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18
Recombinant DNA
and Biotechnology
18 Recombinant DNA and Biotechnology
18.1 What Is Recombinant DNA?
18.2 How Are New Genes Inserted into Cells?
18.3 What Sources of DNA Are Used in
Cloning?
18.4 What Other Tools Are Used to Study
DNA Function?
18.5 What Is Biotechnology?
18.6 How Is Biotechnology Changing Medicine
and Agriculture?
18 Recombinant DNA and Biotechnology
Bioremediation is the use of microorganisms
to remove pollutants.
Some microbes can digest some
components of crude oil, but researchers
are developing genetically modified
organisms that can clean up oil more
rapidly and effectively.
Opening Question:
Are there other uses for microorganisms in
environmental cleanup?
18.1 What Is Recombinant DNA?
Recombinant DNA is a DNA molecule
made in the laboratory using at least
two different sources of DNA.
Restriction enzymes and DNA ligase
are used to cut DNA into fragments
and then splice them together in new
combinations.
18.1 What Is Recombinant DNA?
The first recombinant DNA was made
in 1973 using plasmids from E. coli.
This research was the start of
recombinant DNA technology.
Figure 18.1 Recombinant DNA
18.1 What Is Recombinant DNA?
Some restriction enzymes recognize
palindromic DNA sequences:
5′…….GAATTC……3′
3′…….CTTAAG……5′
Some make straight cuts, others make
staggered cuts, resulting in
overhangs, or sticky ends.
18.1 What Is Recombinant DNA?
Sticky ends can bind by base pairing to
other sticky ends.
Fragments from different sources can
be joined.
Then ligase catalyzes formation of
covalent bonds between adjacent
nucleotides at fragment ends, joining
them to form a single, larger molecule.
Working with Data 18.1: Recombinant DNA
In 1973, the first recombinant plasmid
was made using the restriction
enzyme EcoRI and two plasmids with
resistance to different antibiotics:
• pSC101 had a gene for tetracycline
resistance.
• pSC102 had a gene for kanamycin
resistance.
Working with Data 18.1: Recombinant DNA
Question 1:
In one experiment, some pSC101 was
cut with EcoRI but not sealed with DNA
ligase.
Cut or intact pSC101 were used to
transform E. coli cells, which were grown
on media containing tetracycline or
kanamycin.
What can you conclude from this
experiment?
Working with Data 18.1: Recombinant DNA
Working with Data 18.1: Recombinant DNA
Question 2:
In another experiment, pSC101 and
pSC102 were mixed and treated in
three ways:
Working with Data 18.1: Recombinant DNA
Did treatment with DNA ligase improve
the efficiency of genetic
transformation by the cut plasmids?
What is the quantitative evidence for
your statement?
Working with Data 18.1: Recombinant DNA
Question 3:
How did the antibiotic-resistant
bacteria arise in the “None” DNA
treatment?
Working with Data 18.1: Recombinant DNA
Question 4:
Did the EcoRI + DNA ligase treatment
result in an increase in doublyresistant bacteria over controls?
What data provide evidence for your
statement?
Working with Data 18.1: Recombinant DNA
Question 5:
For the EcoRI + DNA ligase
treatment, compare the number of
transformants that were resistant to
either tetracycline or kanamycin alone
to the number that were doubly
resistant.
What accounts for the large
difference?
Figure 18.2 Cutting, Splicing, and Joining DNA
18.2 How Are New Genes Inserted into Cells?
Recombinant DNA technology can be
used to clone, or make identical
copies, of genes.
Transformation: recombinant DNA is
cloned by inserting it into host cells
(transfection if host cells are from an
animal).
The altered host cell is called
transgenic.
18.2 How Are New Genes Inserted into Cells?
Usually only a few cells are
transformed.
To determine which of the host cells
contain the new sequence, the
recombinant DNA includes selectable
marker genes, such as genes that
confer resistance to antibiotics.
18.2 How Are New Genes Inserted into Cells?
The first host cells used were bacteria,
especially E. coli.
Yeasts (Saccharomyces) are
commonly used as eukaryotic hosts.
Plant cells are also used—they have
the ability to make stem cells
(unspecialized, totipotent cells).
18.2 How Are New Genes Inserted into Cells?
Cultured animal cells can be used to
study expression of human or animal
genes.
Whole transgenic animals can also be
created.
18.2 How Are New Genes Inserted into Cells?
Inserting the recombinant DNA into a
cell:
• Cells may be treated with chemicals
to make plasma membranes more
permeable—DNA diffuses in.
• Electroporation—a short electric
shock creates temporary pores in
membranes, and DNA can enter.
18.2 How Are New Genes Inserted into Cells?
• Viruses can be altered to carry
recombinant DNA into cells.
• Plants are often transformed using a
bacterium that inserts DNA into plant
cells.
• Transgenic animals can be produced
by injecting recombinant DNA into the
nuclei of fertilized eggs.
18.2 How Are New Genes Inserted into Cells?
The new DNA must also replicate as
the host cell divides.
It must become part of a segment with
an origin of replication—a replicon or
replication unit.
18.2 How Are New Genes Inserted into Cells?
New DNA can become part of a
replicon in two ways:
• Inserted near an origin of replication in
host chromosome
• Part of a carrier sequence, or vector,
that already has an origin of
replication
18.2 How Are New Genes Inserted into Cells?
Plasmids make good vectors:
• Small and easy to manipulate
• Have one or more restriction enzyme
recognition sequences that each
occur only once
• Many have genes for antibiotic
resistance that can be used as
selectable markers
18.2 How Are New Genes Inserted into Cells?
• Have a bacterial origin of replication
(ori) and can replicate independently
of the host chromosome
Bacterial cells can contain hundreds
of copies of a recombinant plasmid.
The power of bacterial transformation
to amplify a gene is extraordinary.
In-Text Art, Ch. 18, p. 377 (1)
18.2 How Are New Genes Inserted into Cells?
A plasmid from the soil bacterium
Agrobacterium tumefaciens is used as
a vector for plant cells.
Plasmid Ti (tumor inducing) causes
crown gall.
The plasmid has a region called T
DNA, which inserts copies of itself into
chromosomes of infected plants.
In-Text Art, Ch. 18, p. 377 (2)
18.2 How Are New Genes Inserted into Cells?
T DNA genes are removed and
replaced with foreign DNA.
Altered Ti plasmids transform
Agrobacterium cells, then the
bacterium cells infect plant cells.
Whole plants can be regenerated from
transgenic cells, or germ line cells can
be infected.
18.2 How Are New Genes Inserted into Cells?
Most eukaryotic genes are too large to
be inserted into a plasmid.
Viruses can be used as vectors (e.g.,
bacteriophage).
Because viruses infect cells naturally,
they offer a great advantage over
plasmids.
18.2 How Are New Genes Inserted into Cells?
Usually only a small proportion of host
cells take up the vector, and they may
not have the appropriate sequence.
Host cells with the desired sequence
must be identifiable.
Selectable markers such as antibiotic
resistance genes can be used.
18.2 How Are New Genes Inserted into Cells?
Selectable markers or reporter genes:
genes whose expression is easily
observed.
There are several types:
• Antibiotic resistance in a plasmid or
other vector. A transformed host cell
will grow on medium containing the
antibiotic.
18.2 How Are New Genes Inserted into Cells?
• The lacZ gene codes for an enzyme
that can convert the substrate X-Gal
into a bright blue product.

If foreign DNA is inserted within the
lacZ gene, and the plasmid transforms
bacterial cells, they will not be able to
convert X-Gal, and will produce white
colonies. Untransformed cells produce
blue colonies.
Figure 18.3 Selection for Recombinant DNA
18.2 How Are New Genes Inserted into Cells?
• Green fluorescent protein (GFP),
which normally occurs in a jellyfish,
emits visible light when exposed to
UV light.

The gene for this protein has been
isolated and incorporated into vectors
as a reporter gene.

It has also been modified to produce
other colors.
Figure 18.4 Green Fluorescent Protein as a Reporter
18.3 What Sources of DNA Are Used in Cloning?
DNA fragments used for cloning come
from four sources:
• Gene libraries
• Reverse transcription from mRNA
• Products of PCR
• Artificial synthesis or mutation of DNA
18.3 What Sources of DNA Are Used in Cloning?
A genomic library is a collection of
DNA fragments that comprise the
genome of an organism.
The DNA is cut into fragments by
restriction enzymes, and each
fragment is inserted into a vector,
which is used to produce a colony of
recombinant cells.
Figure 18.5 Constructing Libraries
18.3 What Sources of DNA Are Used in Cloning?
If bacteriophage λ is used as a vector,
about 160,000 “volumes” are required
to store the library.
One petri plate can hold thousands of
phage colonies, or plaques.
DNA in the plaques is screened using
specific probes.
18.3 What Sources of DNA Are Used in Cloning?
Smaller DNA libraries can be made
from complementary DNA (cDNA).
mRNA is extracted from cells, then
cDNA is produced by complementary
base pairing, catalyzed by reverse
transcriptase.
18.3 What Sources of DNA Are Used in Cloning?
mRNAs do not last long in the
cytoplasm and are often present in
small amounts, so a cDNA library is a
“snapshot” of the transcription pattern
of the cell.
cDNA libraries are used to compare
gene expression in different tissues at
different stages of development.
18.3 What Sources of DNA Are Used in Cloning?
RT-PCR: reverse transcriptase and
PCR are used to create and amplify a
specific cDNA sequence.
This is used to study expression of
particular genes in cells and
organisms.
18.3 What Sources of DNA Are Used in Cloning?
Artificial DNA with specific sequences
can be synthesized by PCR.
The process is now fully automated
and is used to create PCR primers
and DNA with specific characteristics,
such as restriction sites or specific
mutations.
Fragments can be pieced together to
form artificial genes.
18.4 What Other Tools Are Used to Study DNA Function?
A way to study a gene and its protein:
express it in cells that do not normally
express the gene or in a different
organism.
The gene must have a promoter and
regulatory sequences for the host cell.
18.4 What Other Tools Are Used to Study DNA Function?
Another way to study a gene:
overexpress it so that more product is
made.
A copy of the coding region is inserted
downstream of a different, stronger
promoter, and cells are transformed
with the recombinant DNA.
18.4 What Other Tools Are Used to Study DNA Function?
Mutations can be created in the
laboratory in synthetic DNA.
Consequences of the mutation can be
observed when the mutant DNA is
expressed in host cells.
18.4 What Other Tools Are Used to Study DNA Function?
Genes can also be studied by
inactivating them (e.g., transposon
mutagenesis) to define the minimal
genome.
In animals, this is called a knockout
experiment.
18.4 What Other Tools Are Used to Study DNA Function?
Homologous recombination can
knock out a specific gene.
Homologous recombination occurs
during meiosis or as part of the DNA
repair process.
18.4 What Other Tools Are Used to Study DNA Function?
The normal allele of a gene is inserted
into a plasmid, with a reporter gene in
the middle of the normal allele.
The recombinant plasmid transfects
mouse embryonic stem cells.
The sequences line up with
homologous sequences, and if
recombination occurs, the normal
allele is lost because the plasmid
cannot replicate in mouse cells.
Figure 18.6 Making a Knockout Mouse
18.4 What Other Tools Are Used to Study DNA Function?
The transfected stem cell is
transplanted into an early mouse
embryo.
The mouse and its progeny will have
the inactive allele in all cells. The mice
are inbred to produce a homozygous
line.
Phenotypic changes provide clues to
the normal allele function.
18.4 What Other Tools Are Used to Study DNA Function?
Complementary RNA:
Translation of mRNA can be blocked
by complementary micro RNAs—
antisense RNA.
Antisense RNA can be synthesized and
added to cells to prevent translation—
the effects of the missing protein can
then be determined.
18.4 What Other Tools Are Used to Study DNA Function?
Interference RNA (RNAi) is a natural
mechanism that blocks translation.
Short, double stranded RNA is
unwound and binds to complementary
mRNA by a protein complex, which
also catalyzes the breakdown of the
mRNA.
Small interfering RNA (siRNA) can be
synthesized in the laboratory to inhibit
gene expression.
Figure 18.7 Using Antisense RNA and siRNA to Block Translation of mRNA
18.4 What Other Tools Are Used to Study DNA Function?
DNA microarray technology provides
a large array of sequences for
hybridization experiments.
DNA sequences are attached to a
glass slide in a precise order.
The slide has microscopic wells which
each contain thousands of copies of
sequences up to 20 nucleotides long.
Figure 18.8 DNA Microarray for Medical Diagnosis
18.4 What Other Tools Are Used to Study DNA Function?
DNA microarrays have been developed
to identify gene expression patterns in
women with a propensity for breast
cancer tumors to recur—a gene
expression signature.
18.5 What Is Biotechnology?
Biotechnology is the use of living cells
or organisms to produce materials
useful to people.
Examples:
• Using yeasts to brew beer and wine
• Using bacteria to make cheese,
yogurt, etc.
• Using microbes to produce antibiotics,
alcohol, and other products
18.5 What Is Biotechnology?
Gene cloning is now used to produce
proteins in large amounts.
Almost any gene can be inserted into
bacteria or yeasts, and the resulting
cells are induced to make large
quantities of the product.
Requires specialized vectors.
18.5 What Is Biotechnology?
Expression vectors include all the
sequences needed for expression of a
transgene in a host cell, including
promoters, termination signals, poly
A–addition sequences, etc.
Figure 18.9 Expression of a Transgene in a Host Cell Produces Large Amounts of Its Protein
Product
18.5 What Is Biotechnology?
Expression vectors may also have:
• Inducible promoters that respond to a
specific signal
• Tissue-specific promoters expressed
only in certain tissues at certain times
• Signal sequences (e.g., a signal to
secrete the product to the extracellular
medium)
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Many medically useful products are
being made using biotechnology.
Example: The manufacture of tissue
plasminogen activator (TPA).
Table 18.1
18.6 How Is Biotechnology Changing Medicine and Agriculture?
After wounds heal, blood clots are
dissolved by plasmin. Plasmin is
stored as an inactive form called
plasminogen.
Conversion of plasminogen is activated
by TPA.
TPA can be used to treat strokes and
heart attacks. The large quantities
needed can be made using
recombinant DNA technology.
Figure 18.10 Tissue Plasminogen Activator
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Pharming: Production of
pharmaceuticals in farm animals or
plants.
Example: Transgenes are inserted next
to the promoter for lactoglobulin—a
protein in milk. The transgenic animal
then produces large quantities of the
protein in its milk.
Figure 18.11 Pharming
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Human growth hormone (for children
suffering deficiencies) can now be
produced by transgenic cows.
Only 15 such cows are needed to
supply all the children in the world
suffering from this type of dwarfism.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Through cultivation and selective
breeding, humans have been altering
the traits of plants and animals for
thousands of years.
Recombinant DNA technology has
several advantages:
• Specific genes can be targeted
• Any gene can be introduced into any
other organism
• New organisms can be generated
Figure 18.12 Genetic Modification of Plants versus Conventional Plant Breeding
Table 18.2
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Crop plants have been modified to
produce their own insecticides:
• The bacterium Bacillus thuringiensis
produces a protein that kills insect
larvae.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Dried preparations of B. thuringiensis
are an alternative to insecticides. The
toxin is easily biodegradable.
Genes for the toxin have been isolated,
cloned, and inserted into plant cells
using the Ti plasmid vector.
Transgenic corn, cotton, soybeans,
tomatoes, and other crops are being
grown. Pesticide use is reduced.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Some transgenic crops are resistant to
herbicides.
• Glyphosate is widely used to kill
weeds.
• Expression vectors have been used to
make plants that synthesize so much
of the target enzyme of glyphosate
that they are unaffected by the
herbicide.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
• The gene has been inserted into corn,
soybeans, and cotton.
• The crops can be sprayed with
glyphosate, and only the weeds will
be killed.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Crops with improved nutritional
characteristics:
• Rice does not have -carotene, but
does have a precursor molecule.
• Genes for enzymes that synthesize βcarotene from the precursor are taken
from daffodils or corn and inserted
into rice by the Ti plasmid.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
• The transgenic rice is yellow, and can
supply -carotene to improve the diets
of many people.
• -carotene is converted to vitamin A in
the body.
Figure 18.13 Transgenic Rice Rich in -Carotene
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Recombinant DNA is also used to
adapt a crop plant to an environment.
Example: Plants that are salt-tolerant.
Genes from a protein that moves
sodium ions into the central vacuole
were isolated from Arabidopsis
thaliana and inserted into tomato
plants.
Figure 18.14 Salt-Tolerant Tomato Plants
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Instead of manipulating the
environment to suit the plant,
biotechnology may allow us to adapt
the plant to the environment.
Some of the negative effects of
agriculture, such as water pollution,
could be reduced.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Concerns over biotechnology:
• Genetic manipulation is an unnatural
interference in nature.
• Genetically altered foods are unsafe
to eat.
• Genetically altered crop plants are
dangerous to the environment.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Advocates of biotechnology point out
that all crop plants have been
manipulated by humans.
Advocates say that since only single
genes for plant function are inserted
into crop plants, they are still safe for
human consumption.
Genes that affect human nutrition may
raise more concerns.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Concern over environmental effects
centers on escape of transgenes into
wild populations.
For example, if the gene for herbicide
resistance made its way into the weed
plants.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Widespread use of glyphosate on fields
of glyphosate-resistant crops has
resulted in the selection of weeds that
are resistant to glyphosate.
More than ten resistant weed species
have appeared in the United States.
18.6 How Is Biotechnology Changing Medicine and Agriculture?
Microorganisms developed to break
down components of crude oil have
not been released into the
environment because of the unknown
effects they might have on natural
ecosystems.
Because of the potential benefits of
biotechnology, scientists believe that it
is wise to proceed with caution.
18 Answer to Opening Question
We use microorganisms to decompose
compost and treat wastewater.
The radiation-resistant bacterium
Deinococcus radiodurans has been
engineered to precipitate heavy
metals and break down crude oil
components.
It may be useful for bioremediation at
radioactively contaminated sites.