Download Lecture 13 - University of Utah

Gene number and organization
Revised gene definition; most genes code for protein,
but some genes code for RNA that is not translated
(tRNA, rRNA small RNAs)
Gene Number varies
E. coli: 4300 protein coding genes
Yeast - 6200 protein coding genes,
Complex eukaryotes (worms, flies, weeds, humans)
20,000 - 35,000 protein coding genes
Today - molecular manipulations that allow us
to study the multitude of genes and use that
information in biotechnology
Lecture 13
Molecular Manipulations: Genes, Genomes
and Biotechnology
Genes and Genomics
1. *Restriction enzymes…
2. Gel electrophoresis of polynucleotides…
3. Detecting specific sequences by hybridization…
4. Cloning and amplification of DNA in bacteria…
5. Genomic and cDNA libraries…
6. Sequencing DNA and genomes
7. Amplifying DNA by the “polymerase chain reaction”…
8. Analyzing gene expression using DNA microarrays…
Biotechnology - genetically modified organisms (GMOs)
Restriction endonucleases are used to cut DNA…
5’
3’
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T
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C
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3’
Hae III
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3’
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+
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3’
5’
“4 cutters” = 1/256
5’
3’
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3’
Alu I
5’
3’
5’
“6 cutters” = 1/4,096
5’
3’
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3’
EcoRI
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3’
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“8-cutters” = 1/65,536
5’
3’
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3’
Not I
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5’
5’
3’
Adapted from ECB figure 10-4
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“Sticky ends”
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+
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Polynucleotides can be separated by gel electrophoresis…
DS DNA
Cut w restriction
enzymes…
Nucleotide pairs (x 1000)
Nucleic acids migrate
in electric field
-
Agarose slab gel
Adapted from ECB 10-5
+
Stain with fluorescent dyes that bind
DNA (ethidium bromide);…
detect radiolabeled polynucleotides by
autoradiography;…
or detect specific sequences by
hybridization …
Similar gels separate ss RNA…
Complementary polynucleotides “hybridize”…
Denature of “melt”
with heat or high pH…
DNA double helices
Renature or “anneal” by
slowly lowering
temperature or pH…
Single strand DNA
in solution
Complementary DNA sequences…
Template strand of DNA to RNA transcript…
“Anti-sense” RNA to “sense” RNA (mRNA)…
ECB figure 10-12
“Renatured” DNA
Hybridization can be used to detect
specific polynucleotide sequences…
“Southern blot:” DNA probes to detect DNA…
“Northern blot:” DNA probes to detect RNA…
Adapted from ECB 10-14
Hybridization can be used to detect
specific polynucleotide sequences…
“anti-sense”
“sense”
“anti-sense”
“anti-sense”
“anti-sense”
“anti-sense”
“sense”
“sense”
ECB figure 10-16
“In situ hybridization ”
detection of DNA sequences in
chromosomes…
“In situ”…
“In situ hybridization” of XMAP215 mRNA
using an “anti-sense” probe and “sense”
controls…
See also ECB 10-17
Bacterial plasmids can be used to “clone” and
amplify specific DNA fragments/sequences…
Bacterial chromosome
MCS contains useful restriction
sites, promoters, and targets for
sequencing primers…
DNA “insert”
cut with Not I
(5 x 106 bp)
“Plasmid”
(2-5 kBP)
Ampr
(ampicillin
resistance)
Nutrient broth
containing Amp…
Plasmid
Multiple
cloning
site
Not I
Replication
origin
Sticky ends facilitate
Plasmid re-introduced (“transformed”) into bacteria…
ligation into plasmid…
Grow in presence of ampicillin (select for Ampr gene in plasmid)…
Can isolate plasmids, or induce transcription and translation and
recombinant protein
See ECB 10-21
isolate
and 10-22
Making a “genomic library”…
2.
3.
4.
1. Isolate genomic DNA from tissue…
2. Digest with restriction enzymes to generate DNA fragments of
convenient size…
3. Ligate into multiple cloning site of plasmid (or l phage) cloning
vector…
4. Transform into (or infect) host bacteria and amplify…
Bacterial culture (or phage) will include sequences corresponding to
entire genome of DNA source…
Adapted from ECB 10-23
cDNA: DNA copies of mRNA…
mRNA population
Hybridize w poly T primer
Tissue
Purifiy mRNA
Make DNA copy with
reverse transcriptase
Ligate linkers to ends…
Cut with restriction enzyme…
Ligate into plasmid vector…
Degrade RNA
with RNAse H
Transform into bacteria host…
Second DNA strand made with DNA
polymerase (RNA fragments act as primers)
See ECB 10-25
DS cDNA copies of original mRNA population
See ECB
Genomic and cDNA libraries are different… figure
10-26
Genomic DNA
A
Exons
Genomic DNA
B
Non-transcribed
region
A
B
Transcription
Introns
Cut with
restriction
enzymes
RNA splicing
Ligate into vector
and transform into
host bacteria
mRNAs
Reverse transcribe
clone into vector/host
B
A
Genomic DNA library
cDNA library
Hybridization can be used to detect bacterial
colonies carrying the desired plasmid insert…
ECB figure 10-24
Sequencing DNA by the “dideoxy” method…
O
O
O
O P -O P -O P O
Base
5’
CH2
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Dideoxyribonucleoside triphosphate
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O P O
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-
A
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CH2
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OH
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O P O
1’
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CH2
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C
d dCTP
A
d dATP
T
d dTTP
G
d dGTP
T
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2’
No 3’ OH to accept next nucleotide…
Polynucleotide chain terminated…
See ECB figure 10-7
3’
Replicate in 5’vitro w XTPs 5’-
+ ddATP 5’5’Template 3’-
5’5’+ ddGTP 5’5’Template 3’5’5’+ ddCTP 5’5’Template 3’5’5’+ ddTTP 5’5’Template 3’-
A*
A*
Primer
A*
A*
AGTCTGCATGCATAGC-5’
G*
G*
G*
G*
AGTCTGCATGCATAGC-5’
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AGTCTGCATGCATAGC-5’
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AGTCTGCATGCATAGC-5’
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ddT
TCAGACGTACGTATCG-3’
AGTCTGCATGCATAGC-5’
ddC
5’
3’
ddG
DS DNA
ddA
Sequencing DNA by the “dideoxy” method…
G
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See ECB 10-7, 10-8
Sequencing entire genomes
Shotgun sequencing small genomes
For larger genomes (human) cut
up genome into large fragments
(100-200 kb each) and clone
into vector
Sequence these clones and align
them based on restriction
patterns
ECB 10-9
ECB 10-11
Polymerase Chain Reaction (PCR)
Yields billions of copies of DNA sequence of interest
in an hour or so
Based on replication
Uses of PCR
1. Forensics/Paternity -- amplify “fingerprint” DNA
to match with suspect
2. Genetic Testing - e.g. Huntington’s disease
3. DNA manipulations - amplification of a region for
later cloning, sequencing, etc.
OLIGONUCLEOTIDE DESIGN
3’
(5’)A G C G T G C A T G C C G A T T G (3’)
5’
G C T A T T C G C A C G T A C G G C T A A C G G T G
C G A T A A G C G T G C A T G C C G A T T G C C A C
5’
3’
Oligos: design with 3’ end oriented toward
the desired direction of synthesis
Typical region to be amplified ~ 1000 bp
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Region desired
to amplify
1st round of synthesis
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2nd round of synthesis
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2n double helices after n rounds
PCR movie
DNA microarrays can be used to
analyze global gene expression…
Collection of gene-specific
cDNA molecules
Amplify by PCR
Red - gene expressed in pop 1 only
Green - ………………………….pop 2 only
Yellow -…………………………..both pops
Robotic “printing”
onto glass slide
Hybridize with
fluorescently-labeled
cDNAs from 2 populations
2
1
Wash
Scan
ECB 10-15
Lecture 13
Molecular Manipulations: Genes, Genomes
and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
*GMO Overview
The Science
Herbicide and insect resistant plants
The major concerns
Herbicide use will increase
Gene pollution
Unintended toxicity to animals
Are GE foods safe?
THOUSANDS OF YEARS OF SELECTIVE
BREEDING
Classical breeding now supplemented
with bioengineering, which can produce
desirable traits much faster
Actually food is an ongoing
experiment; been breeding crops
and animals for millenia
GM foods common in US
An estimated two-thirds of the processed foods in U.S. supermarkets
contain genetically engineered corn, soybeans or other crops.
Yet, 58% of Americans say they have not eaten GM foods (down
from 62% in 2001)
Worldwide145 million acres of GM crops
(2002)
Pro GM Crops - US, Argentina, Canada, Phillipines, recently India
Anti GM - Italy, Austria, France, Great Britian, Japan….
EU has ban on importing or growing GM foods, costs US farmers $$
Farmers continue to increase GM crops
Appears that GM foods are here to stay
MAJOR U.S. GM CROPS
•Crop
•2001 Total
Acreage
•2002 Total
Acreage
•2003 Total Acreage
•Corn
•75,800 (26%)
•79,000 (34%)
•79,066 (40%)
•Soybean •74,105 (68%)
•72,993 (75%)
•73,653 (81%)
•Cotton
•14,151 (71%)
•13,924 (73%)
•15,499 (69%)
Major GM crops
and how they
are modified
Year 2000
Source: Sci. Am. April 2001
Trees too!
Poplars and aspens - genus Populus
Timber, plywood, pulp, paper
Model organism for tree genomics
Fast growth - 7 year old poplar
stand in Oregon
Lecture 13
Molecular Manipulations: Genes, Genomes
and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
GMO Overview
*The Science
Herbicide and insect resistant plants
The major concerns
Herbicide use will increase
Gene pollution
Unintended toxicity to animals
Are GE foods safe?
Most common modifications
Herbicide tolerance - Roundup-ready™ plants
Insect resistance - Bt plants (contain gene from Bacillus thuringiensis)
Glyphosate (Roundup™) blocks shikimate pathway
Shikimate pathway Biosynthesis of aromatic
amino acids (trp, phe, tyr)
Not in animals
Glyphosate binds to and
inhibits EPSP synthase
Glyphosate =
N phosphomethyl glycine
P - CH2 - NH - CH2 - COOSome EPSP synthases from
bacteria are resistant to
glyphosate (single aa change
Gly96 to Ala)
Roundup-ready™ cotton, soybeans - Monsanto
35S promoter
(CMV)
EPSPS(Agrobacterium)
Insert into Ti plasmid with selectable marker
Transform cotton cells in culture
Grow cells in presence of antibiotic
Regenerate plant from transformed cells
Test protein levels and glyphosate resistance
Note that plant will have 3 EPSPS
Ampr
Ti Plasmid
(ampicillin
resistance)
Multiple
cloning
site
Replication
origin
Cotton EPSPS
Agrobacterium EPSPS
(mutation?)
See ECB 10-40
Bt corn
Source Sci. Am. April 2001
Transformation - some cells will take up
plasmids, others use gene gun (biolistics)
Roundup-ready™ soybeans
Untreated - weed infested
Sprayed with Roundup™
Insect resistant plants
BT toxin - pesticide
(several companies)
Protein toxin from Bacillus thuringiensis
Kills larvae of
Lepidopterans (butterflies, moths)
Dipterans (2 winged flies (gnats, mosquitos))
Coleopterans (beetles)
Agricultural importance - Kills corn borer, corn root worm and cotton
bollworm larvae
Corn borer
Corn root worm
Bt Corn from Phillipines
Mechanism of action:
Binds to receptors in insect gut
Ionophore- ion channel that allows ions to flow across
plasma membrane
Note: organic farmers spray crops with intact Bt bacterium
Cotton bollworm
Cotton - #1 pesticide using crop, a
major pollutant environmentally.
Bt cotton has solved this problem.
But raised others, effects on
butterflies………….
Lecture 13
Molecular Manipulations: Genes, Genomes
and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
GMO Overview
The Science
Herbicide and insect resistant plants
*The major concerns
Are GE foods safe?
Herbicide use will increase
Gene pollution
Unintended toxicity to animals
Regulatory oversight
Food and Drug Aministration Safe to eat?
US Department of Agriculture Safe to plant?
Environmental Protection Agency Safe for the environment?
Concern: Are genetically modified foods safe to eat???
Regulatory oversight
Source: USDA website
A new protein not already in diet must be shown to be safe
GRAS - ‘generally recognized as safe’. If protein is not significantly different
from one already in diet. (EPSPS, most Bt)
In consultation, plant must look normal, grow normally, taste normal and have
expected levels of nutrients and toxins
In 2001, request data on bioengineered crops 120 days prior to commercial distribution
To date, no evidence that a GM crop is unsafe to eat. Starlink corn….
Starlink™ corn
In 2000 Starlink™ Bt corn from Aventis was found in Kraft
taco shells
Starlink™ Bt corn had not approved for human consumption
Worse, a watchdog group, not the FDA, found the tainted taco shells
Concern was that Starlink™ Bt corn was an allergen;
but in November 2003, scientists reported that additional
tests had failed to demonstrate the presence of an allergen
in the modified corn
Concern: Introgression (gene pollution)
Gene will be introduced into wild populations when transgenic pollen is
carried to compatible plants
Serious concern for
Cotton and wild relatives in southern US
Corn and teocinte in Mexico and Guatemala
Recent evidence from Mexico that bioengineered gene is in
wild populations
Could result in herbicide resistant weeds and Bt containing plants; 145
weed species known to be herbicide resistant
Possible solutions:
Clone into chloroplast genome which is inherited maternally in most plants
Male sterile plants
Concern: Herbicide use will go up
Source USDA AER 786
Glyphosate up; overall use slightly reduced
Concern: Toxicity to unintended animals
Bt is biggest worry
Toxicity of transgenic pollen
Bt pollen may be carried to nearby plants
(milkweed) and eaten by non-pest (monarch
butterfly)
Risk assessment
Sears et al. (2001) PNAS 98, 11937; “impact of Bt corn pollen from
current commercial hybrids on monarch butterfly populations is negligible.”
Other issues
* US already overproduces food
Major problem in 3rd world is distribution
* Resistance to pesticides (Bt)
GE crops are only short term solutions
* Gene may be transmitted from GM field to organic crops
Almost certainly will happen, Brittish very concerned
* Labeling of foods
Europe and Japan - Label and segregate (if EU lifts current ban)
US - voluntary, although public supports mandatory labeling
* Additional oversight; testing and scientific studies
Agencies currently reviewing their policies
* Enforcement
Starlink™ Bt corn
Future directions
2005-2015
Resistance to herbicides, pests and pathogens
Tolerance to drought, salt, heavy metals and low/high temperature
Improved nutritional quality (proteins, oils, vitamins, minerals)
Improved shelf life of fruits and vegetables
Improved flavors and fragrances
Elimination of allergens
Production of vaccines, human therapeutic proteins, pharmaceuticals
Phytoremediation
Vasil, Nature Biotechnology 21; 849-51 (2003)