Download Golden Rice, or Frankenfood?

PowerLecture:
Chapter 16
Studying and Manipulating
Genomes
Golden Rice, or Frankenfood?
Ordinary daffodil
Fig. 16-1a, p.242
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
Golden Rice, or Frankenfood?
1st generation
2nd generation
Regular rice
Fig. 16-1b, p.242
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
Golden Rice, or Frankenfood?
Today's corn
Ancestral corn
p.243
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
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
5’
G
3’
C
A A T T
T T A A
one DNA fragment
C
3’
G
5’
another DNA fragment
5’
T
C
3’
T T A A
G
5’
C
3’
T A A G
nick
5’
A A T
3’
C
5’
nick
G A A T
3’
C
T
T
DNA ligase action
5’
G A A T
T C
3’
3’
C T
T A A G
5’
Fig. 16-2, p.244
Terms
ligase – seals cuts in DNA
 Recombinant DNA – any molecule
consisting of base sequences from 2 or
more organisms of the same or different
species
 Cloning vector – a plasmid that has
accepted foreign DNA and can be slipped
into host
 DNA
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
Plasmids
Fig. 16-3a, p.244
Using Plasmids
a A restriction enzyme
cuts a specific base
sequence everywhere
it occurs in DNA.
c The same enzyme cuts
the same sequnece in
plasmid DNA.
b The DNA fragments
have sticky ends.
e The DNA
fragments and
the plasmid
DNA are
mixed with
DNA ligase.
f The result? A collection
of recombinant plasmids
that incorporate foreign
DNA fragments.
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
Making cDNA
•Use reverse transcriptase
onto one strand of
complementary DNA
(cDNA)
•DNA polymerase strips
RNA bases
•Copies cDNA into a
second strand
Fig. 16-5, p.245
Gene Libraries
 Bacteria
that contain different
cloned DNA fragments

Genomic library

cDNA library
Use of a Probe

Colonies on plate
You want to find
which bacteria in a
Cells adhere
to filter
library contain a
specific gene

Need a probe for that
Cells are lysed;
DNA sticks
to filter
gene
• A radioisotopelabeled piece of
DNA
• Will base-pair with
gene of interest
Probe is
added
Location where probe binds forms
dark spot on
film, indicates
colony with
gene
Making a Probe
 Make

a primer if the sequence is known.
If not usually DNA from closely related species
Amplifying DNA
 Fragments
can be inserted into
fast-growing microorganisms
 Polymerase
chain reaction (PCR)
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
 Animation
Polymerase
Chain Reaction
Double-stranded
DNA to copy
DNA heated to
90°– 94°C
Primers added to
base-pair with
ends
Mixture cooled;
base-pairing of
primers and ends
of DNA strands
Stepped Art
DNA polymerases
assemble new
DNA strands
Fig. 16-6, p. 256
Polymerase
Chain Reaction
Mixture heated again;
makes all DNA
fragments unwind
Mixture cooled; basepairing between
primers and ends of
single DNA strands
Stepped Art
DNA polymerase
action again
doubles number of
identical DNA
fragments
Fig. 16-6, p. 256
TCCATGGACC
TCCATGGAC
TCCATGGA
Recording
the
Sequence
TCCATGG
TCCATG
TCCAT
TCCA
TCC
•DNA is placed on gel
•Fragments move off
TC
T
electrophoresis
gel
one of the many
fragments of
DNA migrating
through the gel
gel in size order; pass
through laser beam
•Color each fragment
fluoresces is recorded
one of the DNA fragments
passing through a laser beam
after moving through the gel
on printout
T C C A T G G A C C A
Recording the Sequence
http://www.dnalc.org/ddnalc/resources/cycseq.html
Fig. 16-8b, p.248
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
Genome Sequencing
Fig. 16-10a, p.250
Nucleotides for Sequencing
 Standard
 Modified


nucleotides (A, T, C, G)
versions of these nucleotides
Labeled so they fluoresce
Structurally different so that they stop DNA
synthesis when they are added to a strand
Reaction Mixture
 Copies
of DNA to be sequenced
 Primer
 DNA
polymerase
 Standard
 Modified
nucleotides
nucleotides
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
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
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
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
 http://www.dnalc.org/ddnalc/resources/elec
trophoresis.html
Gel
Electrophoresis
Fig. 16-9b, p.249
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
Genomics
 Structural
genomics: actual mapping and
sequencing of genomes of individuals
 Comparative genomics: concerned with
possible evolutionary relationships of
groups of organisms
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
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)
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
Engineered Proteins
 Bacteria
can be used to grow medically
valuable proteins

Insulin, interferon, blood-clotting factors

Vaccines
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
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
p.252
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
Engineered Plants
Fig. 16-12a, p.253
Engineered Plants
Fig. 16-12b, p.253
The Ti plasmid

Researchers
replace tumorcausing genes
with beneficial
genes
 Plasmid
transfers these
genes to
cultured plant
cells
plant cell
foreign gene
in plasmid
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
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
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
Transgenic Mice
Fig. 16-15, p.254
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
Genetically
Modified
Animals
Fig. 16-14a, p.254
Genetically
Modified
Animals
Fig. 16-14b, p.254
Genetically Modified Animals
Fig. 16-14c, p.254
Xenotransplantation
 Researchers
knockout the Ggta1genes in
transgenic piglets
 Ggta1
gene produces proteins that human
antibodies recognize
 Pig’s
organs are less prone to rejection by
a human
Safety
 Superpathogens
 DNA
from pathogenic or toxic organisms
used in recombination experiments
 Hok genes
 NIH guidelines for DNA research
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
Results of Gene Therapy

Modified cells alive in woman’s 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
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
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?