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Lecture 5

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Carleton University
BIOL 1010
James Cheetham

Lecture 5 Tuesday, October 12, 2010 - PCR, then will move to agricultural biotech - Los Angeles Times- first (legal) clinical trial for stem cell therapy (spinal cord damage) o Geron Corporation o First embryonic stem cells used Lecture 4 - Polymerase Chain Reaction o Taq polymerase comes from special bacteria that live in hot springs (high temperature)- very stable o Keeps doubling strands of DNA- in 30 days, could have over one hundred billion samples o Crime scenes- could use a small fragment of person’s DNA and amplify o To stop poachers getting illegal fish, could amplify DNA and see if they’re taking endangered species - Kary Mullis o Cetus sold PCR for $300 million (paid him $10,000) o Thermus aquaticus- Taq o Nucleotides A, T,C, G - Used to have to grind up large amounts of tissue to replicate, too much trouble. With this, could make lots of copies with little material. - Topisomerase could untangle knots- we just heat it up to melt DNA - DNA Polymerase- Taq - Primase- we synthesize primer - dNTP o NTP (ex. GTP, CTP, ATP, TTP) - DNA Microarrays o All 22,000 genes on microscope slide, screen to see if they have mutations, etc. o Want to see what genes get turned on when blastocyst turns into tadpole o During development, what changes in gene expression o Reverse transcriptase- retrovirus- HIV, viruses that cause cancer use this o Lots of cDNA will bind well to a spot o Wash everything off- green is from blastotube, red is from tadpole, yellow is both genes being expressed o Ex. If I give yeast maltose or fructose, would this be better for beer? o Can buy chips and chip readers from various companies, or buy chips and have them analyse for you o Matabolonomics o Pharmacogenomics- 23 and Me Lecture 5: Agricultural Biotechnology - used to do traditional farming not too long ago (and still do in places like Albania)- not too much technology, horses, etc. - Fermentation and brewing have been used for thousands of years, but other aspects of biotechnology are more modern - Farmers have manipulated genetic makeup of plants and animals - Exploitation of natural variation that gave us crops and such - Selected for better plants/animals to domesticate - Breeding - Wild cattle selected to be smaller o Dangerous for humans to be big o More manageable - Phylogenetic trees (for mapping diseases/evolution) - Cats don’t have nearly as much variation as dogs o Phenotypic plasticity o Dog genome has been sequenced, trying to figure out how it’s so variated - Brassica is one of the biggest plant families we’ve bred for human consumption - Karpechenko- Soviet biologist (at time of Stalin) o Vegetable for proletariat o Radish roots + cabbage leaves (but very far apart species-wise) o Radish- eat all the root but waste the tops, cabbage- eat tops but waste all the roots o This way, wouldn’t waste any part of plant o Mitotic accident happened- could produce fertile hybrids o Actually wound up getting radish leaves and cabbage roots (totally unedible) o Inspired research later on hybridization between far away species o Stalin sent him to Siberia- arrested by NKVD for belonging to anti-Soviet group, died in 1971 o Science getting influenced by ideology, scientists getting killed for not doing it right - Green Revolution o Wheat that didn’t grow as tall and didn’t fall in wind to generate wheat strains with greater yields - Marquis wheat- resistant to rust (fungal disease) - Science Advisory Committee part of Green Revolution - Clive James has been studying world food production, poverty, hunger, etc. - Peru- government wants to tear out rainforest to use for agriculture- no room - Can move genes from different species - Gene silencing- proteins that code for allergens - Control gene turning on, where, etc. - Drugs- biopharming o Increase nutrition, pharmaceuticals, etc. - Desired characteristics- will focus on this - Transgenic plants contain gene from another species/genus - GMO used commonly instead of GEO - Spray bacteria on corn- produces toxins to kill off corn ear worm o 1) Cytolysins (blew up cells) o 2) Crystadeltalendotoxins (in guts of bugs- blew them up) o Cyt proteins that target beetles, flies o Cry proteins that target moths and butterflies o Different complements of cyt and cry to kill off different bugs o Scientists got corn to make BT toxin (from bacteria)- better than spraying surface because bug can crawl inside plant and will die o Toxin only affects bugs- they have specific protein it binds to in their gut (we don’t have this) o In cage where monarch butterflies are forced to eat Bt corn, die (environmental criticism). But in actual environment, they avoid this corn. - Agrobacterium tumefaciens: commonly occurring bacteria - Plant cells are hard to get genes into- have tough cell walls, uncooperative- sometimes use gene guns - Plant Genetic Engineering Process o Cut out gene with restriction enzymes o Transformation: introducing foreign DNA into cell - Other than machinery for transformation, low-cost process - TI plasmid- special property- has region of DNA that causes it to be incorporated into genomic DNA of plant cell o Can alter TI plasmid to have gene that you want o Insert into plant- seal with ligase o Gene gets incorporated into DNA (genome of plant) o Each daughter cell has gene of interest- entire transgenic plant o Works well on broad-leaf plants (ie. soy bean plants) - Gene gun technique o Used for corn, rice, wheat (agrobacterium doesn’t work) o Biolistic particle delivery system o Penetrates plant cell walls o DNA incorporated into plant cell with new gene- daughter cells - Electroporation o Remove cell wall with enzymes- make protoplast (cell without cell wall) o Electroporation- make small holes o DNA with gene of interest gets into cell, genome o Cell wall grows back - Marker gene to make sure plant has gene of interest - Plant genes have control regions too - Transgenic technology- gene from wide range of species (bacteria --> corn) - Corn, soybean, papaya, squash --> GMOs - GMO: transgenic organism - Herbicide tolerance- resistant to glyphosphate (herbicide for weeds) - Plastic potat
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