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Today: Biotechnology •Exam #2 W 7/9 in class Comparative genomics also has been used to identify recently mobilized transposons in genetically diverse humans. For example, over 600 recent transposon insertions were identified by examining DNA resequencing traces from 36 genetically diverse humans. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Tbl 1 Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in Genetics 23: 183-191 DNA fingerprinting using RFLPs Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2 Fig 18.1 Restriction Enzymes cut DNA at specific sequences tbl 18.3 Examples of some restriction enzymes… Recognition Enzyme Sequence EcoRI 5'GAATTC 3'CTTAAG BamHI 5'GGATCC 3'CCTAGG HindIII 5'AAGCTT 3'TTCGAA TaqI 5'TCGA 3'AGCT AluI 5'AGCT 3'TCGA Cut 5'---G AATTC---3' 3'---CTTAA G---5' 5'---G GATCC---3' 3'---CCTAG G---5' 5'---A AGCTT---3' 3'---TTCGA A---5' 5'---T CGA---3' 3'---AGC T---5' 5'---AG CT---3' 3'---TC GA---5' Fig 20.5+.6 Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2 Separating DNA on a gel by size Fig 20.6 • Gel electrophoresis Fig 24.21 Visualizing differences in DNA sequence by RFLPs Fig 22.23 DNA fingerprinting DNA fingerprinting DNA fingerprinting Can DNA be obtained from hair? How can DNA be obtained from such a small sample? The inventor of PCR Fig 18.6 Polymerase Chain Reaction: amplifying DNA Polymerase Chain Reaction Fig 18.6 Fig 18.6 Polymerase Chain Reaction: Primers allow specific regions to be amplified. The inventor of PCR PCR animation http://www.dnalc.org/ddnalc/resources/pcr.html Areas of DNA from very small samples can be amplified by PCR, and then cut with restriction enzymes for RFLP analysis. Genetic Engineering: Direct manipulation of DNA Fig 18.2 Bacteria can be modified or serve as intermediates Fig 18.2 a typical bacteria Bacterial DNA plasmid DNA tbl 18.2 A typical bacterial plasmid used for genetic engineering Fig 18.2 Moving a gene into bacteria via a plasmid What problems exist for expressing eukaryotic gene in bacteria? Bacterial DNA plasmid DNA Fig 18.4 Reverse transcriptase can be used to obtain coding regions without introns. Fig 18.6 After RT, PCR will amplify the gene or DNA Fig 18.2 Moving a gene into bacteria via a plasmid RT and PCR Fig 18.1 Restriction Enzymes cut DNA at specific sequences Fig 18.1 Restriction enzymes cut DNA at a specific sequence Fig 18.1 Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends A typical bacterial plasmid used for genetic engineering Using sticky ends to add DNA to a bacterial plasmid Fig 18.1 If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself. Fig 18.1 The plasmid is treated with phosphatase to remove the 5’-P, preventing selfligation Fig 18.1 Transformation of bacteria can happen via several different methods. tbl 6.1 Bacteria can take up DNA from the environment Fig 9.2 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Tbl 6.1 •Electroporation •Heat shock •Osmotic Stress Fig 18.1 How can you know which bacteria have been transformed, and whether they have the insert? Resistance genes allow bacteria with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic Fig 18.1 Fig 20.5 Is the insert present? Plasmids with the MCS in the lacZ gene can be used for blue/white screening… A typical bacterial plasmid used for genetic engineering Intact lacZ makes a blue color when expressed and provided X-galactose When the lacZ gene is disrupted, the bacteria appear white Blue/white screening: Fig 18.1 Transformed bacteria plated on antibiotic and Xgal plates. Each colony represents millions of clones of one transformed cell. Fig 18.1 Successful transformation will grow a colony of genetically modified bacteria RT and/or PCR Fig 18.1 Inserting a gene into a bacterial plasmid Millions of Hectares Bacteria can be used to transform plants Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html Texas = 70 ha Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15b Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15 By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant. Fig 19.16 The generation of a transgenic plant Grown on herbicide Fig 19.16 *Not covered in class; will NOT be on the exam; check it out if you are interested.* How do you know whether the gene you want to express has the correct sequence? DNA sequencing *Not covered in class; will NOT be on the exam; check it out if you are interested.* • The structure of 2’,3’-dideoxynucleotides Fig 18.14 Fig 18.15 *Not covered in class; will NOT be on the exam; check it out if you are interested.* The dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.* The dideoxy sequencing method Fig 18.15 *Not covered in class; will NOT be on the exam; check it out if you are interested.* Gel produced by the dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.* Computerized sequencers use a similar method Fig 18.16 …now enjoy making Frakencritters. What is the largest scale experiment that is being currently performed? Genetically Modified Organisms Human resource production and use Worldwide Grain Production per Person http://www.earth-policy.org/Updates/2008/Update69.htm World Grain Stocks as Days of Consumption http://www.earth-policy.org/Updates/2008/Update69.htm ~60 percent of the world grain harvest is consumed as food, ~36 percent as feed, and ~3 percent as fuel. While the use of grain for food and feed grows by roughly 1 percent per year, that used for fuel is growing by over 20 percent per year. http://www.earth-policy.org/Updates/2008/Update69.htm Corn prices late 1999 - Dec 2007 Wheat prices http://www.earthpolicy.org/Updates/2008/ Update69.htm http://www.earth-policy.org/Updates/2008/Update69.htm Increasing oil prices drive up potential profit, and cost of food crops Cost of oil----Profitable cost of corn for ethanol $50 ---- $4/bushel $100 ---- $7/bushel $140 ---- $10/bushel Current corn price: ~$8/bushel http://www.farmdoc.uiuc.edu/manage/newsletters/fefo07_11/fefo07_11.html http://www.earth-policy.org/Updates/2008/Update69.htm Whereas previous dramatic rises in world grain prices were weather-induced, this one is policy-induced and can be dealt with by policy adjustments. If the entire U.S. grain harvest were converted into ethanol, it would satisfy scarcely 18 percent of our automotive fuel needs. http://www.earth-policy.org/Updates/2008/Update69.htm Land use in USA, 1997 Artificial Selection (breeding) HunterGatherer Industrial Agricultural Modern cows were bred from earlier bovine species. from Teosinte Maize By artificial selection that began ~10,000 years ago. Different foods from one species of plant. Created by artificial selection. Wild member of Brassica oleracea: small side buds Artificial selection Number of Individuals 1. Select individuals that have the largest side buds and breed them. 2. Of the offspring, select individuals that have the largest side buds and breed them. 3. Of the offspring, select individuals that have the largest side buds and breed them. 4. After several generations, bud size increases dramatically. 0 Brussels sprouts: extremely large side buds 1 2 Size of buds (cm) 3 Agricultural HunterGatherer Industrial increased technology and understanding of molecular biology Genetic Engineering: Direct manipulation of DNA Fig 18.1 Millions of Hectares Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html Texas = 70 ha Tbl 19.5 Why Change a Plant’s DNA? • Can change plant so that it has new or different characteristics – Produce needed protein – Insect resistance (Bt toxin) – Herbicide resistance (Round-up) – Drought or other stress resistance % of Total US Acres Common GM Crops in the U.S. http://blog.wired .com/wiredscien ce/2007/09/mon santo-ishap.html Types of Genetically Modified Crops Herbicide Tolerance; Insect Resistance; Viral Resistance % of Total US Acres Common GM Crops in the U.S. http://blog.wired .com/wiredscien ce/2007/09/mon santo-ishap.html Millions of Hectares Is changing an organsisms DNA a good idea? http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html Texas = 70 ha Is Changing an Organism’s DNA a Good or Bad Idea? • Genes flow within species and between species. • Are GM foods safe for the environment? • Are GM foods profitable for: – Consumers, Farmers, and Companies? Pollution in Gulf of Mexico from run-off in the Mississippi river QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. AAL 25.17 Pollen is easily transferred from plant to plant, and to related species. Genetic information moves. GM crops found in Oaxaca and other MX states GM crops illegal Genetic information moves, and is self-replicating. GM crops found in Oaxaca and other MX states GM crops illegal Many Questions, but Few Answers? • Are herbicide or insect resistant plants safe to eat? • Are GM foods safe for the environment? • Are GM foods profitable for: – Consumers, Farmers, and Companies? Types of Genetically Modified Crops Herbicide Tolerance; Insect Resistance; Viral Resistance Patents give 17 year monopoly for inventor Examples of current patents: •Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Examples of current patents: •Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties •Evolutec has patents on proteins in tick saliva for use as anti-inflammatory Examples of current patents: •Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties •Evolutec has patents on proteins in tick saliva for use as anti-inflammator •GTG in Australia has patents on non-coding human DNA for detecting risk of various diseases Examples of current patents: •Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties •Evolutec has patents on proteins in tick saliva for use as anti-inflammatory •GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases •Harvard patented cancer susceptible mouse Examples of current patents: •Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties •Evolutec has patents on proteins in tick saliva for use as anti-inflammatory •GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases •Harvard patented cancer susceptible mouse •Over 70 U.S. patents for use of Indian neem tree Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree One was rejected due to previous use in indigenous Indian culture Concerns of developing nations vs. industrialized countries Bioprospecting Lack of research on third world problems. (tuberculosis, malaria, cholera, dysentery… Not profitable. Where does the information about genetically modified foods come from? Millions of Hectares Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html Texas = 70 ha Today: Biotechnology •Exam #2 W 7/9 in class