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Genetics of Viruses
and Bacteria
Microbial Model Systems
• Viruses called bacteriophages can infect and set in motion a
genetic takeover of bacteria, such as Escherichia coli
• E. coli and phage model systems frequently use by researchers in
studies that reveal broad biological principles
• Viruses and bacteria have unique genetic mechanisms
Figure 18.1
0.5 m
Characteristics of Viruses
• Recall that bacteria are prokaryotes with cells much
smaller and more simply organized than those of
eukaryotes
• Viruses are smaller and simpler still
•
Smallest viruses are only 20 nm in diameter
•
The virus particle, or virion, is just nucleic acid enclosed by a
protein coat
Virus
Bacterium
Animal
cell
Animal cell nucleus
0.25 m
Characteristics of Viruses
•
A virus has a genome but can reproduce only within a host cell
•
Scientists detected viruses indirectly long before they could see them
•
The story of how viruses were discovered begins in the late 1800s
•
Tobacco mosaic disease stunts growth of tobacco plants and gives their
leaves a mosaic coloration
•
In the late 1800s, 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)
Characteristics of Viruses
• Viruses are very small infectious particles consisting of
•
Nucleic acid - genome
•
Protein coat which encloses the genome
•
And in some cases, a membranous envelope
• Viral genomes may consist of
•
Double- or single-stranded DNA
•
Double- or single-stranded RNA
Capsids
• A capsid is the protein shell
that encloses the viral genome,
it can have various structures
• May be rod-shaped, polyhedral or
complex
Capsomere
of capsid
RNA
Capsomere
DNA
Glycoprotein
70–90 nm (diameter)
18  250 mm
• Composed of capsomeres –
protein subunits; from one or a
few types of proteins
• Spikes or glycoproteins like the
herpes shown
20 nm
50 nm
(a) Tobacco mosaic virus (b) Adenoviruses
Membranous Envelope
• Some viruses have envelopes
which are membranous coverings
derived from the membrane of the
host cell
• Maybe a single layer or double
layer envelope
• Bilipid bilayer with glycoproteins
spikes protruding from the outer
layer
Membranous Envelope
• Many animal viruses have a
membranous envelope
• The membrane cloaks the viral
capsid, helps viruses infect their
host
Membranous
envelope
Capsid
RNA
Glycoprotein
80–200 nm (diameter)
• Derived from host cell membrane
which is usually virus-modified
• Viral glycoproteins on the envelope
bind to specific receptor molecules
on the surface of a host cell
50 nm
(c) Influenza viruses
Bacteriophages
Head
• Also called phages (T2, T4, T6)
have the most complex capsids
found among viruses
• Icosohedral head encloses the
genetic material; the protein
tailpiece w/tail fibers attaches
the phage to its bacterial host
and injects its DNA into the
bacterium
Tail
sheath
DNA
Tail
fiber
80  225 nm
50 nm
(d) Bacteriophage T4
Viral Reproductive Cycles
•
Although a virus has a genome it can only reproduce within a host cell
•
Viruses are obligate intracellular parasites
•
Each virus has a host range - a limited number of host cells that it can
infect
•
Recognize host cells by a complementary fit between external viral
proteins and specific cell surface receptor sites
•
Viruses use enzymes, ribosomes, and small molecules of host cells to
synthesize progeny viruses
Viral Reproduction
DNA
Capsid
Entry into cell and
uncoating of DNA
VIRUS
Transcription
Replication
HOST CELL
Viral DNA
mRNA
Viral DNA
Capsid
proteins
Self-assembly of new
virus particles and
their exit from cell
Reproductive Cycles of Phages
• Phages are the best understood of all viruses
• They through two alternative reproductive
mechanisms: the lytic cycle and the lysogenic cycle
•
Lytic cycle - culminates in the death of the host
•
Lysogenic cycle - replicates the phage genome without
destroying the host
The Lytic Cycle
•
A phage reproductive cycle that culminates in the death of the host cell
•
Produces new phages and digests 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
5
Attachment. The T4 phage uses
its tail fibers to bind to specific
receptor sites on the outer
surface of an E. coli cell.
2
Release. The phage directs production
of an enzyme that damages the bacterial
cell wall, allowing fluid to enter. The cell
swells and finally bursts, releasing 100
to 200 phage particles.
Entry of phage DNA
and degradation of host DNA.
The sheath of the tail contracts,
injecting the phage DNA into
the cell and leaving an empty
capsid outside. The cell’s
DNA is hydrolyzed.
Phage assembly
4 Assembly. Three separate sets of proteins
self-assemble to form phage heads, tails,
and tail fibers. The phage genome is
packaged inside the capsid as the head forms.
Head
Tails
Tail fibers
3
Synthesis of viral genomes
and proteins. The phage DNA
directs production of phage
proteins and copies of the phage
genome by host enzymes, using
components within the cell.
The Lysogenic Cycle
•
The lysogenic cycle replicates the phage genome without destroying the host
•
The viral DNA molecule is incorporated by genetic recombination 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 it to the daughter cells
•
Phages that use both the lytic and lysogenic cycles are called temperate phages
Phage
DNA
The phage attaches to a
host cell and injects its
DNA.
Phage DNA
circularizes
Phage
Occasionally, a prophage
exits the bacterial chromosome,
initiating a lytic cycle.
Bacterial
chromosome
Lytic cycle
The cell lyses, releasing phages.
Lysogenic
cycle
Certain factors
determine whether
Lytic cycle
Lysogenic cycle
or
is induced
is entered
New phage DNA and
proteins are synthesized
and assembled into phages.
Prophage
Many cell divisions
produce a large
population of bacteria
infected with the
prophage.
The bacterium reproduces
normally, copying the prophage
and transmitting it to daughter cells.
Phage DNA integrates into
the bacterial chromosome,
becoming a prophage.
Viral Classification
• The nature of the genome is
the basis for the common
classification of animal
viruses
3 patterns of viral replication
1. DNA  DNA: If viral DNA is double-stranded, DNA
replication resembles that of cellular DNA, and the
virus uses DNA polymerase produced by the host.
2. RNA  RNA: Since host cells lack the enzyme to
copy RNA, most RNA viruses contain a gene that
codes for RNA replicase, an enzyme that uses viral
RNA as a template to produce complementary RNA.
3. RNA  DNA  RNA: Some RNA viruses encode
reverse transcriptase, an enzyme that transcribes
DNA from a RNA template.
RNA As Genetic Material - Retroviruses / Proviruses
•
The broadest variety of RNA genomes is found among the viruses that
infect animals
•
Retroviruses, such as HIV, use the enzyme reverse transcriptase to
copy their RNA genome into DNA
•
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
Glycoprotein
Viral envelope
Capsid
Reverse
transcriptase
RNA
(two identical
strands)
The Reproductive Cycle Of An Enveloped RNA Virus
•
The host’s 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
1 Glycoproteins on the viral envelope
bind to specific receptor molecules
(not shown) on the host cell,
promoting viral entry into the cell.
Capsid
RNA
Envelope (with
glycoproteins)
2 Capsid and viral genome
enter cell
HOST CELL
Viral genome (RNA)
Template
Complementary RNA
5
strands also
function as mRNA,
which is translated into both
capsid proteins (in the cytosol)
and glycoproteins for the viral
envelope (in the ER).
The viral genome (red)
3
functions as a template for
synthesis of complementary
RNA strands (pink) by a viral
enzyme.
mRNA
Capsid
proteins
ER
Glycoproteins
6Vesicles transport
envelope glycoproteins to
the plasma membrane.
A
7 capsid assembles
around each viral
genome molecule.
Copy of
genome (RNA)
4 New copies of viral
genome RNA are made
using complementary RNA
strands as templates.
8 New virus
The Reproductive Cycle Of HIV, A Retrovirus
HIV
Membrane of
white blood cell
1 The virus fuses with the
cell’s plasma membrane.
The capsid proteins are
removed, releasing the
viral proteins and RNA.
2 Reverse transcriptase
catalyzes the synthesis of a
DNA strand complementary
to the viral RNA.
HOST CELL
3 Reverse transcriptase
catalyzes the synthesis of
a second DNA strand
complementary to the first.
Reverse
transcriptase
Viral RNA
RNA-DNA
hybrid
4 The double-stranded
DNA is incorporated
as a provirus into the cell’s
DNA.
0.25 µm
HIV entering a cell
DNA
NUCLEUS
Chromosomal
DNA
RNA genome
for the next
viral generation
Provirus
5 Proviral genes are
transcribed into RNA molecules,
which serve as genomes for the
next viral generation and as
mRNAs for translation into viral
proteins.
mRNA
6
The viral proteins include capsid
proteins and reverse transcriptase
(made in the cytosol) and envelope
glycoproteins (made in the ER).
New HIV leaving a cell
9 New viruses bud
off from the host cell.
8 Capsids are
assembled around
viral genomes and
reverse transcriptase
molecules.
7 Vesicles transport the
glycoproteins from the ER to
the cell’s plasma membrane.
Viral Diseases in Animals
•
•
•
Viruses, viroids, and prions are formidable pathogens in animals and plants
Viruses may damage or kill cells by causing the release of hydrolytic enzymes from
lysosomes
Some viruses cause infected cells to produce toxins that lead to disease symptoms
•
Emerging viruses are those that appear suddenly or suddenly come to the attention of
medical scientists
•
Outbreaks of “new” viral diseases in humans are usually caused by existing viruses that
expand their host territory
•
Severe acute respiratory syndrome (SARS) recently appeared in China
(a) Young ballet students in Hong Kong
wear face masks to protect themselves
from the virus causing SARS.
(b) The SARS-causing agent is a coronavirus
like this one (colorized TEM), so named for the
“corona” of glycoprotein spikes protruding from
the envelope.
Bacterial Genetics
• Rapid reproduction, mutation, and genetic
recombination contribute to the genetic diversity of
bacteria
• Bacteria allow researchers to investigate molecular
genetics in the simplest true organisms
• The bacterial chromosome is usually a circular DNA
molecule with few associated proteins
• In addition to the chromosome, many bacteria have
plasmids, smaller circular DNA molecules that can
replicate independently of the bacterial chromosome
Mutation and Genetic Recombination
•
Since bacteria can reproduce rapidly new mutations can quickly increase a population’s
genetic diversity
•
Further genetic diversity can arise by recombination of the DNA from two different
bacterial cells
EXPERIMENT Researchers had two mutant strains, one that could make arginine but not tryptophan (arg+ trp–) and one
that could make tryptophan but not arginine (arg trp+). Each mutant strain and a mixture of both strains were grown in a
liquid medium containing all the required amino acids. Samples from each liquid culture were spread on plates containing a
solution of glucose and inorganic salts (minimal medium), solidified with agar.
Mixture
Mutant
strain
arg+ trp–
Mutant
strain
arg trp+
Only the samples from the mixed culture, contained cells that gave rise to colonies on
RESULTS
minimal medium, which lacks amino acids.
Mixture
Mutant
strain
arg+ trp–
Mutant
strain
arg– trp+
No
colonies
(control)
Colonies
grew
No
colonies
(control)
Because only cells that can make both arginine and tryptophan (arg+ trp+ cells) can grow into colonies on
CONCLUSION
minimal medium, the lack of colonies on the two control plates showed that no further mutations had occurred restoring this
ability to cells of the mutant strains. Thus, each cell from the mixture that formed a colony on the minimal medium must have
acquired one or more genes from a cell of the other strain by genetic recombination.
Mechanisms of Gene Transfer and Genetic
Recombination in Bacteria
• Three processes bring bacterial DNA from different
individuals together
•
Transformation - Is the alteration of a bacterial cell’s
genotype and phenotype by the uptake of naked,
foreign DNA from the surrounding environment
•
Transduction - Phages carry bacterial genes from one
host cell to another
•
Conjugation - Is the direct transfer of genetic material
between bacterial cells that are temporarily joined
Transformation
• Transformation is the alteration of a bacterial cell’s
genotype and phenotype by the uptake of naked,
foreign DNA from the surrounding environment
• For example, harmless Streptococcus pneumoniae
bacteria can be transformed to pneumonia-causing
cells
Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells (control)
Mixture of heat-killed
S cells and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample
Transduction
Phage DNA
1
Phage infects bacterial cell that has alleles A+ and B+
2
Host DNA (brown) is fragmented, and phage DNA
and proteins are made. This is the donor cell.
A+ B+
A+ B +
Donor
cell
3
A bacterial DNA fragment (in this case a fragment with
the A+ allele) may be packaged in a phage capsid.
A+
4
A+
Phage with the
allele from the donor cell infects
a recipient A–B– cell, and crossing over (recombination)
between donor DNA (brown) and recipient DNA
(green) occurs at two places (dotted lines).
Crossing
over
A+
A– B–
Recipient
cell
5
The genotype of the resulting recombinant cell (A+B–)
differs from the genotypes of both the donor (A+B+) and
the recipient (A–B–).
A+ B–
Recombinant cell
Conjugation and Plasmids
• Conjugation is the direct transfer of genetic material between
bacterial cells that are temporarily joined
• The transfer is one-way: One cell (“male”) donates DNA, and its
“mate” (“female”) receives the genes
• “Maleness,” the ability to form a sex pilus and donate DNA,
results from an F (for fertility) factor as part of the chromosome
or as a plasmid
• Plasmids, including the F plasmid, are small, circular, selfreplicating DNA molecules
The F Plasmid and Conjugation
•
•
•
Cells containing the F plasmid, designated F+ cells, function as DNA
donors during conjugation
F+ cells transfer DNA to an F recipient cell
Chromosomal genes can be transferred during conjugation when the
donor cell’s F factor is integrated into the chromosome
1 A cell carrying an F plasmid
(an F+ cell) can form a
mating bridge with an F– cell
and transfer its F plasmid.
F plasmid
2
A single strand of the
F plasmid breaks at a
specific point (tip of blue
arrowhead) and begins to
move into the recipient cell.
As transfer continues, the
donor plasmid rotates
(red arrow).
3 DNA replication occurs in
both donor and recipient
cells, using the single
parental strands of the
F plasmid as templates
to synthesize complementary
strands.
4 The plasmid in the
recipient cell
circularizes. Transfer
and replication result
in a compete F plasmid
in each cell. Thus, both
cells are now F+.
Bacterial chromosome
F+ cell
Mating
bridge
F– cell
F+ cell
F+ cell
Bacterial
chromosome
Conjunction and transfer of an F plasmid from and F+ donor to an F– recipient
The F Plasmid and Conjugation
•
A cell with a built-in F factor is called an Hfr cell
•
The F factor of an Hfr cell brings some chromosomal DNA along when transferred to an
F– cell
•
Thr transfer of part of the bacterial chromosome from an Hfr donor to an F– recipient
results in recombination
Hfr cell
F+ cell
F factor
+
1 The circular F plasmid in an F cell
can be integrated into the circular
chromosome by a single crossover
event (dotted line).
B+
Hfr cell
A+
C+
D+
C+
B+
D+
2 The resulting cell is called an Hfr cell
(for High frequency of recombination).
D+
A+
C+
B+
A+
D+
C+
B+
A+
A+
B+
F– cell
B–
C–
A–
D–
B–
3 Since an Hfr cell has all
4
the F-factor genes, it can
form a mating bridge with
an F– cell and transfer DNA.
C–
A–
A+
D–
B–
C–
B+
D–
A–
A+
A single strand of the F factor 5 The location and orientation
of the F factor in the donor
breaks and begins to move
chromosome determine
through the bridge. DNA
the sequence of gene transfer
replication occurs in both donor
during conjugation. In this
and recipient cells, resulting in
example, the transfer sequence
double-stranded DNA
for four genes is A-B-C-D.
Temporary
partial
diploid
B+
B–
A+
7 Two crossovers can result
in the exchange of similar
(homologous) genes between
the transferred chromosome fragment
(brown) and the recipient cell’s
chromosome (green).
C–
A–
B–
D–
A+
B+
C–
A–
D–
B–
C–
D–
A–
6 The mating bridge
usually breaks well
before the entire
chromosome and
the rest of the
F factor are transferred.
Recombinant F–
bacterium
8 The piece of DNA ending up outside the
bacterial chromosome will eventually be
degraded by the cell’s enzymes. The recipient
cell now contains a new combination of genes
but no F factor; it is a recombinant F– cell.
R plasmids and Antibiotic Resistance
• R plasmids confer resistance to various antibiotics
• When a bacterial population is exposed to an
antibiotic, individuals with the R plasmid will survive
and increase in the overall population
Transposition of Genetic Elements
• The DNA of a cell can also undergo recombination
due to movement of transposable elements within the
cell’s genome
• Transposable elements:
•
Can move around within a cell’s genome
•
Are often called “jumping genes”
•
Contribute to genetic shuffling in bacteria
Insertion Sequences
• The simplest transposable elements, called insertion
sequences, exist only in bacteria
• An insertion sequence contains a single gene for transposase,
an enzyme that catalyzes movement of the insertion sequence
from one site to another within the genome
Insertion sequence
3
A T C C G G T…
A C C G G A T…
3
5
TAG G C CA…
TG G C CTA…
5
Transposase gene
Inverted
Inverted
repeat
repeat
(a) Insertion sequences, the simplest transposable elements in bacteria, contain a single gene that
encodes transposase, which catalyzes movement within the genome. The inverted repeats are
backward, upside-down versions of each other; only a portion is shown. The inverted repeat
sequence varies from one type of insertion sequence to another.
Figure 18.19a
Transposons
• Transposable elements called transposons are longer
and more complex than insertion sequences
• In addition to DNA required for transposition,
transposons have extra genes that “go along for the
ride,” such as genes for antibiotic resistance
Transposon
Insertion
sequence
Antibiotic
resistance gene
Insertion
sequence
5
5
3
3
Inverted repeats
Transposase gene
(b) Transposons contain one or more genes in addition to the transposase gene. In the transposon
shown here, a gene for resistance to an antibiotic is located between twin insertion sequences.
The gene for antibiotic resistance is carried along as part of the transposon when the transposon
is inserted at a new site in the genome.
Figure 18.19b
Control of Gene Expression
• Every cell contains thousands of genes which code for
proteins.
• However, every gene is not actively producing proteins at all
times.
• To be expressed, a gene must be transcribed into m-RNA, the
m-RNA must be translated into a protein, and the protein must
become active.
• Gene regulation can theoretically occur at any step in this
process
Control of Gene Expression
• Two categories of gene regulation:
•
Transcriptional controls - factors that regulate
transcription
•
Post-transcriptional controls – factors that regulate
any step in gene expression after transcription is
complete
• It is most efficient to regulate genes during
transcription.
• Both prokaryotes and eukaryotes rely primarily on
transcriptional controls.
Regulating Prokaryotic Gene Expression
•
Prokaryotes can quickly turn genes on and off in response to
environmental conditions.
•
This metabolic control occurs on two levels
•
Adjusting the activity of metabolic enzymes already present
•
Regulating the genes encoding the metabolic enzymes
(a) Regulation of enzyme
activity
Precursor
Feedback
inhibition
(b) Regulation of enzyme
production
Enzyme 1
Gene 1
Enzyme 2
Gene 2
Enzyme 3
Gene 3
Regulation
of gene
expression
–
Enzyme 4 Gene 4
–
Enzyme 5
Tryptophan
Gene 5
Prokaryotic Gene Regulation
•
•
Response is facilitated by:
•
Simultaneous transcription and translation
•
Short-lived m-RNAs
•
Operons
Functionally related genes are often located next to each other and
are transcribed as a unit.
• For example E. coli,
•
5 different enzymes are needed to synthesize the amino acid
tryptophan
•
The genes that code for these enzymes are located together
Operons: The Basic Concept
• In bacteria, genes are often clustered into operons,
composed of
•
An operator, an “on-off” switch
•
A promoter
•
Genes for metabolic enzymes
• An operon
•
Is usually turned “on”
•
Can be switched off by a protein called a repressor
Prokaryotic Gene Regulation
• A single promoter serves all 5 genes. (region where RNA
polymerase binds to DNA and begins transcription)
• The genes are transcribed as a unit, - one long mRNA
molecule which contains the code to make all 5 enzymes
trp operon
Promoter
DNA
Regulatory
gene
mRNA
5
Promoter
Genes of operon
trpD
trpC
trpE
trpR
3
Operator
RNA
Start codon
polymerase mRNA 5
Inactive
repressor
trpA
Stop codon
E
Protein
trpB
D
C
B
A
Polypeptides that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at
the
promoter and transcribes the operon’s genes.
Figure 18.21a
Prokaryotic Gene Regulation
•
There is also a single regulatory switch, called the operator.
•
The operator is positioned within the promoter, or between the
promoter and the protein coding genes. It controls access of RNA
polymerase to the genes.
trp operon
Promoter
DNA
Regulatory
gene
mRNA
5
Promoter
Genes of operon
trpD
trpC
trpE
trpR
3
trpA
Operator
Start codon Stop
RNA
codon
polymerase mRNA 5
E
Protein
trpB
Inactive
repressor
D
C
B
A
Polypeptides that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at
the promoter and transcribes the operon’s genes.
Prokaryotic Gene Regulation
• Transcription of the 5 coding genes in the tryptophan operon is
blocked when a transcriptional repressor binds to the operator.
• The repressor binds to the operator only when there is a high
level of tryptophan present:
DNA
No RNA made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off. As tryptophan
accumulates, it inhibits its own production by activating the repressor protein.
Two Types of Negative Gene Regulation
• In a repressible operon, binding of a specific repressor
protein to the operator shuts off transcription
• Repressible enzymes usually function in anabolic
pathways
• In an inducible operon, binding of an inducer to a
repressor inactivates the repressor and turns on
transcription
• Inducible enzymes usually function in catabolic
pathways
Prokaryotic Gene Regulation: Inducible Operon
•
The lactose operon in E. coli is an inducible operon
•
It controls the production of 3 enzymes needed to digest lactose
(catabolism of a disaccharide made of glucose and galactose)
•
When lactose is absent, the repressor is active and the operon is off.
•
The lac repressor is innately active, and in the absence of lactose it
switches off the operon by binding to the operator.
Promoter
Regulatory
gene
DNA
mRNA
Protein
(a)
Operator
lacl
5
lacZ
3 RNA
polymerase
Active
repressor
No
RNA
made
lac Operon
•
•
If lactose is present, the repressor is inactivated and the operon is on
Allolactose, an isomer of lactose, turns on the operon by inactivating
the repressor. In this way, the enzymes for lactose utilization are
induced.
lac operon
DNA
lacl
lacz
3
mRNA
lacY
lacA
mRNA 5'
5
RNA
mRNA
polymerase
5
-Galactosidase
Protein
Allolactose
(inducer)
Inactive
repressor
Permease
Transacetylase
Positive Gene Regulation
• Regulation of both the trp and lac operons involves
the negative control of genes, because the operons
are switched off by the active form of the repressor
protein
• Some operons are also subject to positive control via
a stimulatory activator protein, such as catabolite
activator protein (CAP)
• CAP (catabolite activator protein) stimulates
transcription of genes that allow E. coli to use other
food sources when glucose is not present such as
lactose
Positive Transcriptional Control
•
In E. coli, when glucose is scarce, the lac operon is activated by the
binding of a regulatory protein, catabolite activator protein (CAP)
•
Low levels of glucose lead to high levels of cAMP
•
cAMP binds to CAP, CAP binds to CAP binding site, and
transcription of lac mRNA is stimulated for catabolism of lactose
Promoter
DNA
lacl
lacZ
CAP-binding site
cAMP
Inactive
CAP
RNA
Operator
polymerase
can bind
Active
and transcribe
CAP
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.
If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces
large amounts of mRNA for the lactose pathway.
Positive Transcriptional Control
• When the glucose level is high, cAMP is low. CAP is
not activated and transcription is not stimulated:
Promoter
lacl
• WhenDNA
glucose levels
in an E. coli cell increase,lacZ
CAP
detaches from
the lac
CAP-binding
site operon, turning it off
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.
When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.
Lab 9A/B - How Are Plasmids Used In
Recombinant DNA Technology
Recombinant DNA
• Formed by joining DNA from 2 different individuals into
a single molecule.
• Various natural mechanisms can combine DNA from 2
individuals of the same species
• Scientists have also developed techniques to combine
DNA from any 2 individuals.
Recombinant DNA
•
Two key enzymes are used to make artificially
recombined DNA.
•
•
Restriction enzymes (also called restriction endonucleases)
cut DNA into fragments – so called “molecular scissors”

Each one recognizes and cuts DNA only where a specific
sequence of base pairs occurs

A restriction enzyme will usually make many cuts in a DNA
molecule yielding a set of restriction fragments

The most useful restriction enzymes cut DNA in a staggered
way leaving unpaired bases at both ends.

These fragments are called “sticky ends” and can bond with
complementary “sticky ends” of other fragments
DNA ligase is used to join DNA fragments together. This is the
“molecular glue”
Procedure for Recombining DNA
•
•
•
•
Restriction site
Isolate DNA from 2 different sources
Cut the DNA from both sources into
fragments using the same restriction
enzyme.
Mix the DNA fragments together.
Since they were cut with the same
restriction enzyme, fragments from
different sources will have the same
“sticky ends” and can pair up.
Use the enzyme DNA ligase to join
the paired fragments together
DNA 5
3
3
5
GAATTC
CTTAAG
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow
G
G
Sticky end
2 DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
G AATT C
C TTAA G
G
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATTC
CTTAA G
One possible combination
3 DNA ligase
seals the strands.
Recombinant DNA molecule
Recombinant Plasmids
• Recombinant DNA technology can be used to
create recombinant plasmids (or other agents such
as viruses) used to insert foreign genes into
recipient cells.
• Plasmids (or other recombinant agents) used to
insert foreign DNA into recipient cells are called
vectors
• Recombinant plasmids can then be used to
produce multiple copies of the DNA fragment
Lab 9-A
•
Transformation – bacteria absorb fragments of DNA from
surrounding media
•
Transform E. Coli with 3 unknown media samples
•
One solution contains no DNA at all
•
One solutuion contains normal pUC18 plasmid
•
•
Gene for ampicillin resistance
•
Lac Z gene which codes for -galactosidase, lactose
digestion enzyme
One solution contains recombinant pUC18
•
Contains a fragment of foreign DNA from  phage
•
Inserted in to middle of Lac Z gene, inactivating it
Transformation Procedure
• Add E. Coli to all three unknown solutions
• Chill then “heat shock” samples to facilitate uptake of
plasmid
• Incubate then inoculate agar plates
• Agar plates contain nutrients, ampicillin, Xgal (analog
of lactose that release blue color when digested)
• Results?
Using Restriction Enzyme EcoRI
• Procedure will cut the plasmids in the three unknown
samples with the restriction enzyme EcoRI
• Add EcoRI to the three unknown plasmid stock
solutions and incubate
• Separate the DNA fragments using gel electrophresis
•
Small fragments move faster farther
•
Similar to proteins except instead of MW we use base
pairs (bp) to reference size
• Results?
Plasmid pUC18
• 2686 base pairs in size
bp 1
Laz Z gene: bp 236 - 469
EcoRI site: bp 396
Ampicillin resistance
gene: bp 1626 - 2486
bp 2014
bp 671
bp 1343
Plasmid pUC18
• 2686 base pairs in size
Laz Z gene: bp 236 - 469
bp 1
Phage DNA inserted
Ampicillin resistance
gene: bp 1626 - 2486
(Not to scale!)
bp 2014
bp 671
bp 1343