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Biotechnology Chapter 17 Biotechnology • Generally implies the genetic manipulation of organisms to give them new capabilities or improved characteristics • “bio” – life • “technology” – application of science to creation of products for human use, processes, and services Plasmids • Discovered in 1960s • Small pieces of DNA – Separate from main bacterial chromosome – Generally not required for survival of bacterial cell – May carry genes that help cell survive in unusual environments – May carry information about antibiotic resistance Plasmids – Can be replicated in cell just like main chromosome – Useful because easy to purify and work with • Have fewer genes than main chromosome • More stable in test tube • Easier to analyze – Bacterial cells can be induced to take up plasmids from surrounding solution • Process called transformation Recombinant DNA • Microbiologists discovered in 1960s that bacteria contain enzymes capable of cutting DNA at specific base sequences – Restriction endonucleases or restriction enzymes • Function to protect cell by restricting invasion of cell by foreign DNA • Different restriction enzymes recognize different sequences of bases in DNA Recombinant DNA • Restriction enzymes – Allow scientists to cut purified plasmid DNA in specific, reproducible places – Cuts can be reversed – Many make cuts with sticky ends • Overlapping regions of complementary DNA strands • At lower temperatures, ends stick together, and DNA can be covalently connected (ligated) using DNA ligase Recombinant DNA • Can combine DNA pieces from different sources because sticky ends formed by particular restriction enzyme all have same base sequence – Forms recombinant DNA molecule – If process inserts new gene and DNA molecule becomes circular, new gene can be taken up with plasmid by receptive bacterium Recombinant DNA • Key to genetic engineering is selecting desired combination of ligated pieces of DNA through procedure known as cloning Cloning • Clone – Colony or group of cells or organisms – All members of group have same genes • Cloning – Replication of cells in the colony – Simple method of separating and eventually characterizing individual molecules of DNA – Individual molecule inserted into single bacterial cell can be replicated many times as cell divides – Cells in colony makes hundreds of thousands of copies of the same molecule Cloning • Cloning example – Recombinant DNA molecules formed from plasmid and specific gene – Plasmid (pUC19) has two genes • Gene for resistance to ampicillin • Gene for making enzyme β-galactosidase – Treat plasmid with restriction enzyme • Restriction enzyme makes cut in middle of βgalactosidase gene Cloning – Add new gene cut with same enzyme and ligate – Combine mixture of DNA molecules with suspension of bacterial cells in way so that each cell takes up only one DNA molecule – Spread bacteria on Petri dish containing nutrient agar, ampicillin, and chemical that turns blue in presence of β-galactosidase – Bacteria without plasmid will not grow on medium • Ampicillin kills cells Cloning – Bacteria with plasmids (ampicillin resistance) survive and grow into colonies • Colonies with β-galactosidase gene turn blue • Colonies with gene inserted in middle of βgalactosidase gene remain white – Check white colonies to verify that they contain desired gene Reverse Transcriptase and cDNA • Reverse transcriptase – Enzyme that can produce DNA using RNA template • Extract mRNAs and reproduce base sequences in DNA molecules • Starting with – extracted mRNA – a “primer” (small piece of DNA complementary in base sequence to mRNAs) – substrates (nucleoside triphosphates) Reverse Transcriptase and cDNA • Reverse transcriptase adds nucleotides to primer to form – Single strands of DNA with base sequences complementary to mRNA templates • Result is mixture of “complementary” or “copy” DNAs – Abbreviated cDNAs Polymerase Chain Reaction • PCR – Method to produce multiple copies of desired gene • Reaction combines – cDNAs with oligonucleotides (serve as “primers”) – Nucleoside triphosphates – DNA polymerase • Enzyme that synthesizes DNA Polymerase Chain Reaction • Flexible technique – Can be used to • Detect traces of animal or plant genes in criminal investigations • Synthesize a gene with added restriction sites at ends – Useful for transforming plants – Allows gene to be inserted into plasmid and cloned in bacteria Polymerase Chain Reaction • Steps in reaction cycle – Heat reaction solution almost to boiling • Separates complementary strands of DNA – Each strand is potential template – Cool reaction solution • Allows primers to bind to ends of any DNA with complimentary base sequences Polymerase Chain Reaction – Heat reaction solution to optimum temperature for DNA polymerase • Allows synthesis of new DNA by addition of nucleotides to primers Genomics • Genome – Genetic material in a cell • Genomics – Study of genome structure, function and evolution – Provides information useful in identifying genes • Genes with similar functions have similar base sequences Genomics • Information obtained also teaches how networks of genes are regulated Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Scientists focused on condition called crown gall disease – Caused by Agrobacterium tumefaciens – Bacteria attach to plant cell walls and cause cells to begin dividing – Plant cells continue to divide even after bacteria have been killed with antibiotics Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens – Shows bacteria transform plant cells • Turns off normal mechanism for limiting cell division – Result much like an animal cancer – Mechanism involved • Infectious strains of A. tumefaciens have large plasmid, Ti (tumor-inducing) plasmid Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Bacterium injects part of plasmid into plant cells – Region injected (T-DNA) contains three genes that cause cells to divide and grow » Two genes code for enzymes that make auxin » One gene codes for a cytokinin (isopentenyl adenine) – Another gene is for enzyme that synthesizes amino acid called an opine » Opines out leak into intercellular spaces » Bacteria growing in intercellular spaces of tumor make enzyme allowing them to take up and metabolize opines Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • In order to use Ti plasmid to carry genes into plant cells – Begin with T-DNA that has lost genes for auxin and cytokinin synthesis • Will not cause tumors in plant – Insert gene of interest • Controlled by promoter that regulates when and in what tissues it is turned on, and “reporter” gene that allows selection for cells that incorporate T-DNA – Recombinant T-DNA, usually in form of miniplasmid, transferred to A. tumefaciens cell with Ti plasmid lacking its own T-DNA Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens – Spread on cut surface of piece of leaf – Bacteria transfer recombinant T-DNA to plant cells – Transfer leaf to medium containing antibiotics to kill bacterial cells – Engineers then select for plant cells that have incorporated reporter gene in T-DNA – Regenerate new plants using tissue culture techniques – Plants with new genetic information transgenic plants Biolistics • Method for adding new genetic material to plant cells • Uses gene gun • DNA containing gene is absorbed onto surface of small particles (subcellularsized) of gold or tungsten • Particles pressed onto front of bullet – Loaded into gun – Fired at plant tissue Biolistics • Metal plate with hole smaller than bullet stops bullet • Particles penetrate cells • Absorbed DNA dissolves into cell cytoplasm – Used as template for RNA synthesis – Genetic information expressed Electroportation • Another method for getting DNA into plant cell • Based on discovery that short, highvoltage charge of electricity can produce temporary holes in plasma membrane without permanently harming cell Electroportation • Make protoplasts by removing cell walls from recipient plant cells • Place protoplasts between two electrodes in ice-cold solution that contains the DNA • A few pulses of electricity produce membrane holes • Some DNA enters cells Electroportation • Culture protoplasts under proper conditions – Protoplasts regenerate cell walls – Start dividing – Regenerate whole plants that express genes of DNA that entered protoplasts Use of Viruses to Inject Genes Into Plants • Method does not produce permanently transformed plant – Viral and introduced genes not incorporated into plant’s nuclear DNA • Genes are not passed to seed formed by infected plant • Proteins made by infected plant in response to introduced genes – Often very useful Applications of Biotechnology • Examples of proteins produced through genetic engineering – Insulin – Somatotropin – Erythropoietin – Clotting factors – Interferon Applications of Biotechnology • Enzymes produced from genetically engineered bacteria (or yeasts) – Laundry detergent additives – Restriction enzymes – DNA polymerases Applications of Biotechnology • Plants are being genetically engineered to produce vaccines – Designing and testing food plants that contain genes for proteins from pathogens • Banana (Musa sapientum) – Makes protein from hepatitis B vaccine • Alfalfa (Medicago saliva) sprout – Contains part of the cholera toxin Development of New Plant Varieties • Produced plants with additional enzymes in anthocyanin pathway – Results are flowers with unusual colors or patterns – Hope to produce blue rose Pest Resistance • Classical genetic techniques – Inefficient • Require many cycles of back crossing and selection • Modern molecular techniques – Use of Bacillus thuringiensis to control pests • Bacterium B. thuringiensis produces protein toxin that kills insects • Gene for toxin inserted into important crop plants – Potato, tomato, corn, cotton • Plants synthesize toxins and kill insects that graze on them Pest Resistance – Insertion of gene for viral coat protein of tobacco mosaic virus TMV infects plants such as tomato, potato, eggplant, green pepper • Insertion of gene into these plants makes plant resistant to infection by virus – Development of crops resistant to herbicides • Resistant crop allows farmer to use herbicides to kill weeds in middle of field of crop plants – Allows more discriminating use of safer herbicides Improved Quality of Fruit After Harvest • Large portion of harvested crops never reach consumers due to spoilage – First bioengineered food approved in United States • “FlavrSavr “ tomato • Contains gene that blocks synthesis of polygalacturonase (needed to soften tomato as it rots) • Lack of enzyme delays senescence (aging) Improved Quality of Fruit After Harvest – Genes inserted into cantaloupes reduce synthesis of ethylene (ripening hormone) Improved Nutrition • Some dietary staples are not most nutritious – Example: corn low in essential amino acids lysine and tryptophan • High lysine varieties of corn have been developed • Varieties of rice developed – One type produces seed with endosperm rich in β-carotene • β-carotene precursor for vitamin A • Help prevent blindness due to this deficiency Improved Nutrition – Another type of rice rich in ferritin • Help prevent iron deficiency which results in anemia – Modification of canola (Brassica napus) • Given gene for fungal enzyme phytase • Enzyme phytase improves nutrition when included in feed for pigs and chickens – Releases phosphate from phytic acid – Helps animals grow faster and stronger Improved Tolerance to Environmental Stress • Resistance to some stresses thought to depend on several genes • Research directed toward identifying genes that differ between stress-tolerant and stress-sensitive varieties Is Biotechnology Safe? • Scientific issues to be evaluated in the approval of a genetically engineered food – Does the product contain any new allergenic material that might affect especially sensitive groups? – Are new toxic compounds introduced into the food supply, or are existing toxins increased to unacceptable levels? Is Biotechnology Safe? – Are nutrient levels adversely affected? – Will the use of genes for antibiotic resistance (used to indicate when a plant has been stably transformed) compromise the use of important therapeutic drugs? Is Biotechnology Safe? • Environmental effects – Impact of new plants on wildlife – Possibility that new genes from desired recipient species could be transferred to a related wild, weedy species • Concern when new gene confers protection against natural pests or chemical herbicides Is Biotechnology Safe? • Field of biotechnology is growing • Research is key – The more we understand about plant and animal physiology and ecology, the more safely and effectively we can use biotechnology to improve our lives.