins properly
• In such cases, cultured mammalian or insect
cells may be used to express and study proteins
• One method of introducing recombinant DNA into eukaryotic cells is
electroporation, applying a brief electrical pulse to create temporary
holes in plasma membranes
• Alternatively, scientists can inject DNA into cells using microscopically
thin needles
• Once inside the cell, the DNA is incorporated into the cell’s DNA by
natural genetic recombination
Cross-Species Gene Expression and
Evolutionary Ancestry
• The remarkable ability of bacteria to express some eukaryotic
proteins underscores the shared evolutionary ancestry of living
species
• For example, Pax-6 is a gene that directs formation of a vertebrate
eye; the same gene in flies directs the formation of an insect eye
(which is quite different from the vertebrate eye)
• The Pax-6 genes in flies and vertebrates can substitute for each other
Concept 20.2: Biologists use DNA technology
to study gene expression and function
• Analysis of when and where a gene or group of genes is expressed
can provide important clues about gene function
Analyzing Gene Expression
• The most straightforward way to discover which genes are expressed
in certain cells is to identify the mRNAs being made
Studying the Expression of Single Genes
• mRNA can be detected by nucleic acid hybridization with
complementary molecules
• These complementary molecules, of either DNA or RNA, are nucleic
acid probes
• In situ hybridization uses fluorescent dyes attached to probes to
identify the location of specific mRNAs in place in the intact organism
Figure 20.10
5′
3′
TAACGGTTCCAGC
CTCAAGTTGCTCT
ATTGCCAAGGTCG
5′
5′
3′
GAGTTCAACGAGA
3′
5′
3′
en mRNA
wg mRNA
Cells
expressing
the wg gene
Head
50 µm
Cells
expressing
the en gene
Thorax
T1
T2
Abdomen
T3 A1 A2
A3
Segment
boundary
Head
Thorax
Abdomen
A4 A5
Figure 20.10a
5′
3′
5′
3′
TAACGGTTCCAGC
CTCAAGTTGCTCT
ATTGCCAAGGTCG
GAGTTCAACGAGA
3′
5′
3′
5′
en mRNA
wg mRNA
Cells
expressing
the wg gene
Head
50 µm
Cells
expressing
the en gene
Thorax
T1
T2
Abdomen
T3 A1 A2
A3
A4 A5
Figure 20.10b
Head
50 µm
Thorax
T1
T2
Abdomen
T3 A1 A2
A3
Segment
boundary
Head
Thorax
Abdomen
A4 A5
Figure 20.10c
Thorax
Head
50 µm
T1
T2
Abdomen
T3
A1
A2
A3
A4 A5
• Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful
for comparing amounts of specific mRNAs in several samples at the
same time
• Reverse transcriptase is added to mRNA to make complementary
DNA (cDNA), which serves as a template for PCR amplification of the
gene of interest
• The products are run on a gel and the mRNA of interest is identified
Figure 20.11-1
DNA in
nucleus
mRNAs in
cytoplasm
Figure 20.11-2
DNA in
nucleus
mRNAs in
cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
5′
3′
A A A A A A 3′
T T T T T 5′
DNA
Primer
strand (poly-dT)
Figure 20.11-3
DNA in
nucleus
mRNAs in
cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
5′
3′
A A A A A A 3′
T T T T T 5′
DNA
Primer
strand (poly-dT)
5′
3′
A A A A A A 3′
T T T T T 5′
Figure 20.11-4
DNA in
nucleus
mRNAs in
cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
5′
A A A A A A 3′
T T T T T 5′
3′
DNA
Primer
strand (poly-dT)
A A A A A A 3′
T T T T T 5′
5′
3′
5′
3′
DNA
polymerase
3′
5′
Figure 20.11-5
DNA in
nucleus
mRNAs in
cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
5′
A A A A A A 3′
T T T T T 5′
3′
DNA
Primer
strand (poly-dT)
A A A A A A 3′
T T T T T 5′
5′
3′
5′
3′
3′
5′
DNA
polymerase
5′
3′
3′
5′
cDNA
Figure 20.12
Technique
1 cDNA synthesis
mRNAs
cDNAs
Primers
2 PCR amplification
Specific
gene
3 Gel electrophoresis
Results
Embryonic stages
1 2 3 4 5 6
Studying the Expression of Interacting Groups
of Genes
• Automation has allowed scientists to measure the expression of
thousands of genes at one time using DNA microarray assays
• DNA microarray assays compare patterns of gene expression in
different tissues, at different times, or under different conditions
Figure 20.13
Each dot is a well containing identical copies
of DNA fragments that carry a specific gene.
Genes expressed
in first tissue.
Genes expressed
in second tissue.
Genes expressed
in both tissues.
DNA microarray
(actual size)
Genes expressed
in neither tissue.
►
Figure 20.13a
Each dot is a well containing identical copies
of DNA fragments that carry a specific gene.
• With rapid and inexpensive sequencing methods available,
researchers can also just sequence cDNA samples from different
tissues or embryonic stages to determine the gene expression
differences between them
• By uncovering gene interactions and clues to gene function DNA
microarray assays may contribute to understanding of disease and
suggest new diagnostic targets
Determining Gene Function
• One way to determine function is to disable the gene and observe the
consequences
• Using in vitro mutagenesis, mutations are introduced into a cloned
gene, altering or destroying its function
• When the mutated gene is returned to the cell,
the normal gene’s function might be determined by examining the
mutant’s phenotype
• Gene expression can also be silenced using RNA interference (RNAi)
• Synthetic double-stranded RNA molecules matching the sequence of
a particular gene are used to break down or block the gene’s mRNA
• In humans, researchers analyze the genomes of many people with a
certain genetic condition to try to find nucleotide changes specific to
the condition
• These genome-wide association studies test for genetic markers,
sequences that vary among individuals
• SNPs (single nucleotide polymorphisms), single nucleotide variants,
are among the most useful genetic markers
• SNP variants that are found frequently associated with a particular
inherited disorder alert researchers to the most likely location for the
disease-causing gene
• SNPs are rarely directly involved in the disease; they are most often in
noncoding regions of the genome
Figure 20.14
A
DNA
T
Normal allele
SNP
C
G
Disease-causing
allele
Concept 21.5: Duplication, rearrangement, and
mutation of DNA contribute to genome evolution
• The basis of change at the genomic level is mutation,
which underlies much of genome evolution
• The earliest forms of life likely had only those genes
necessary for survival and reproduction
• The size of genomes has increased over evolutionary
time, with the extra genetic material providing raw
material for gene diversification
Duplication of Entire Chromosome Sets
• Accidents in meiosis can lead to one or more extra sets of
chromosomes, a condition known as polyploidy
• The genes in one or more of the extra sets can diverge by
accumulating mutations; these variations may persist if the organism
carrying them survives and reproduces
• In this way genes with novel functions can evolve
Alterations of Chromosome Structure
• Humans have 23 pairs of chromosomes, while chimpanzees have 24
pairs
• Following the divergence of humans and chimpanzees from a
common ancestor, two ancestral chromosomes fused in the human
line
• Duplications and inversions result from mistakes during meiotic
recombination
• Comparative analysis between chromosomes of humans and seven
mammalian species paints a hypothetical chromosomal evolutionary
history
Figure 21.11
Human
chromosome
Chimpanzee
chromosomes
Telomere
sequences
Centromere
sequences
Telomere-like
sequences
12
Centromere-like
sequences
2
13
Figure 21.12
Human chromosome
16
Mouse chromosomes
7
8
16
17
• The rate of duplications and inversions seems to have accelerated
about 100 million years ago
• This coincides with when large dinosaurs went extinct and mammals
diversified
• Chromosomal rearrangements are thought to contribute to the
generation of new species
Duplication and Divergence of Gene-Sized
Regions of DNA
• Unequal crossing over during prophase I of meiosis can result in one
chromosome with a deletion and another with a duplication of a
particular region
• Transposable elements can provide sites for crossover between
nonsister chromatids
Figure 21.13
Nonsister
Gene
chromatids
Incorrect pairing
of two homologs
during meiosis
Crossover
point
and
Transposable
element
Evolution of Genes with Related Functions:
The Human Globin Genes
• The genes encoding the various globin proteins evolved from one
common ancestral globin gene, which duplicated and diverged about
450–500 million years ago
• After the duplication events, differences between the genes in the
globin family arose from the accumulation of mutations
Figure 21.14
Ancestral globin gene
Evolutionary time
Duplication of
ancestral gene
Mutation in
both copies
α
Transposition to
different chromosomes
Further duplications
and mutations
α
β
α
ζ
ζ
β
ζ α α α2 α1 yθ
1
2
α-Globin gene family
on chromosome 16
ϵ
β

ϵ
G A
β

β
β-Globin gene family
on chromosome 11
• Subsequent duplications of these genes and random mutations gave
rise to the present globin genes, which code for oxygen-binding
proteins
• The similarity in the amino acid sequences of the various globin
proteins supports this model of gene duplication and mutation
Evolution of Genes with Novel Functions
• The copies of some duplicated genes have diverged so much in
evolution that the functions of their encoded proteins are now very
different
• For example the lysozyme gene was duplicated and evolved into the
gene that encodes
-lactalbumin in mammals
• Lysozyme is an enzyme that helps protect animals against bacterial
infection
• -lactalbumin is a nonenzymatic protein that plays a role in milk
production in mammals
Figure 21.15
(a) Lysozyme
Lysozyme
1
α–lactalbumin
1
Lysozyme
51
α–lactalbumin
51
Lysozyme
(b) α–lactalbumin
101
α–lactalbumin 101
(c) Amino acid sequence alignments of lysozyme and α–lactalbumin
Rearrangements of Parts of Genes: Exon
Duplication and Exon Shuffling
• The duplication or repositioning of exons has contributed to genome
evolution
• Errors in meiosis can result in an exon being duplicated on one
chromosome and deleted from the homologous chromosome
• In exon shuffling, errors in meiotic recombination lead to some mixing
and matching of exons, either within a gene or between two
nonallelic genes
Figure 21.16
EGF
EGF
EGF
EGF
Epidermal growth
factor gene with multiple
EGF exons
F
F
F
Exon
shuffling
Exon
duplication
F
Fibronectin gene with multiple
“finger” exons
F
EGF
K
K
K
Plasminogen gene with a
“kringle” exon
Portions of ancestral genes
Exon
shuffling
TPA gene as it exists today
How Transposable Elements Contribute to
Genome Evolution
• Multiple copies of similar transposable elements may facilitate
recombination, or crossing over, between different chromosomes
• Insertion of transposable elements within a protein-coding sequence
may block protein production
• Insertion of transposable elements within a regulatory sequence may
increase or decrease protein production
• Transposable elements may carry a gene or groups of genes to a new
position
• Transposable elements may also create new sites for alternative
splicing in an RNA transcript
• In all cases, changes are usually detrimental but may on occasion
prove advantageous to an organism