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’
Major GM crops
and how they are
modified
Year 2000
Source: Sci. Am. April 2001
Trees too!
Poplars and aspens - genus Populus
Fast growth - 7 year old poplar
stand in Oregon
Model organism for tree genomics
Timber, plywood, pulp, paper
Engineering wood (cell wall) for
better pulp quality, etc.
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, 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)
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)
Transform cotton cells in culture, plasmid inserts in
genome
Grow cells in presence of antibiotic
Ampr
Regenerate plant from transformed cells
Ti Plasmid
(ampicillin
resistance)
Multiple
cloning
site
Test protein levels and glyphosate resistance
Note that plant will have 3 EPSPS
Replication
origin
Cotton EPSPS
Agrobacterium EPSPS
See ECB 10-40
‘Plant cells are totipotent’
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
Clone gene coding for 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 toxin 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
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: 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) will be selected for
GE crops are only short term solutions
* Gene may be transmitted from GM field to organic crops
Almost certainly will happen, British 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)
Golden rice - engineered to synthesize b-carotene,
vitamin A precursor
Vit. A deficiency causes blindness
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)
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
C-terminal
domains fold
N-terminal domains fold
Completed protein
released from ribosome (a
few minutes after translation
began)
mRNA
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’t
From MBoC (4) figure 6-81 © Garland Publishing
“Heat shock (HSP)” proteins aid protein folding
HSP70 family ATPases act as “chaperones” to aid protein folding
Synthesis of chaperones increases dramatically at elevated T
HSP70 binds
hydrophobic regions
Correctly
folded protein
HSP70 released
ATP
ATP
ADP
+ Pi
ATP hydrolysis
ADP
Incorrectly
folded protein
Exchange
HSP60 family of chaperones tries to
re-fold mis-folded proteins…
ATP +
“Proteosome”
+ ADP + Pi
Adapted from MBoC(4)
figures 6-83 and 6-84
HSP60 family
Correctly
folded protein
Death of a protein: mis-folded, damaged, or
unneeded proteins are degraded in proteosomes
Active sites
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.”
20S
proteosome
19S cap = ‘gate’
The proteosome ECB 7-36
ubiquitin (?)
Incorrectly folded or
damaged protein
ATP
AMP+
2 Pi
Ubiquitinated protein
marked for degradation
“Proteosome”
Peptides
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
ECB panel 1-2 and
figure 15-2
Nucleus (DNA replication, transcription Mitochondria and chloroplasts (ATP
and RNA processing)
synthesis and carbon fixation)
ER (lipid metabolism; synthesis of
secretory and membrane proteins)
Golgi (processing and sorting of
secretory and membrane proteins)
Endosomes (endocytosis)
Lysosomes (recycling)
Peroxisomes (detoxification)
Cytosol (lots of things)
Relative numbers and volumes of some membranebounded compartments in a hepatocyte (liver cell)
CompartmentNumber/cell Relative volume (%)
Nucleus
Mitochondria
Function
1
1700
6
22
Sequesters genome.
TCA, resp., ox phos etc
ER
1
12
Golgi
1
3
Peroxisomes
Lysosomes
Endosomes
Cytosol
400
300
200
1
1
1
1
54
Lipid synthesis. Synthesis
of secreted and membrane
proteins.
Processing andorting
s
membrane/secreted
proteins.
Oxidative detoxification.
Degradation and recycling.
Sorting.
Metabolism and protein
synthesis.
Adapted from ECB Tables 15-1 and 15-2
Plant cell - most of volume is vacuole, dozens to 100s of chloroplast
Qs for next few lectures
How are proteins targeted to the correct compartments?
How do these organelles communicate with each other?
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
We will begin with import
into nucleus and then
consider chloroplast and
mitochondria
_05_import_proteins.jpg
ECB 15-5
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
(NLS)
Typically 15-60 aa long, usually removed after import
Often not a specific sequence but hydrophobicity or placement of
charged amino acids
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
Promoter
Test protein
GFP
Construct is transformed into cells where it is transcribed and translated
Import into nucleus
Virus protein::GFP
virus protein(-NLS)::GFP
NLS::GFP
Cell
GFP
merge
End L14 2004