Download Chapter 13 from book

13
Biotechnology
Chapter 13 Biotechnology
Key Concepts
• 13.1 Recombinant DNA Can Be Made in
the Laboratory
• 13.2 DNA Can Genetically Transform Cells
and Organisms
• 13.3 Genes and Gene Expression Can Be
Manipulated
• 13.4 Biotechnology Has Wide Applications
Chapter 13 Opening Question
How is biotechnology used to
alleviate environmental problems?
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
It is possible to modify organisms with
genes from other, distantly related
organisms.
Recombinant DNA is a DNA molecule
made in the laboratory that is derived from
at least two genetic sources.
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Three key tools:
• Restriction enzymes for cutting DNA into
fragments
• Gel electrophoresis for analysis and
purification of DNA fragments
• DNA ligase for joining DNA fragments
together in new combinations
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Restriction enzymes recognize a specific
DNA sequence called a recognition
sequence or restriction site.
5′…….GAATTC……3′
3′…….CTTAAG……5′
Each sequence forms a palindrome: the
opposite strands have the same sequence
when read from the 5′ end.
Figure 13.1 Bacteria Fight Invading Viruses by Making Restriction Enzymes
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Some restriction enzymes cut DNA leaving
a short sequence of single-stranded DNA
at each end.
Staggered cuts result in overhangs, or
“sticky ends;” straight cuts result in “blunt
ends.”
Sticky ends can bind complementary
sequences on other DNA molecules.
Methylases add methyl groups to restriction
sites and protect the bacterial cell from its
own restriction enzymes.
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Many restriction enzymes with unique
recognition sequences have been purified.
In the lab they can be used to cut DNA
samples from the same source.
A restriction digest combines different
enzymes to cut DNA at specific places.
Gel electrophoresis analysis can create a
map of the intact DNA molecule from the
formed fragments.
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
DNA fragments cut by enzymes can be
separated by gel electrophoresis.
A mixture of fragments is placed in a well in
a semisolid gel, and an electric field is
applied across the gel.
Negatively charged DNA fragments move
towards the positive end.
Smaller fragments move faster than larger
ones.
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
DNA fragments separate and give three
types of information:
• The number of fragments
• The sizes of the fragments
• The relative abundance of the fragments,
indicated by the intensity of the band
Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
After separation on a gel, a specific DNA
sequence can be found with a singlestranded probe.
The gel region can be cut out and the DNA
fragment removed.
The purified DNA can be analyzed by
sequence or used to make recombinant
DNA.
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
DNA ligase is an enzyme that catalyzes the
joining of DNA fragments, such as Okazaki
fragments during replication.
With restriction enzymes to cut fragments
and DNA ligase to combine them, new
recombinant DNA can be made.
Figure 13.3 Cutting, Splicing, and Joining DNA
Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Recombinant DNA was shown to be a
functional carrier of genetic information.
Sequences from two E.coli plasmids, each
with different antibiotic resistance genes,
were recombined.
The resulting plasmid, when inserted into
new cells, gave resistance to both of the
antibiotics.
Figure 13.4 Recombinant DNA (Part 1)
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Recombinant DNA technology can be used
to clone (make identical copies) 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.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Usually only a few cells exposed to
recombinant DNA are actually
transformed.
To determine which of the host cells are
transgenic, the recombinant DNA includes
selectable marker genes, such as genes
that confer resistance to antibiotics.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Most research has been done using model
organisms:
• Bacteria, especially E. coli
• Yeasts (Saccharomyces), commonly used
as eukaryotic hosts
• Plant cells, able to make stem cells—
unspecialized, totipotent cells
• Cultured animal cells, used for expression
of human or animal genes—whole
transgenic animals can be created
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Methods for 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.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
• Viruses and bacteria can be altered to
carry recombinant DNA into cells.
• Transgenic animals can be produced by
injecting recombinant DNA into the nuclei
of fertilized eggs.
• “Gene guns” can “shoot” the host cells with
particles of DNA.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
The new DNA must also replicate as the
host cell divides.
DNA polymerase does not bind to just any
sequence.
The new DNA must become part of a
segment with an origin of replication—a
replicon or replication unit.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
New DNA can become part of a replicon in
two ways:
• Inserted near an origin of replication in
host chromosome
• It can be part of a carrier sequence, or
vector, that already has an origin of
replication
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
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
which can be selectable markers
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
• 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. 13, p. 249
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
A plasmid from the soil bacterium
Agrobacterium tumefaciens is used as a
vector for plant cells.
A. tumefaciens contains a plasmid called Ti
(for tumor-inducing).
The plasmid has a region called T DNA,
which inserts copies of itself into
chromosomes of infected plants.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
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.
In-Text Art, Ch. 13, p. 250
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Most eukaryotic genes are too large to be
inserted into a plasmid.
Viruses can be used as vectors—e.g.,
bacteriophage. The genes that cause host
cells to lyse can be cut out and replaced
with other DNA.
Because viruses infect cells naturally they
offer an advantage over plasmids.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Usually only a small proportion of host cells
take up the vector (1 cell in 10,000) 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.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
If a vector carrying genes for resistance to
two different antibiotics is used, one
antibiotic can select cells carrying the
vector.
If the other antibiotic resistance gene is
inactivated by the insertion of foreign DNA,
then cells with the desired DNA can be
identified by their sensitivity to that
antibiotic.
Figure 13.5 Marking Recombinant DNA by Inactivating a Gene
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Selectable markers are a type of reporter
gene—a gene whose expression is easily
observed.
Green fluorescent protein, 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.
Figure 13.6 Green Fluorescent Protein as a Reporter
Concept 13.3 Genes and Gene Expression Can Be Manipulated
DNA fragments used for cloning come from
three sources:
• Gene libraries
• Reverse transcription from mRNA
• Products of PCR
• Artificial synthesis or mutation of DNA
Concept 13.3 Genes and Gene Expression Can Be Manipulated
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.
A vector is taken up by host cells which
produce a colony of recombinant cells.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
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.
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.
Figure 13.7 Constructing Libraries
Concept 13.3 Genes and Gene Expression Can Be Manipulated
DNA can be synthesized by PCR if
appropriate primers are available.
The amplified DNA can then be inserted into
plasmids to create recombinant DNA and
cloned in host cells.
Artificial synthesis of DNA is now fully
automated.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
Synthetic oligonucleotides are used as
primers in PCR reactions.
Primers can create new sequences to
create mutations in a recombinant gene.
Longer synthetic sequences can be used to
construct an artificial gene.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
Synthetic DNA can be manipulated to create
specific mutations in order to study the
consequences of the mutation.
Mutagenesis techniques have revealed
many cause-and-effect relationships (e.g.,
determining signal sequences).
Concept 13.3 Genes and Gene Expression Can Be Manipulated
A knockout experiment inactivates a gene
so that it is not transcribed and translated
into a functional protein.
In mice, homologous recombination
targets a specific gene.
The normal allele of a gene is inserted into a
plasmid—restriction enzymes are used to
insert a reporter gene into the normal
gene.
The extra DNA prevents functional mRNA
from being made.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
The recombinant plasmid is used to
transfect mouse embryonic stem cells.
Stem cells—unspecialized cells that divide
and differentiate into specialized cells
The original gene sequences line up with
their homologous sequences on the
mouse chromosome.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
The transfected stem cell is then
transplanted into an early mouse embryo.
The knockout technique has been important
in determining gene functions and studying
human genetic diseases.
Many diseases have a knockout mouse
model.
Figure 13.8 Making a Knockout Mouse
Concept 13.3 Genes and Gene Expression Can Be Manipulated
Complementary RNA:
Translation of mRNA can be blocked by
complementary microRNAs—antisense
RNA.
Antisense RNA can be synthesized and
added to cells to prevent translation—the
effects of the missing protein can then be
determined.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
RNA interference (RNAi) is a rare natural
mechanism that blocks translation.
RNAi occurs via the action of small
interfering RNAs (siRNAs).
An sRNA is a short, double stranded RNA
that is unwound to single strands by a
protein complex, which also catalyzes the
breakdown of the mRNA.
Small interfering RNA (siRNA) can be
synthesized in the laboratory.
Figure 13.9 Using Antisense RNA and siRNA to Block the Translation of mRNA
Concept 13.3 Genes and Gene Expression Can Be Manipulated
DNA microarray technology provides a large
array of sequences for hybridization
experiments.
A series of DNA sequences are attached to
a glass slide in a precise order.
The slide has microscopic wells, each
containing thousands of copies of
sequences up to 20 nucleotides long.
Concept 13.3 Genes and Gene Expression Can Be Manipulated
DNA microarrays can be used to identify
specific single nucleotide polymorphisms
or other mutations.
Microarrays can be used to examine gene
expression patterns in different tissues in
different conditions.
Example: Women with a propensity for
breast cancer tumors to recur have a gene
expression signature.
Figure 13.10 Using DNA Microarrays for Clinical Decision-Making
Concept 13.4 Biotechnology Has Wide Applications
Almost any gene can be inserted into
bacteria or yeasts and the resulting cells
induced to make large quantities of a
product.
Requires specialized expression vectors
with extra sequences needed for the
transgene to be expressed in the host cell.
Figure 13.11 A Transgenic Cell Can Produce Large Amounts of the Transgene’s Protein Product
Concept 13.4 Biotechnology Has Wide Applications
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
Concept 13.4 Biotechnology Has Wide Applications
Many medically useful products are being
made using biotechnology.
The two insulin polypeptides are
synthesized separately along with the βgalactosidase gene.
After synthesis the polypeptides are
cleaved, and the two insulin peptides
combined to make a functional human
insulin molecule.
Figure 13.12 Human Insulin: From Gene to Drug (Part 1)
Figure 13.12 Human Insulin: From Gene to Drug (Part 2)
Concept 13.4 Biotechnology Has Wide Applications
Before giving it to humans, scientists had to
be sure of its effectiveness:
• Same size as human insulin
• Same amino acid sequence
• Same shape
• Binds to the insulin receptor on cells and
stimulates glucose uptake
Concept 13.4 Biotechnology Has Wide Applications
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 13.13 Pharming
Concept 13.4 Biotechnology Has Wide Applications
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.
Concept 13.4 Biotechnology Has Wide Applications
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 quickly
Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 1)
Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 2)
Table 13.2 Potential Agricultural Applications of Biotechnology
Concept 13.4 Biotechnology Has Wide Applications
Crop plants have been modified to produce
their own insecticides:
• The bacterium Bacillus thuringiensis
produces a protein that kills insect larvae
• Dried preparations of B. thuringiensis are
sold as a safe alternative to synthetic
insecticides. The toxin is easily
biodegradable.
Concept 13.4 Biotechnology Has Wide Applications
• Genes for the toxin have been isolated,
cloned, and modified, 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.
Concept 13.4 Biotechnology Has Wide Applications
Crops with improved nutritional
characteristics:
• Rice does not have β-carotene, but does
have a precursor molecule
• Genes for enzymes that synthesize βcarotene from the precursor are taken
from daffodils and inserted into rice by the
Ti plasmid
Concept 13.4 Biotechnology Has Wide Applications
• The transgenic rice is yellow and can
supply β-carotene to improve the diets of
many people
• β-carotene is converted to vitamin A in the
body
Figure 13.15 Transgenic Rice Rich in -Carotene
Concept 13.4 Biotechnology Has Wide Applications
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 13.16 Salt-tolerant Tomato Plants (Part 1)
Figure 13.16 Salt-tolerant Tomato Plants (Part 2)
Concept 13.4 Biotechnology Has Wide Applications
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.
Concept 13.4 Biotechnology Has Wide Applications
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
Concept 13.4 Biotechnology Has Wide Applications
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.
Concept 13.4 Biotechnology Has Wide Applications
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
• Beneficial insects can also be killed from
eating plants with B. thuringiensis genes
Answer to Opening Question
Bioremediation is the use, by humans, of
organisms to remove contaminants from
the environment.
Composting and wastewater treatment use
bacteria to break down large molecules,
human wastes, paper, and household
chemicals.
Recombinant DNA technology has
transformed bacteria to help clean up oil
spills.
Figure 13.17 The Spoils of War