View
215
Download
0
Category
Tags:
Preview:
Citation preview
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
Recommended