Recombinant%20DNA%20and%20Biotechnology

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Recombinant DNA and Biotechnology
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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?

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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?
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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.

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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.

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Figure 18.1 Recombinant DNA
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18.1 What Is Recombinant DNA?

• Some restriction enzymes recognize palindromic
  DNA sequences
• 5'.GAATTC3'
• 3'.CTTAAG5'
• Some make straight cuts, others make staggered
  cuts, resulting in overhangs, or sticky ends.

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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.

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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.

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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?

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Working with Data 18.1 Recombinant DNA
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Working with Data 18.1 Recombinant DNA

• Question 2
• In another experiment, pSC101 and pSC102 were
  mixed and treated in three ways

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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?

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Working with Data 18.1 Recombinant DNA

• Question 3
• How did the antibiotic-resistant bacteria arise
  in the None DNA treatment?

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Working with Data 18.1 Recombinant DNA

• Question 4
• Did the EcoRI DNA ligase treatment result in
  an increase in doubly-resistant bacteria over
  controls?
• What data provide evidence for your statement?

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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?

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Figure 18.2 Cutting, Splicing, and Joining DNA
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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.

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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.

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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 usedthey have the ability
  to make stem cells (unspecialized, totipotent
  cells).

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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.

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

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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.

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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 replicationa replicon or replication unit.

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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

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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

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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.

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In-Text Art, Ch. 18, p. 377 (1)
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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.

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In-Text Art, Ch. 18, p. 377 (2)
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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.

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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.

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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.

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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.

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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.

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Figure 18.3 Selection for Recombinant DNA
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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.

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Figure 18.4 Green Fluorescent Protein as a
Reporter
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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

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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.

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Figure 18.5 Constructing Libraries
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18.3 What Sources of DNA Are Used in Cloning?

• If bacteriophage ? 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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Figure 18.6 Making a Knockout Mouse
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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.

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18.4 What Other Tools Are Used to Study DNA
Function?

• Complementary RNA
• Translation of mRNA can be blocked by
  complementary micro RNAsantisense 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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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.

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Figure 18.7 Using Antisense RNA and siRNA to
Block Translation of mRNA
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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.

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Figure 18.8 DNA Microarray for Medical Diagnosis
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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 recura
  gene expression signature.

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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

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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.

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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 Aaddition sequences, etc.

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Figure 18.9 Expression of a Transgene in a Host
Cell Produces Large Amounts of Its Protein Product
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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)

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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).

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Table 18.1
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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.

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Figure 18.10 Tissue Plasminogen Activator
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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 lactoglobulina protein in milk. The
  transgenic animal then produces large quantities
  of the protein in its milk.

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Figure 18.11 Pharming
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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.

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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 quickly

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Figure 18.12 Genetic Modification of Plants
versus Conventional Plant Breeding
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Table 18.2
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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.

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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.

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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.

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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.

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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 ß-carotene from
  the precursor are taken from daffodils or corn
  and inserted into rice by the Ti plasmid.

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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.

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Figure 18.13 Transgenic Rice Rich in ?-Carotene
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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.

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Figure 18.14 Salt-Tolerant Tomato Plants
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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.

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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.

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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.

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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.

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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.

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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.

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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.