Production of Protein from Cloned Genes OrR. Why you need to express protein via cloned genes?
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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
Revised gene definition; most genes code for protein,but some genes code for RNA that is not translated
(tRNA, rRNA small RNAs)
Gene number and organization
Today - molecular manipulations that allow us to study the multitude of genes and use
that information in biotechnology
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Lecture 13
Molecular Manipulations: Genes, Genomes and Biotechnology
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…
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
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Restriction endonucleases are used to cut DNA…
Adapted from ECB figure 10-4
G
C
G
C
A T C C A G
T A G G T C
3’5’
5’3’
Hae III G
C
G
C
A T
T A
C C A G
G G T C+
5’
3’ 5’
3’
G
C
A
T
A T C T A G
T A G A T C
3’5’
5’3’
Alu I G
C
A
T
A T
T A
C T A G
G A T C+
5’
3’ 5’
3’
EcoRIA
T
A
T
A T T G
T C
G
A A G
C
C
3’5’
5’3’
A
T C
G
AA T T G
G
C
C
3’
5’
TT A A
5’
3’
Not IG
C
G
C
G C C C
C G
C
G G C
G
G
3’5’
5’3’
G
C G
C
GG C C C
C
G
G
3’
5’
CC G G
5’
3’
“Sticky ends”
“4 cutters” = 1/256
“6 cutters” = 1/4,096
“8-cutters” = 1/65,536
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Polynucleotides can be separated by gel electrophoresis…
DS DNA
Cut w restriction enzymes…
Agarose slab gel
Nuc
leot
ide
pairs
(x
1000
)
+
-N
ucle
ic a
cids
m
igra
te in
ele
ctric
fie
ld
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…
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Complementary polynucleotides “hybridize”…
Denature of “melt” with heat or high pH…
Single strand DNA in solution “Renatured” DNA
Renature or “anneal” by slowly lowering temperature or pH…
DNA double helices
ECB figure 10-12
Complementary DNA sequences…
Template strand of DNA to RNA transcript…
“Anti-sense” RNA to “sense” RNA (mRNA)…
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Hybridization can be used to detect specific polynucleotide sequences…
Adapted from ECB 10-14“Southern blot:” DNA probes to detect DNA…
“Northern blot:” DNA probes to detect RNA…
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Hybridization can be used to detect specific polynucleotide sequences…
See also ECB 10-17
“In situ hybridization ” detection of DNA sequences in chromosomes…
ECB figure 10-16
“In situ”… “In situ hybridization” of XMAP215 mRNA using an “anti-sense” probe and “sense” controls…
“anti-sense”“anti-sense”
“anti-sense”
“sense”“sense”
“anti-sense”“anti-sense”
“sense”
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Plasmid re-introduced (“transformed”) into bacteria…
Grow in presence of ampicillin (select for Ampr gene in plasmid)…
Can isolate plasmids, or induce transcription and translation and isolate recombinant protein
Bacterial chromosome
(5 x 106 bp)
Bacterial plasmids can be used to “clone” and amplify specific DNA fragments/sequences…
Plasmid
Ampr
(ampicillin resistance)
Replication origin
MCS contains useful restriction sites, promoters, and targets for sequencing primers…
DNA “insert” cut with Not I
“Plasmid”(2-5 kBP)
Nutrient broth containing Amp…
See ECB 10-21 and 10-22
Not I
Sticky ends facilitate ligation into plasmid…
Multiple cloning
site
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Making a “genomic library”…
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 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…
2. 3. 4.
Adapted from ECB 10-23
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cDNA: DNA copies of mRNA…
Hybridize w poly T primer
DS cDNA copies of original mRNA population
Second DNA strand made with DNA polymerase (RNA fragments act as primers)
Make DNA copy with reverse transcriptase
Degrade RNA with RNAse H
TissuePurifiy mRNA
See ECB 10-25
Ligate linkers to ends…
Cut with restriction enzyme…
Ligate into plasmid vector…
Transform into bacteria host…
mRNA population
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Genomic and cDNA libraries are different…
cDNA libraryGenomic DNA library
A B
Genomic DNA Genomic DNA
A B
Cut with restriction enzymes
Ligate into vector and transform into
host bacteria
Transcription
See ECB figure 10-26
Exons IntronsNon-transcribed region
RNA splicing
mRNAs
AB
Reverse transcribeclone into vector/host
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Hybridization can be used to detect bacterial colonies carrying the desired plasmid insert…
ECB figure 10-24
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OH
Sequencing DNA by the “dideoxy” method…
1’
2’3’
4’
5’-O
P O
O
-OO
CH2
Base-O
P O
O
-O
-O
P O
O
-O
C
T dTTP
A
G dGTPd
dATPd
d
dCTPd
3’
1’4’
5’
O
P O
O
-OO
CH2
T
2’
deoxyribonucleoside triphosphateDi
1’
2’3’
4’
5’O
P O
O
-OO
CH2
A
No 3’ OH to accept next nucleotide…Polynucleotide chain terminated…
See ECB figure 10-7
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AGTCTGCATGCATAGC-5’Template 3’-
T*5’-T*5’-
T*5’-T*5’-
AGTCTGCATGCATAGC-5’Template 3’-
C*5’-C*5’-
C*5’-C*5’-
AGTCTGCATGCATAGC-5’Template 3’-
A*5’-A*5’-
A*5’-A*
Sequencing DNA by the “dideoxy” method…
AGTCTGCATGCATAGC-5’Template 3’-
G*5’-G*5’-
G*5’-G*5’-
See ECB 10-7, 10-8
3’
GCTATGCATGCAGACT5’
ddT
ddC
ddG
ddA
AGTCTGCATGCATAGC-5’TCAGACGTACGTATCG-3’5’
3’
+ ddATP
+ ddGTP
+ ddCTP
+ ddTTPT
C
A
G
A
C
G
T
A
C
G
T
A
T
C
GReplicate in vitro w XTPs
5’- Primer
DS DNA
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Sequencing entire genomes
Shotgun sequencing small genomesFor 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
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Based on replication
Polymerase Chain Reaction (PCR)
Yields billions of copies of DNA sequence of interest in an hour or so
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.
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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
3’ 5’
5’ 3’
(5’)A G C G T G C A T G C C G A T T G (3’)
Oligos: design with 3’ end oriented toward the desired direction of synthesis
OLIGONUCLEOTIDE DESIGN
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Region desired to amplify
3’5’
5’3’
Typical region to be amplified ~ 1000 bp
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5’
3’
3’
5’
5’ 3’
3’ 5’
1st round of synthesis
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5’ 3’
3’ 5’
5’ 3’
3’ 5’
3’ 5’
5’ 3’
5’ 3’
3’ 5’
PCR movie
2nd round of synthesis
2n double helices after n rounds
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DNA microarrays can be used to analyze global gene expression…
Collection of gene-specific cDNA molecules
Scan
Robotic “printing” onto glass slide
Amplify by PCR
Wash
ECB 10-15
Hybridize with fluorescently-labeled
cDNAs from 2 populations
1 2
Red - gene expressed in pop 1 onlyGreen - ………………………….pop 2 onlyYellow -…………………………..both pops
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Lecture 13Molecular Manipulations: Genes,
Genomes and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
*GMO Overview
The ScienceHerbicide and insect resistant plants
The major concernsHerbicide use will increaseGene pollutionUnintended toxicity to animals Are GE foods safe?
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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
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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)
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Worldwide145 million acres of GM crops (2002)
Pro GM Crops - US, Argentina, Canada, Phillipines, recently IndiaAnti GM - Italy, Austria, France, Great Britian, Japan….EU has ban on importing or growing GM foods, costs US farmers $$
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Farmers continue to increase GM crops
Appears that GM foods are here to stay
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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 •15,499 (69%) •14,151 (71%) •13,924 (73%)
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Source: Sci. Am. April 2001
Major GM crops and how they are modified
Year 2000
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Model organism for tree genomicsTimber, plywood, pulp, paper
Fast growth - 7 year old poplar stand in Oregon
Trees too!
Poplars and aspens - genus Populus
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Lecture 13Molecular Manipulations: Genes,
Genomes and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
GMO Overview
*The ScienceHerbicide and insect resistant plants
The major concernsHerbicide use will increaseGene pollutionUnintended toxicity to animals Are GE foods safe?
![Page 31: Gene Number varies E. coli: 4300 protein coding genes Yeast - 6200 protein coding genes, Complex eukaryotes (worms, flies, weeds, humans) 20,000 - 35,000.](https://reader035.fdocuments.in/reader035/viewer/2022062322/56649ea95503460f94baddf7/html5/thumbnails/31.jpg)
Most common modifications
Herbicide tolerance - Roundup-ready™ plants
Insect resistance - Bt plants (contain gene from Bacillus thuringiensis)
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Glyphosate (Roundup™) blocks shikimate pathway
Shikimate pathway - Biosynthesis of aromatic
amino acids (trp, phe, tyr)
Glyphosate binds to and inhibits EPSP synthase
Not in animals
Glyphosate = N phosphomethyl glycine P - CH2 - NH - CH2 - COO-Some EPSP synthases from bacteria are resistant to glyphosate (single aa change Gly96 to Ala)
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35S promoter(CMV)
EPSPS(Agrobacterium)
Insert into Ti plasmid with selectable markerTransform cotton cells in cultureGrow cells in presence of antibioticRegenerate plant from transformed cellsTest protein levels and glyphosate resistance
Ti PlasmidAmpr
(ampicillin resistance)
Replication origin
Multiple
cloning site
Cotton EPSPS
Agrobacterium EPSPS(mutation?)
Note that plant will have 3 EPSPS
Roundup-ready™ cotton, soybeans - Monsanto
See ECB 10-40
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Source Sci. Am. April 2001Bt corn
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Transformation - some cells will take up plasmids, others use gene gun
(biolistics)
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Roundup-ready™ soybeans
Untreated - weed infested Sprayed with Roundup™
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BT toxin - pesticide (several companies)
Protein toxin from Bacillus thuringiensisKills 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
Insect resistant plants
Corn borerCorn root worm
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Bt Corn from Phillipines
Mechanism of action:Binds to receptors in insect gutIonophore- ion channel that allows ions to flow across plasma membrane
Note: organic farmers spray crops with intact Bt bacterium
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Cotton bollworm
Cotton - #1 pesticide using crop, a major pollutant environmentally.
Bt cotton has solved this problem.
But raised others, effects on butterflies………….
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Lecture 13Molecular Manipulations: Genes,
Genomes and Biotechnology
Genes and Genomics
Biotechnology - genetically modified organisms (GMOs)
GMO Overview
The ScienceHerbicide and insect resistant plants
*The major concernsAre GE foods safe?Herbicide use will increaseGene pollutionUnintended toxicity to animals
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Regulatory oversight
Environmental Protection Agency - Safe for the environment?
US Department of Agriculture -Safe to plant?
Food and Drug Aministration - Safe to eat?
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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 haveexpected 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….
Source: USDA website
Concern: Are genetically modified foods safe to eat???
Regulatory oversight
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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
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Gene will be introduced into wild populations when transgenic pollen is carried to compatible plants
Serious concern for Cotton and wild relatives in southern USCorn 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: Introgression (gene pollution)
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Glyphosate up; overall use slightly reduced
Source USDA AER 786
Concern: Herbicide use will go up
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Concern: Toxicity to unintended animals
Bt is biggest worry
Toxicity of transgenic pollenBt pollen may be carried to nearby plants (milkweed) and eaten by non-pest (monarch butterfly)
Risk assessmentSears et al. (2001) PNAS 98, 11937; “impact of Bt corn pollen from current commercial hybrids on monarch butterfly populations is negligible.”
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* US already overproduces foodMajor 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 cropsAlmost certainly will happen, Brittish very concerned
* Labeling of foodsEurope and Japan - Label and segregate (if EU lifts current ban)US - voluntary, although public supports mandatory labeling
* Additional oversight; testing and scientific studiesAgencies currently reviewing their policies
* EnforcementStarlink™ Bt corn
Other issues
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Future directions2005-2015
Resistance to herbicides, pests and pathogensTolerance to drought, salt, heavy metals and low/high temperatureImproved nutritional quality (proteins, oils, vitamins, minerals)Improved shelf life of fruits and vegetablesImproved flavors and fragrancesElimination of allergensProduction of vaccines, human therapeutic proteins, pharmaceuticalsPhytoremediation
Vasil, Nature Biotechnology 21; 849-51 (2003)