Finish Genetically Modified (GM) Foods

Yesterday - UK approved first GM crop for planting (with strict guidelines

Herbicide-tolerant corn

‘8,000,000 farmers in 18 countries are now growing GM crops’

Source: Sci. Am. April 2001

Major GM crops Major GM crops and how they and how they are modifiedare modified

Year 2000

Model organism for tree genomics

Timber, plywood, pulp, paper

Fast growth - 7 year old poplar stand in Oregon

Trees too!

Poplars and aspens - genus Populus

Engineering wood (cell wall) for better pulp quality, etc.

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, contain gene that makes plant resistant to herbicides

Insect resistance - Bt plants, contain toxin gene from Bacillus thuringiensis that kills larvae

Glyphosate (Roundup™; Monsanto) 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)

Transform cotton cells in culture, plasmid inserts in genome

Grow cells in presence of antibiotic

Regenerate plant from transformed cells

Test protein levels and glyphosate resistance

Ti PlasmidAmpr

(ampicillin resistance)

Replication origin

Multiple

cloning site

Cotton EPSPS

Agrobacterium EPSPS

Note that plant will have 3 EPSPS

Roundup-ready™ cotton, soybeans - Monsanto

See ECB 10-40

Source Sci. Am. April 2001

Bt corn‘Plant cells are totipotent’

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

(biolistics)

Roundup-ready™ soybeans

Untreated - weed infested Sprayed with Roundup™

Clone gene coding for 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 toxin 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….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

Evidence from Mexico that bioengineered gene is in wild populations

Could result in herbicide resistant weeds and Bt containing wild plants

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) will be selected forGE crops are only short term solutions

* Gene may be transmitted from GM field to organic cropsAlmost certainly will happen, British 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)

Golden rice - engineered to synthesize -carotene, vitamin A precursorVit. A deficiency causes blindness

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)

Beyond the central dogma

Central dogma culminates with synthesis of protein in cytoplasm

But can’t mix proteins, polysaccharides, lipids and nucleotides together and get a living cell

Formation of a cell requires the context of a pre-existing cell

Cell structures (organelles; mitochondria, chloroplasts, Golgi, ER) and organization must be inherited, just like

DNA

Epigenetics

Lecture 14-17 Endomembrane System

•Protein targeting, secretion, and vesicle traffic–Targeting proteins to cytoplasmic organelles–Targeting and translocating proteins into the ER–Vesicle trafficking, budding and fusion (ER, Golgi,lysosome–Endocytosis

Today L14, protein targeting to cytoplasmic organelles

•Protein folding and degradation•Intro to targeting•Import into nucleus•Import into chloroplast and mitochondria

Nascent proteins must fold to the correct IIo and IIIo conformation

Folding of the nascent polypeptide begins during translation

Information for folding is in amino acid sequence: fold to minimum energy configuration

Some proteins can fold (and can refold) spontaneously

Some proteins can’tFrom MBoC (4) figure 6-81 © Garland Publishing

mRNA

N-terminal domains fold

C-terminal domains fold

Completed protein released from ribosome

(a few minutes after translation began)

ATP +

+ ADP + Pi

HSP60 family

“Heat shock (HSP)” proteins aid protein foldingHSP70 family ATPases act as “chaperones” to aid protein folding

Adapted from MBoC(4)figures 6-83 and 6-84

Correctly folded protein

Incorrectly folded protein

ATP ADP+ Pi

ADP

ATP hydrolysis Exchange

HSP70 binds hydrophobic regions

HSP70 released

Correctly folded protein

“Proteosome”

HSP60 family of chaperones tries to re-fold mis-folded proteins…

ATP

Synthesis of chaperones increases dramatically at elevated T

Death of a protein: mis-folded, damaged, or unneeded proteins are degraded in

proteosomes

Cytoplasmic enzymes recognize mis-folded (up to 1/3 of newly synthesized proteins), damaged, or short-lived proteins

…and “tag” those proteins for destruction by covalently linking ubiquitin (76 aa polypeptide) to lysine side chains.

Short-lived proteins may contain specific “destruction” sequences that target them for rapid ubiquitination

Tagged proteins are then degraded in “proteosomes.”

ubiquitin (?)

ATP AMP+ 2 Pi Incorrectly folded or

damaged protein

? ?

?

?

Ubiquitinated protein marked for degradation

“Proteosome” Peptides

The proteosome ECB 7-36

20S proteosome

19S cap = ‘gate’

Active sites

Lecture 14

Protein folding and degradation

Intro to protein import into organelles

Import into the nucleus

Import into mitochondria and chloroplasts

Review: Prokaryotes have few “compartments”

Nucleoid (packaged DNA)

Cytoplasm

ECB figure 1-11

…in contrast to eukaryotic cells, which have many compartments

Nucleus (DNA replication, transcription and RNA processing)

ER (lipid metabolism; synthesis of secretory and membrane proteins)

Golgi (processing and sorting of secretory and membrane proteins)

Mitochondria and chloroplasts (ATP synthesis and carbon fixation)

Endosomes (endocytosis)

Lysosomes (recycling)

Peroxisomes (detoxification)

Cytosol (lots of things)

ECB panel 1-2 and figure 15-2

Relative numbers and volumes of some membrane-bounded compartments in a

hepatocyte (liver cell)

Qs for next few lecturesHow are proteins targeted to the correct compartments?How do these organelles communicate with each other?

Nucleus 1 6 Sequesters genome.Mitochondria 1700 22 TCA, resp., ox phos etc

ER 1 12 Lipid synthesis. Synthesis of secreted and membrane proteins.

Golgi 1 3 Processing and sorting membrane/secreted proteins.

Peroxisomes 400 1 Oxidative detoxification.

Lysosomes 300 1 Degradation and recycling.

Endosomes 200 1 Sorting.

Cytosol 1 54 Metabolism and protein synthesis.

Adapted from ECB Tables 15-1 and 15-2

CompartmentNumber/cell Relative volume (%) Function

Plant cell - most of volume is vacuole, dozens to 100s of chloroplast

Origin of nucleus and ER

Invagination of plasma membrane

Nucleus surrounded by double membrane

Outer nuclear membrane is contiguous with ER

ECB 15-3

Origin of mitochondria and chloroplasts

ECB 15-4

Surrounded by double membrane and contain own DNA, but codes for very few proteins! (a few dozen)

Instead, most genes from prokaryotic ancestor have been transferred to the nucleus, so proteins must be imported

Three ways organelles import proteins

15_05_import_proteins.jpg

ECB 15-5

We will begin with import into nucleus and then consider chloroplast and mitochondria

Then import into ER and protein transport to Golgi, lysosomes etc. via vesicles

Import into organelle from cytoplasm is

directed by sequence in protein Specific aa sequence for each organelle, often near amino terminus

Typically 15-60 aa long, usually removed after import

Often not a specific sequence but hydrophobicity or placement of charged amino acids

(NLS)

Signal sequence is both necessary and sufficient for import

Necessary

Sufficient

Study import into organelles using molecular tools

Green fluorescent protein (GFP) is cloned onto protein of interest

GFP Promoter

Test protein

Construct is transformed into cells where it is transcribed and translated

Virus protein::GFP

virus protein(-NLS)::GFP

NLS::GFP

Cell GFP mergeImport into nucleus

End L14 2004