Studying and Manipulating Genomes

1
Chapter 16

• Studying and Manipulating Genomes

2
Impacts, Issues Golden Rice, or Frankenfood?

• 124 million children around the world have
  vitamin A deficiencies
• Golden rice
• Rice plants engineered with genes from daffodils
  allowing it to produce beta-carotine in its seeds
  (rice)
• Beta carotine is the precursor to Vitamin A
• Rice is the main food for 3 billion people

3
Impacts, Issues Golden Rice, or Frankenfood?

• Many crops plants have been modified, including
  corn, beets, potatoes, and cotton
• Potentially less harmful to the environment than
  current agricultural practices

4
Golden Rice, or Frankenfood?
Fig. 16-1a, p.242
5
Golden Rice, or Frankenfood?
Fig. 16-1b, p.242
6
Golden Rice, or Frankenfood?
p.243
7
Genetic Changes

• Humans have been changing the genetics of other
  species for thousands of years
• Artificial selection of plants and animals
• Natural processes also at work
• Mutation, crossing over

8
Discovery of Restriction Enzymes

• Hamilton Smith was studying how Haemophilus
  influenzae defend themselves from bacteriophage
  attack
• Discovered bacteria have an enzyme that chops up
  viral DNA

9
Specificity of Cuts

• Restriction enzymes cut DNA at a specific
  sequence
• Number of cuts made in DNA will depend on number
  of times the target sequence occurs

10
Making Recombinant DNA
5
G
A A T T C
3
C T T A A
G
one DNA fragment
another DNA fragment
5
G
A A T T C
3
C T T A A
G
5
3
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Making Recombinant DNA
nick
5
3
G
A A T T C
3
C T T A A
G
5
nick
DNA ligase action
G
A A T T C
C T T A A
G
12
Fig. 16-2, p.244
13
Stepped Art
Fig. 16-2, p.244
14
Using Plasmids

• Plasmid is small circle of bacterial DNA
• Foreign DNA can be inserted into plasmid
• Forms recombinant plasmids
• Plasmid is a cloning vector
• Can deliver DNA into another cell

15
Plasmids
Fig. 16-3a, p.244
16
Plasmids
Fig. 16-3b, p.244
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Restriction enzyme cuts molecule of chromosomal
DNA or cDNA
Foreign DNA, plasmid DNA, and modification enzyme
s are mixed
DNA or cDNA fragments with sticky ends
recombinant plasmids containing foreign DNA
Same enzyme cuts same sequence in plasmid DNA
plasmid DNA with sticky ends
host cells containing recombinant plasmids
Stepped Art
Fig. 16-4, p.245
18
Using Plasmids
e The DNA fragments and the plasmid DNA are
mixed with DNA ligase.
a A restriction enzyme cuts a specific base
sequence everywhere it occurs in DNA.
b The DNA fragments have sticky ends.
f The result? A collection of recombinant
plasmids that incorporate foreign DNA
fragments.
c The same enzyme cuts the same sequnece in
plasmid DNA.
d The plasmid DNA also has sticky ends
g Host cells that can divide rapidly take up the
recombinant plasmids.
Fig. 16-4, p.245
19
Making cDNA
Fig. 16-5, p.245
20
Gene Libraries

• Bacteria that contain different cloned DNA
  fragments
• Genomic library
• cDNA library

21
Using a Probe to Find a Gene

• You want to find which bacteria in a library
  contain a specific gene
• Need a probe for that gene
• A radioisotope-labeled piece of DNA
• Will base-pair with gene of interest

22
Use of a Probe
Colonies on plate
Cells adhere to filter
Cells are lysed DNA sticks to filter
Probe is added
Location where probe binds forms
dark spot on film, indicates colony with gene
23
Use of a Probe
a Bacterial colonies, each derived from a single
cell, grow on a culture plate. Each colony is
about 1 millimeter across.
b A nitrocellulose or nylon filter is placed on
the plate. Some cells of each colony adhere to
it. The filter mirrors how the colonies are
distributed on the culture plate.
c The filter is lifted off and put into a
solution. Cells stuck to it rupture the cellular
DNA sticks to the filter.
d The DNA is denatured to single strands at each
site. A radioactively labeled probe is added to
the filter. The probe binds to DNA with a
complementary base sequence.
e The probes location is identified by exposing
the filter to x-ray film. The image that forms on
the film reveals the colony that has the gene of
interest.
Fig. 16-6, p.246
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Familial Hypercholesterolemia

• Gene encodes protein that serves as cells LDL
  receptor
• Two normal alleles for the gene keep blood level
  of LDLs low
• Two mutated alleles lead to abnormally high
  cholesterol levels heart disease

25
Example of Gene Therapy

• Woman with familial hypercholesterolemia
• Part of her liver was removed
• Virus used to insert normal gene for LDL receptor
  into cultured liver cells
• Modified liver cells placed back in patient

26
Amplifying DNA

• Fragments can be inserted into fast-growing
  microorganisms
• Polymerase chain reaction (PCR)

27
Polymerase Chain Reaction

• Sequence to be copied is heated
• Primers are added and bind to ends of single
  strands
• DNA polymerase uses free nucleotides to create
  complementary strands
• Doubles number of copies of DNA

28
Polymerase Chain Reaction
Double-stranded DNA to copy
Stepped Art
Fig. 16-6, p. 256
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Polymerase Chain Reaction
Mixture heated again makes all DNA fragments
unwind
Stepped Art
Fig. 16-6, p. 256
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Fig. 16-7, p.247
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Recording the Sequence
T C C A T G G A C C
T C C A T G G A C
T C C A T G G A
T C C A T G G
T C C A T G
T C C A T
T C C A
electrophoresis gel
T C C

• DNA is placed on gel
• Fragments move off gel in size order pass
  through laser beam
• Color each fragment fluoresces is recorded on
  printout

T C
one of the many fragments of DNA
migrating through the gel
T
one of the DNA fragments passing through a laser
beam after moving through the gel
T C C A T G G A C C A
32
Recording the Sequence
p.248
33
Recording the Sequence
electrophoresis gel
one of many fragments of DNA migrating through
the gel
one of the DNA fragments passing through a laser
beam after moving through the gel
Fig. 16-8a, p.248
34
Recording the Sequence
Fig. 16-8b, p.248
35
DNA Fingerprints

• Unique array of DNA fragments
• Inherited from parents in Mendelian fashion
• Even full siblings can be distinguished from one
  another by this technique

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Tandem Repeats

• Short regions of DNA that differ substantially
  among people
• Many sites in genome where tandem repeats occur
• Each person carries a unique combination of
  repeat numbers

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RFLPs

• Restriction fragment length polymorphisms
• DNA from areas with tandem repeats is cut with
  restriction enzymes
• Because of the variation in the amount of
  repeated DNA, the restriction fragments vary in
  size
• Variation is detected by gel electrophoresis

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Gel Electrophoresis

• DNA is placed at one end of a gel
• A current is applied to the gel
• DNA molecules are negatively charged and move
  toward positive end of gel
• Smaller molecules move faster than larger ones

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Gel Electrophoresis
Fig. 16-9a, p.249
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Gel Electrophoresis
Fig. 16-9b, p.249
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Analyzing DNA Fingerprints

• DNA is stained or made visible by use of a
  radioactive probe
• Pattern of bands is used to
• Identify or rule out criminal suspects
• Identify bodies
• Determine paternity

42
Genome Sequencing

• 1995 - Sequence of bacterium Haemophilus
  influenzae determined
• Automated DNA sequencing now main method
• Draft sequence of entire human genome determined
  in this way

43
Genome Sequencing
Fig. 16-10a, p.250
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Genome Sequencing
Fig. 16-10b, p.250
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Nucleotides for Sequencing

• Standard nucleotides (A, T, C, G)
• Modified versions of these nucleotides
• Labeled so they fluoresce
• Structurally different so that they stop DNA
  synthesis when they are added to a strand

46
Reaction Mixture

• Copies of DNA to be sequenced
• Primer
• DNA polymerase
• Standard nucleotides
• Modified nucleotides

47
Genomics

• Structural genomics actual mapping and
  sequencing of genomes of individuals
• Comparative genomics concerned with possible
  evolutionary relationships of groups of organisms

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Reactions Proceed

• Nucleotides are assembled to create complementary
  strands
• When a modified nucleotide is included, synthesis
  stops
• Result is millions of tagged copies of varying
  length

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DNA Chips

• Microarrays of thousands of gene sequences
  representing a large subset of an entire genome
  (p251)
• Stamped onto a glass plate the size of a small
  business card (p251)

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DNA Chips
Fig. 16-11, p.251
51
Videos CNN

• Ask your Thomson Sales Representative for these
  volumes on CD or VHS
• Genetics, 2003, Vol. 1, Transgenic Tobacco (200)

52
Genetic Engineering

• Genes are isolated, modified, and inserted into
  an organism
• Made possible by recombinant technology
• Cut DNA up and recombine pieces
• Amplify modified pieces

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Engineered Proteins

• Bacteria can be used to grow medically valuable
  proteins
• Insulin, interferon, blood-clotting factors
• Vaccines

54
Cleaning Up the Environment

• Microorganisms normally break down organic wastes
  and cycle materials
• Some can be engineered to break down pollutants
  or to take up larger amounts of harmful materials

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Can Genetically Engineered Bacteria Escape?

• Genetically engineered bacteria are designed so
  that they cannot survive outside lab
• Genes are included that will be turned on in
  outside environment, triggering death

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p.252
57
Engineered Plants

• Cotton plants that display resistance to
  herbicide
• Aspen plants that produce less lignin and more
  cellulose
• Tobacco plants that produce human proteins
• Mustard plant cells that produce biodegradable
  plastic

58
Engineered Plants
Fig. 16-12a, p.253
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Engineered Plants
Fig. 16-12b, p.253
60
The Ti plasmid

• Researchers replace tumor-causing genes with
  beneficial genes
• Plasmid transfers these genes to cultured plant
  cells

plant cell
foreign gene in plasmid
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The Ti plasmid
b The bacterium infects a plant and transfers the
Ti plasmid into it.
a A bacterial cell contains a Ti plasmid (purple)
that has a foreign gene (blue).
e Young plants with a fluorescent gene product.
c The plant cell divides.
d Transgenic plants.
Fig. 16-13, p.253
62
First Engineered Mammals

• Experimenters used mice with hormone deficiency
  that leads to dwarfism
• Fertilized mouse eggs were injected with gene for
  rat growth hormone
• Gene was integrated into mouse DNA
• Engineered mice were 1-1/2 times larger than
  unmodified littermates

63
Transgenic Mice
Fig. 16-15, p.254
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Cloning Dolly

• 1997 - A sheep cloned from an adult cell
• Nucleus from mammary gland cell was inserted into
  enucleated egg
• Embryo implanted into surrogate mother
• Sheep is genetic replica of animal from which
  mammary cell was taken

65
Designer Cattle

• Genetically identical cattle embryos can be grown
  in culture
• Embryos can be genetically modified
• create resistance to mad cow disease
• engineer cattle to produce human serum albumin
  for medical use

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Genetically Modified Animals
Fig. 16-14a, p.254
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Genetically Modified Animals
Fig. 16-14b, p.254
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Genetically Modified Animals
Fig. 16-14c, p.254
69
Videos CNN

• Ask your Thomson Sales Representative for these
  volumes on CD or VHS
• Environmental Science, 2003, Vol. 6, Organic
  Farming (137)

70
Safety

• Superpathogens
• DNA from pathogenic or toxic organisms used in
  recombination experiments

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Safety

• Hok genes
• NIH guidelines for DNA research

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The Human Genome Initiative

• Goal - Map the entire human genome
• Initially thought by many to be a waste of
  resources
• Process accelerated when Craig Ventner used bits
  of cDNAs as hooks to find genes
• Sequencing was completed ahead of schedule in
  early 2001

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Results of Gene Therapy

• Modified cells alive in womans liver
• Blood levels of LDLs down 20 percent
• No evidence of atherosclerosis
• Cholesterol levels remain high
• Remains to be seen whether procedure will prolong
  her life

74
Using Human Genes

• Even with gene in hand it is difficult to
  manipulate it to advantage
• Viruses usually used to insert genes into
  cultured human cells but procedure has problems
• Very difficult to get modified genes to work
  where they should

75
Ethical Issues

• Who decides what should be corrected through
  genetic engineering?
• Should animals be modified to provide organs for
  human transplants?
• Should humans be cloned?