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 and how they are 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 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) Glyphosate binds to and inhibits EPSP synthase Not in animals Glyphosate = N phosphomethyl glycine P - CH 2 - NH - CH 2 - COO - Some EPSP synthases from bacteria are resistant to glyphosate (single aa change Gly 96 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 Plasmid Amp r (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 infestedSprayed with Roundup™

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 Insect resistant plants 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 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 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…. 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 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: 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 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.”

* 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 Other issues

Future directions 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  -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; (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 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 II o and III o 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’t From 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 folding HSP70 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 ATPADP + 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 ( ? ) ATPAMP+ 2 Pi Incorrectly folded or damaged protein ? ? ? ? Ubiquitinated protein marked for degradation “Proteosome” Peptides The proteosome ECB S 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 lectures How are proteins targeted to the correct compartments? How do these organelles communicate with each other? Nucleus16Sequesters genome. Mitochondria170022TCA, resp., ox phos etc ER112Lipid synthesis. Synthesis of secreted and membrane proteins. Golgi13Processing and sorting membrane/secreted proteins. Peroxisomes4001Oxidative detoxification. Lysosomes3001Degradation and recycling. Endosomes2001Sorting. Cytosol154Metabolism and protein synthesis. Adapted from ECB Tables 15-1 and 15-2 CompartmentNumber/cellRelative 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 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 CellGFPmerge Import into nucleus

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