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The Living World - Chapter 10 by dbfQd67


									Lecture 11
A Scientific Revolution

 Genetic engineering is the process of moving genes from one
  organism to another
    Having a major impact on agriculture & medicine
    Restriction Enzymes

 Restriction enzymes bind to
  specific short sequences
  (usually 4- to 6- bases long) on
  the DNA
      The nucleotide sequence on both
       DNA strands is identical when GAATTC
       read in opposite directions   CTTAAG
   Most restriction enzymes cut the
    DNA in a staggered fashion
      This generates “sticky” ends
          These ends can pair with
           any other DNA fragment
           generated by the same
               The pairing is aided
                 by DNA ligase

    Play   Restriction Enzymes
4 Stages of a Genetic Engineering Experiment
      All gene transfer experiments share four distinct stages

1.     Cleaving DNA

2.     Producing recombinant

3.     Cloning

4.     Screening

     Play   Steps in cloning a gene
Stage 1
 Cleaving the DNA
   The large number of fragments produced are separated by

                                                           appear as
                                                          bands under
                                                        fluorescent light
Stages 2 & 3

 Producing Recombinant DNA
   Fragments of source DNA are inserted into vectors
   Vectors are plasmids or viruses that carry foreign DNA into the host
       Vector DNA is cut with the same enzyme as the source DNA, thus
        allowing the joining of the two

 Cloning
   Host cells are usually bacteria
   As each bacterial cell reproduces, it forms a clone of cells
    containing the fragment-bearing vector
       Together all clones constitute a clone library
 Stage 4
 Screening
     A preliminary screen of the
      clone library eliminates
       1. Clones without vectors
       2. Clones with vectors
          that do not contain

     The vector employed
      usually has genes for
        Antibiotic resistance
            This eliminates the
             first type of clones
             because they are
             sensitive to antibiotics
        b-galactosidase
            This eliminates the
             second type of clones
             based on X-gal
             metabolism and color
Stage 4 (cont.)
 Screening
   To find the gene of interest, the clone library is screened by a process termed
       The cloned genes form base pairs with complementary sequences on another
        nucleic acid, termed the probe
   The bacterial colonies are first grown on agar
       They are then transferred to a filter
       The filter is treated with a radioactive probe
       The filter is then subjected to autoradiography
Working with DNA

 Key techniques used by today’s genetic engineers include

    PCR amplification
       Used to increase the amounts of DNA
    cDNA formation
       Used to build genes from their mRNA
    DNA fingerprinting
       Used to identify particular individuals
    PCR Amplification                                       Target sequence

                                                                            1 Denaturation
    The polymerase chain reaction               1                          2 Annealing of primers
     (PCR) requires primers                                          DNA polymerase
                                                       2 copies      Free nucleotides
          Short single-stranded
           sequences complementary to                                       3 Primer extension
           regions on either side of the
           DNA of interest                                           Heat

    PCR consists of three basic       Cycle
     steps                               2       4 copies            Cool

     1. Denaturation
     2. Primer annealing                                             Heat
     3. Primer extension                                             Cool
                                                 8 copies

    Play   Polymerase Chain Reaction
 cDNA Formation

 The primary mRNA
  transcript contains
  exons and introns

 The processed
  mRNA contains only
    It is used as a
     template to create a
     single strand of
     DNA termed
     DNA (cDNA)
    cDNA is then
     converted to a
  DNA Fingerprinting

 This is a process that is
  used to determine if two
  DNA samples are from the
  same source

 The DNA from the two
  sources is fragmented
  using restriction enzymes
    The fragments are
     separated using gel
    They are transferred to a
    The filters are screened
     with radioactive probes
    Then subjected to

 Play   DNA Fingerprinting
Genetic Engineering and Medicine

    Genetic engineering has been used in many medical applications

     1. Production of proteins to treat illnesses
     2. Creation of vaccines to combat infections
     3. Replacement of defective genes
  Making “Magic Bullets”

 In diabetes, the body is unable to
  control levels of sugar in the blood
  because of lack of insulin

     Diabetes can be cured if the body
      is supplied with insulin
     The gene encoding insulin has
      been introduced into bacteria
 Other genetically engineered drugs
     Anticoagulants
        Used to treat heart attack
     Factor VIII
        Used to treat hemophilia
     Human growth hormone (HGH)
        Used to treat dwarfism
Piggyback Vaccines

 Genetic engineering has also been used to create subunit vaccines
  against viruses
 A gene encoding a viral
 protein is put into the DNA of
 a harmless virus and
 injected into the body
 The viral protein will elicit
 antibody production in the
 A novel kind of vaccine was
 introduced in 1995
 The DNA vaccine uses
 plasmid vectors
      It elicits a cellular immune
      response, rather than
      antibody production
Genetic Engineering of Farm Animals

    In 1994, the recombinant hormone bovine
     somatotropin (BST) became commercially
       Dairy farmers used BST as a supplement to
        enhance milk production in cows

    Consumers are concerned about the presence of
     the hormone in milk served to children
       This fear has not been supported by research data
    Genetic Engineering of Crop Plants
    Pest resistance
       Leads to a reduction in the
        use of pesticides
       Bt, a protein produced by
        soil bacteria, is harmful to
        pests but not to humans
            The Bt gene has been
             introduced into tomato
             plants, among others

    Herbicide resistance
       Crop plants have been
        created that are resistant to

    Herbicide resistance offers two main advantages
       Leads to a reduction in the use of pesticides
       Lowers the cost of producing crops
       Reduces plowing and conserves the top soil
  Genetic Engineering of Crop Plants

 More Nutritious Crops
    Worldwide, two major deficiencies are iron and vitamin A
    Deficiencies are especially severe in developing countries where the
     major staple food is rice
    Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden”
     rice to solve this problem
Potential Risks of Genetically Modified (GM) Crops

   The promise of genetic engineering is very much in evidence
       However, it has generated considerable controversy and protest
        Are genetic engineers “playing God” by tampering with the genetic
   Two sets of risks need to be considered
    1. Are GM foods safe to eat?
           The herbicide glyphosate blocks the synthesis of aromatic amino acids
              Humans don’t make any aromatic amino acids, so glyphosate doesn’t
               hurt us
           However, gene modifications that render plants resistant to glyphosate
            may introduce novel proteins
              Moreover, introduced proteins may cause allergies in humans
    2. Are GM foods safe for the environment?
           Three legitimate concerns are raised
             1. Will other organisms be harmed unintentionally?
             2. Will pests become resistant to pesticides?
             3. What if introduced genes will pass from GM crops to their wild or
                weedy relatives?
Potential Risks of Genetically Modified (GM) Crops

 Should GM foods be labeled?
Every serious scientific investigation
has concluded that GM foods are
    So there is no health need for a GM
However, people have a right to know
what is in their food
    So there may be a need for label
    after all
    Cloning Higher Organisms
     The successful embryos (about 30 in 277 tries) were transplanted into
      surrogate mother sheep
         On July 5, 1996, “Dolly” was born
         Only 1 of 277 tries succeeded
         However, Wilmut proved that reproductive cloning is possible
   Since Dolly,
    scientists have
    successfully cloned
    sheep, mice, cattle,
    goats and pigs
       However, problems
        and complications
        arise, leading to
        premature death
       Dolly died in 2002,
        having lived only
        half a normal
        sheep life span
    Embryonic Stem Cells

    The blastocyst, an early
     embryo, consists of
       A protective outer layer that
        will form the placenta
       Inner cell mass that will
        form the embryo
    The inner cell mass consists of
     embryonic stem cells
       These are pluripotent
            Capable of forming the
             entire organism
                                           The research in human embryonic stem cells is
    As development proceeds,               associated with two serious problems
     cells lose their pluripotency            Finding a source: harvesting them from
                                               discarded embryos raises ethical issues
       They become committed to
                                              Immunological rejection: Implanted stem
          one type of tissue                   cells will likely be rejected by the immune
       They are then called adult             system of the individual
          stem cells
  Stem Cells

 Embryonic stem cells
  could be used to restore
  tissues lost or damaged
  due to accident or
    Experiments have
     already been tried
     successfully in mice
    Damaged spinal
     neurons have been
     partially repaired

 The course of
  development is broadly
  similar in all mammals
    Therefore, the
     experiments in mice
     are very promising
Grappling with the Ethics of Stem Cell Research

   Stem cells offer enormous promise for treating a wide range
    of diseases
     However, the research involves ethical issues
        1. Destruction of human embryos
           When does human life begin?
       2. Possibility of future abuse or misuse
           Is human reproductive cloning next?
       3. Alternative sources of stem cells
           Are adult stem cells equally effective?
  Gene Therapy

 Gene therapy involves the
  introduction of “healthy”
  genes into cells that lack
 It was first used successfully
  in 1990
     Two girls were cured of a
      rare blood disorder caused
      by a defective adenosine
      deaminase gene
     The girls stayed healthy

 In 1999, AAV successfully cured anemia in rhesus monkeys
 AAV was also used to cure dogs of a hereditary disorder leading to retinal
  degeneration & blindness

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