Biotechnology

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Biotechnology
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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

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Chapter 13 Opening Question

• How is biotechnology used to alleviate
  environmental problems?

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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.

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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

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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'.GAATTC3'
• 3'.CTTAAG5'
• Each sequence forms a palindrome the opposite
  strands have the same sequence when read from the
  5' end.

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Figure 13.1 Bacteria Fight Invading Viruses by
Making Restriction Enzymes
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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.

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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.

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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.

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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

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Figure 13.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
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Figure 13.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
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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 single-stranded
  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.

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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.

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Figure 13.3 Cutting, Splicing, and Joining DNA
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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.

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Figure 13.4 Recombinant DNA (Part 1)
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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.

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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.

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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 cellsunspecialized
  , totipotent cells
• Cultured animal cells, used for expression of
  human or animal geneswhole transgenic animals
  can be created

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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 permeableDNA diffuses in.
• Electroporationa short electric shock creates
  temporary pores in membranes, and DNA can enter.

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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.

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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 replicationa replicon or replication
  unit.

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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

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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

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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.

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In-Text Art, Ch. 13, p. 249
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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.

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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.

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In-Text Art, Ch. 13, p. 250
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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 vectorse.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.

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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.

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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.

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Figure 13.5 Marking Recombinant DNA by
Inactivating a Gene
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Concept 13.2 DNA Can Genetically Transform Cells
and Organisms

• Selectable markers are a type of reporter genea
  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.

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Figure 13.6 Green Fluorescent Protein as a
Reporter
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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

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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.

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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.

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Figure 13.7 Constructing Libraries
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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.

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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.

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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).

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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
  plasmidrestriction enzymes are used to insert a
  reporter gene into the normal gene.
• The extra DNA prevents functional mRNA from being
  made.

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Concept 13.3 Genes and Gene Expression Can Be
Manipulated

• The recombinant plasmid is used to transfect
  mouse embryonic stem cells.
• Stem cellsunspecialized cells that divide and
  differentiate into specialized cells
• The original gene sequences line up with their
  homologous sequences on the mouse chromosome.

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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.

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Figure 13.8 Making a Knockout Mouse
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Concept 13.3 Genes and Gene Expression Can Be
Manipulated

• Complementary RNA
• Translation of mRNA can be blocked by
  complementary microRNAsantisense RNA.
• Antisense RNA can be synthesized and added to
  cells to prevent translationthe effects of the
  missing protein can then be determined.

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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.

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Figure 13.9 Using Antisense RNA and siRNA to
Block the Translation of mRNA
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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.

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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.

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Figure 13.10 Using DNA Microarrays for Clinical
Decision-Making
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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.

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Figure 13.11 A Transgenic Cell Can Produce Large
Amounts of the Transgenes Protein Product
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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 sequencese.g., a signal to secrete the
  product to the extracellular medium

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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 ß-galactosidase gene.
• After synthesis the polypeptides are cleaved, and
  the two insulin peptides combined to make a
  functional human insulin molecule.

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Figure 13.12 Human Insulin From Gene to Drug
(Part 1)
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Figure 13.12 Human Insulin From Gene to Drug
(Part 2)
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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

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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 lactoglobulina protein in milk. The
  transgenic animal then produces large quantities
  of the protein in its milk.

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Figure 13.13 Pharming
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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.

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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

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Figure 13.14 Genetic Modification of Plants
versus Conventional Plant Breeding (Part 1)
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Figure 13.14 Genetic Modification of Plants
versus Conventional Plant Breeding (Part 2)
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Table 13.2 Potential Agricultural Applications
of Biotechnology
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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.

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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.

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Concept 13.4 Biotechnology Has Wide Applications

• Crops with improved nutritional characteristics
• Rice does not have ß-carotene, but does have a
  precursor molecule
• Genes for enzymes that synthesize ß-carotene from
  the precursor are taken from daffodils and
  inserted into rice by the Ti plasmid

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Concept 13.4 Biotechnology Has Wide Applications

• The transgenic rice is yellow and can supply
  ß-carotene to improve the diets of many people
• ß-carotene is converted to vitamin A in the body

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Figure 13.15 Transgenic Rice Rich in ?-Carotene
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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.

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Figure 13.16 Salt-tolerant Tomato Plants (Part 1)
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Figure 13.16 Salt-tolerant Tomato Plants (Part 2)
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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.

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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

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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.

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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

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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.

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Figure 13.17 The Spoils of War