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The Living World
Fifth Edition
George B. Johnson
Jonathan B. Losos
Chapter 14
Gene Technology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
14.1 A Scientific Revolution
• genetic engineering involves moving
genes from one organism to another
 this process is having a major impact on
medicine and agriculture
Figure 14.1 Examples of genetic
engineering.
14.2 Restriction Enzymes
• in any genetic engineering experiment, the gene
of interest must first be isolated
 restriction endonucleases are special enzymes that
bind to specific short sequences of DNA and make a
cut
• these short sequences are typical 4 to 6 nucleotides long
• these sequences are symmetrical in that the DNA double
helix has the identical sequence running in opposite
directions
14.2 Restriction Enzymes
• the cut made by restriction enzymes is also
unusual
 the cut is not made in the center of the sequence but
to one side
• this creates a break with short single strands of DNA
dangling from each end
• these overhangs are called “sticky ends”
• the same sticky ends are always produced from cuts with the
same restriction enzymes
14.2 Restriction Enzymes
• sticky ends make possible recombination
of fragments generated by cut restriction
enzymes
 fragments from the same DNA source could
pair up and be resealed by DNA ligase
 fragments from different sources of DNA cut
by the same restriction enzymes could also
be resealed together
Figure 14.2 How restriction enzymes produce DNA
fragments with sticky ends.
14.2 Restriction Enzymes
• where do restriction enzymes come from?
 some bacteria have the ability to prevent infection by
bacterial viruses
• these bacteria use restriction enzymes to cut up the foreign
(viral) DNA
• hundreds of different restriction enzymes have been
discovered
– each kind always cuts in one kind of sequence and always
makes a cut at the same place
14.3 The Four Stages of a Genetic
Engineering Experiment
•
three “ingredients” are necessary for
genetic engineering
1. a source of DNA that contains the gene you want
to transfer
2. a restriction enzyme to cut the DNA
3. a vehicle to carry the source DNA into the host
cell
14.3 The Four Stages of a Genetic
Engineering Experiment
 DNA library is a collection of DNA fragments
representing all of the DNA from an organism
• the source DNA used for genetic engineering is
often obtained from a DNA library
• restriction enzymes are used to cut the fragment
containing the gene of interest
 vector is the term for the vehicle that can
carry the gene of interest
• common vectors include both bacteria and viruses
14.3 The Four Stages of a Genetic
Engineering Experiment
•
all genetic transfer experiments share four
distinct stages
1. cleaving DNA
2. producing recombinant DNA
3. cloning
4. screening
14.3 The Four Stages of a Genetic
Engineering Experiment
• during the cleaving DNA stage, a restriction
enzyme is used to cut the source DNA into
fragments
 many different-sized fragments will be generated
 only some fragments will contain the gene of interest
 the fragments can be separated from each other,
based on size, by a process called electrophoresis
14.3 The Four Stages of a Genetic
Engineering Experiment
• during electrophoresis, fragments of DNA migrate
through a gel in response to an electrical current
 DNA is negatively charged and moves towards the + end of the
gel
 smaller-sized fragments move faster than larger-sized ones
 bands containing fragments of an appropriate size for the gene
of interest can then be cut from the gel for use in later stages
Figure 14.4 Using restriction enzymes to cleave DNA and
electrophoresis to separate the fragments.
14.3 The Four Stages of a Genetic
Engineering Experiment
• the second stage of a genetic engineering
experiment, the source DNA fragments are
allowed to mix with vector DNA
 the vector DNA has been cut with the identical
restriction enzyme used on the source
 the combination of vector and source DNA
into one DNA molecule is an example of
recombinant DNA
14.3 The Four Stages of a Genetic
Engineering Experiment
• recombinant DNA is carried into a host cell using
different types of vectors
 plasmids are tiny circles of bacterial DNA that can
replicate outside the main bacterial chromosome
• plasmids are introduced into a bacterial host cell by a
process called transformation
 viruses introduce recombinant DNA into a host cell
by infection
14.3 The Four Stages of a Genetic
Engineering Experiment
•
as each host cell (normally a bacterium) reproduces, it forms
clones that contain the DNA introduced by the vector
•
the clones can be any of four types
1.
a clone that has taken up a recombinant vector with the gene of
interest
2.
a clone that has taken up a recombinant vector without the gene of
interest
3.
a clone that has taken up a non-recombinant vector
4.
a clone that did not take up any vector
14.3 The Four Stages of a Genetic
Engineering Experiment
• clone library is a collection of separate clones
containing fragments of source DNA cut from the
entire genome of an organism
• genetic engineers need to identify which clone in
a library contains the gene of interest
 this is often the most challenging part of a genetic
engineering experiment
14.3 The Four Stages of a Genetic
Engineering Experiment
• genetic engineers first employ preliminary
screens in order to eliminate
 clones that do not contain any vectors
 or clones that do not contain recombinant
vectors
14.3 The Four Stages of a Genetic
Engineering Experiment
• clones without any vector can be identified
by a simple screening process
 the vector used in the experiment usually
contains a gene that confers antibiotic
resistance
 clones are grown in a medium that contains
that antibiotic
 only clones that have the resistant gene will
be able to grow
14.3 The Four Stages of a Genetic
Engineering Experiment
• screening can also eliminate clones that contain
recombinant vectors, but without the gene of
interest
 vectors are used that, in addition to the antibiotic
resistance gene, contain the gene lacZ’
• the lacZ’ gene encodes for the enzyme β-galactosidase
• β-galactosidase breaks down the sugar X-gal
– metabolism of X-gal results in the formation of a blue reaction
product
14.3 The Four Stages of a Genetic
Engineering Experiment
• using a restriction enzyme whose recognition
sequence lies within the lacZ’ gene, the gene
will be cleaved when recombinants are formed
 clones with vectors with non-recombinant DNA will
appear blue
 clones with vectors with recombinant DNA will appear
colorless
Figure 14.5 Using antibiotic resistance and X-gal as
preliminary screens of restriction fragment clones.
14.3 The Four Stages of a Genetic
Engineering Experiment
• once the preliminary screening has
occurred, the remaining clones that
contain the gene of interest must be
identified
 hybridization is a common method that uses
a probe consisting of a complementary
nucleic acid sequence to that of the gene of
interest
14.3 The Four Stages of a Genetic
Engineering Experiment
• hybridization typically involves
 growing previously screened clones on agar
 pressing a special filter onto the colonies to
create a replica of some of the cells
 treating the filter with a solution to denature
the DNA into single strands
 washing the filter in a solution of radioactivelylabeled probe
14.3 The Four Stages of a Genetic
Engineering Experiment
• hybridization continued…
 the probe only hybridizes with DNA from colonies that
contain the gene of interest
 the filter with hybridized DNA is overlaid on
photographic film
 any radioactivity will be revealed as a black spot on
the film
 the position of the black spot can be compared to the
original master plate of colonies
 the colonies containing the genes are then identified
Figure 14.6 Using hybridization to
identify the gene of interest.
Figure 14.3 How a genetic
engineering experiment works.
Review of the four stages of a genetic engineering experiment.
14.4 Working with DNA
• polymerase chain reaction (PCR) is a
technique to generate multiple copies of
DNA
 short sequences of DNA, called primers, are
first synthesized
 the primers sequences occur on either side of
the DNA region to be amplified
 the PCR technique is a way to generate a lot
of DNA of interest quickly, rather than rely on
bacteria to produce copies
14.4 Working with DNA
•
there are three steps involved in PCR
1. Denaturation
2. Primer annealing
3. Primer extension
14.4 Working with DNA
• the DNA target sequence, primers, polymerase,
and a supply of all four nucleotides are first
combined together in a solution
 the solution is heated to about 95°C
 the polymerase used is a special heat-resistant
variety call Taq polymerase
 the heat causes the DNA to denature into single
strands
14.4 Working with DNA
• as the denatured solution cools, the primers bind to their
complementary sequence
• the polymerase then uses the primer as a starting point
to move down the strand and lengthen the entire DNA
fragment
 because both strands behave this way, by the end of the process
there are 2 copies of the original fragment
• the PCR process is repeated many times resulting in the
desired level of amplification of DNA
Figure 14.7 How the polymerase
chain reaction works.
14.4 Working with DNA
• recall that, in eukaryotes, genes are encoded in
both translated and non-translated segments
• the primary mRNA transcript produced by RNA polymerase
contains both the coding regions (exons) and non-coding
regions (introns)
• the introns must be cut out from the primary transcript before
the mRNA can be translated
• the remaining exon fragments are stitched together to form
the final RNA transcript, the processed mRNA, which is
eventually translated in the cytoplasm
14.4 Working with DNA
• it is desirable for genetic engineers to transfer
DNA that is ready to be translated
 one reason is that bacteria, as prokaryotes, lack the
enzymes to process mRNA
 to obtain DNA without introns, genetic engineers
isolate first the processed mRNA corresponding to a
particular gene
 the enzyme reverse transcriptase produces a DNA
version of this mRNA, called complementary DNA
(cDNA)
Figure 14.8 cDNA: producing an intron-free version of a
eukaryotic gene for genetic engineering.
14.4 Working with DNA
• 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 unique DNA sequences found in non-coding
regions of human DNA that are highly variable among individuals
 the chances that any two individuals, other than identical twins,
having the same restriction pattern for these sequences varies
from 1 in 800K to 1 in 1 billion, depending on the number of
probes used
14.4 Working with DNA
• DNA fingerprints consist of
autoradiographs depicting rows of parallel
bars on X-ray film
 each bar represents the position of a DNA
restriction endonuclease fragment that
complementarily binds to a probe
Figure 14.9 Two of the DNA profiles
that led to conviction.
14.4 Working with DNA
• 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 (i.e., 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
14.5 Genetic Engineering and
Medicine
• much of the promise of genetic engineering lies in
improving medicine
 specifically, to aid in curing and preventing illnesses
• one such application comes in the form of “magic bullets”
 many genetic disorders, such as diabetes, involve a failure to
make a critical protein
 genetic engineering can supply persons suffering from the
disease with the protein they lack by engineering another
organism, usually a bacterium, to do it
 the donated protein is like a “magic bullet” to the disorder
Table 14.1 Genetically Engineered
Drugs
Figure 14.10 Genetically engineered
human growth hormone.
14.5 Genetic Engineering and
Medicine
• recombinant viruses are produced by genetic
engineering against common viruses, such as herpes
and hepatitis
 genes encoding part of the protein coat of these viruses are
spliced into a harmless fragment of the vaccinia (cowpox) virus
 the vaccinia virus acts as a vector for introducing the viral genes
and, after translation, proteins into a human
 the human develops immunity against the viruses prior to
exposure to the true form
 the utilization of one vaccine to introduce genes from another
virus is called a piggyback vaccine
Figure 14.11 Constructing a subunit, or piggyback,
vaccine for the herpes simplex virus.
14.6 Genetic Engineering of Farm
Animals
• gene technology is having a major impact on the
breeding and rearing of agricultural animals
 use of genetically engineered hormones
• recombinant bovine somatotropin (BST) has been used in cows to
increase milk production
 use of transgenic animals
• these animals have been engineered to have specific desirable
genes
• this precludes having to wait through several generations of
selective breeding
Figure 14.12 The production of bovine somatropin
(BST) through genetic engineering.
14.7 Genetic Engineering of Crop
Plants
• genes in crop plants have been successfully
manipulated through genetic engineering in
order to
 make plants more resistant to diseases caused by
insects
 make plants resistant to herbicides
 improve their nutritional balance and protein content
 make plants hardier against environmental stress
14.7 Genetic Engineering of Crop
Plants
• engineering crops to be resistant to insect
pests can reduce pesticide use
 Bacillus thuringiensis (Bt) is a soil bacterium
that produces a protein that is toxic when
eaten by crop pests
 inserting the gene producing Bt protein into
crop plant chromosomes makes the crop pest
resistant
14.7 Genetic Engineering of Crop
Plants
• a big success in genetic engineering has been the
creation of crop plants that are resistant to the herbicide
glyphosphate
 glyphosphate is used in orchards and agricultural fields to control
weeds
 plants cannot make aromatic amino acids needed for protein
production
 genetic engineers found a gene in bacteria that made aromatic
amino acids in the presence of glyphosphate
 this gene was then inserted into plant genomes using a DNA
particle gun, or gene gun
Figure 14.13 Shooting gene into
cells.
Figure 14.14 Genetically
engineered herbicide resistance.
14.7 Genetic Engineering of Crop
Plants
• 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
 these benefits translate into reduced prices
for consumers because cultivating GM crops
is cheaper and more efficient
14.7 Genetic Engineering of Crop
Plants
• the real promise of plant genetic engineering is
to prevent GM plants with desirable traits that
directly benefit the consumer
 “golden” rice is a solution from genetic engineering to
nutrient deficiencies common to regions of the world
where the major staple food is rice
 rice eaters often are deficient in two major
micronutrients, iron and vitamin A
14.7 Genetic Engineering of Crop
Plants
• the development of transgenic, “golden,” rice
involved the splicing of genes from different
sources
Figure 14.15 Transgenic “golden” rice.
Table 14.2 Genetically Modified
Crops
14.7 Genetic Engineering of Crop
Plants
• are there any risks associated with genetic
engineering of crops?
 the potential for risks have alarmed many activists
and scientists
 two sets of risks need to be considered
• is eating genetically modified food dangerous?
• are GM crops actually harmful to the environment?
Inquiry & Analysis
• Does the gene conferring
resistance to herbicide
pass to other plants of
this species, A.
stolonifera?
• What general statement
can be made about the
effect of distance on the
likelihood that the
herbicide resistance gene
will pass to another
plant?
p. 258, graph of frequency of GM
Sentinel plants versus distance