C HAPTER 16 Studying and Manipulating Genomes. I MPACTS, I SSUES : G OLDEN R ICE, OR F RANKENFOOD ?...
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Transcript of C HAPTER 16 Studying and Manipulating Genomes. I MPACTS, I SSUES : G OLDEN R ICE, OR F RANKENFOOD ?...
CHAPTER 16
Studying and Manipulating Genomes
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
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
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
MAKING RECOMBINANT DNA
5’
3’
G
C T T A A
A A T T C
G
G A A T T C
C T T A A G3’
5’
one DNA fragment another DNA fragment
3’
5’
MAKING RECOMBINANT DNA
nick
5’
3’
3’
5’
G A A T T C
C T T A A G
nick
GA A T T C
C T T A AG
DNA ligase action
Fig. 16-2, p.244
Stepped ArtGC T T A A
A A T T CG
3’
5’ 3’
5’
C T T A A
A A T T CG
cut fragments
G
DNA ligase action
nick
nickGC T T A A
A A T T CG
3’
5’ 3’
5’
another DNA fragment
A A T T C
3’
5’ 3’
5’G5’
one DNA fragment
3’
3’
5’ G
C T T A A
3’
5’
enzyme recognition site
GC T T A A
AAT T CG 3’
5’
USING PLASMIDS
Plasmid is small circle of bacterial DNA
Foreign DNA can be inserted into plasmid
Forms recombinant plasmidsPlasmid is a cloning vectorCan deliver DNA into another
cell
Fig. 16-3b, p.244
Plasmids
Restriction enzyme cuts molecule of chromosomal DNA or cDNA
recombinant plasmids containing foreign DNA
host cells containing recombinant plasmids
Same enzyme cuts same sequence in plasmid DNA
Foreign DNA, plasmid DNA, and modificationenzymes are mixed
DNA or cDNAfragments with sticky ends
plasmid DNA with sticky ends
Stepped Art
Fig. 16-4, p.245
a A restriction enzymecuts a specific base sequence everywhere it occurs in DNA.
c The same enzyme cuts the same sequnece in plasmid DNA.
d The plasmid DNA also has 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.
g Host cells that can divide rapidly take up the recombinant plasmids.
b The DNA fragmentshave sticky ends.
Fig. 16-4, p.245
Using Plasmids
MAKING CDNA
Fig. 16-5, p.245
GENE LIBRARIES
Bacteria that contain different cloned
DNA fragments
Genomic library
cDNA library
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
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. Somecells 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 probe’s 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
Use of a Probe
FAMILIAL HYPERCHOLESTEROLEMIA
Gene encodes protein that serves as cell’s 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
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
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
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
DNA polymerasesassemble new DNA strands
Fig. 16-6, p. 256
Stepped Art
POLYMERASE CHAIN REACTION
Stepped Art
Mixture heated again; makes all DNA fragments unwind
Mixture cooled; base-pairing between primers and ends of single DNA strands
DNA polymerase action again doubles number of identical DNA fragments
Fig. 16-6, p. 256
RECORDING THE SEQUENCE
T C C A T G G A C CT 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
T C C
T C
T
electrophoresisgel
one of the many fragments of DNA migratingthrough the gel
one of the DNA fragmentspassing through a laser beam after moving through the gel
T C C A T G G A C C A
•DNA is placed on
gel•Fragments move off
gel in size order;
pass through laser
beam•Color each
fragment fluoresces
is recorded on
printout
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
Fig. 16-9b, p.249
GEL ELECTROPHO
RESIS
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
BOZEMAN VIDEO—DNA FINGERPRINTING
http://www.youtube.com/watch?v=DbR9xMXuK7c
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
GENOMICS
Structural genomics: actual mapping and sequencing of genomes of individuals
Comparative genomics: concerned with possible evolutionary relationships of groups of organisms
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
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
THE TI PLASMID
Researchers replace tumor-causing genes with beneficial genes
Plasmid transfers these genes to cultured plant cells
foreign genein plasmid
plant cell
Fig. 16-13, p.253
a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).
b The bacterium infects a plant and transfers the Ti plasmid into it.
c The plant cell divides.
d Transgenic plants.
e Young plants with a fluorescent
gene product.
The Ti plasmid
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
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
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
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
Fig. 16-14c, p.254
Genetically Modified Animals
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
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?