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

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)

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

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…

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)…

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…

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”

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

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

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

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

Hybridization can be used to detect bacterial colonies carrying the desired plasmid insert…

ECB figure 10-24

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

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

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

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.

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

Region desired to amplify

3’5’

5’3’

Typical region to be amplified ~ 1000 bp

5’

3’

3’

5’

5’ 3’

3’ 5’

1st round of synthesis

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

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

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?

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 IndiaAnti 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 •15,499 (69%) •14,151 (71%) •13,924 (73%)

Source: Sci. Am. April 2001

Major GM crops and how they are modified

Year 2000

Model organism for tree genomicsTimber, plywood, pulp, paper

Fast growth - 7 year old poplar stand in Oregon

Trees too!

Poplars and aspens - genus Populus

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?

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)

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)

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

Source Sci. Am. April 2001Bt corn

Transformation - some cells will take up plasmids, others use gene gun

(biolistics)

Roundup-ready™ soybeans

Untreated - weed infested Sprayed with Roundup™

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

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

Cotton bollworm

Cotton - #1 pesticide using crop, a major pollutant environmentally.

Bt cotton has solved this problem.

But raised others, effects on butterflies………….

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

Regulatory oversight

Environmental Protection Agency - Safe for the environment?

US Department of Agriculture -Safe to plant?

Food and Drug Aministration - Safe to eat?

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

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

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)

Glyphosate up; overall use slightly reduced

Source USDA AER 786

Concern: Herbicide use will go up

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.”

* 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

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)