Chapter Outline
INTRODUCTION New Techniques Developed to Manipulate DNA Techniques Can Be Applied to Alter an Organism`s Genes fig 19.1 PLASMIDS AND THE NEW GENETICS First Human Gene Inserted into Bacteria Interferon Increases resistance to viral infection Rare, purification of small quantities is very expensive Bacterial cells made to produce protein at high rate Masses of cells cloned from original cells Each cell a miniature interferon factory Insulin produced in the same manner Beginning of Genetic Engineering Ability to cut up DNA into pieces and rearrange them Recognize and cleave specific nucleotide sequences Segments inserted via plasmids or viruses RESTRICTION ENZYMES Bacteria Are Natural Source of Enzymes Viruses infect bacteria, multiply within and release progeny Bacteria have enzymes that chop up invading viruses Enzymes are restriction endonucleases Bacterial DNA not damaged because it is modified Recognize sequence, bind to DNA and cleave strand Methylase enzymes recognize bacterial DNA Bind to same bacterial sites Add methyl groups to nucleotides Restriction enzymes do not recognize methylated sites Bacterial DNA protected from fragmentation Endonucleases recognize sites Recognize a variety of four to six nucleotide sequences Segments possess two-fold rotational symmetry fig 19.2 Nucleotides at one end are complementary to those at other end Enzyme cleaves both strands of DNA at same time Results in one strand with longer tail than other end Restriction enzymes effectively cut DNA in half Site where DNA is cut has offset ends fig 19.3 Hundreds of Different Restriction Enzymes Each enzyme always cuts at same sequence Fragments always have same ends that are complementary to other ends Sets of nucleotides called "sticky ends" Ends can pair with each other Two fragments can be glued together by DNA ligase Fragments can be from entirely different organisms CONSTRUCTING CHIMERIC GENOMES Mythological Chimera Composed of Parts of Several Animals Biological Chimeras Are Made of Different Kinds of DNA Cohen and Boyer: First Artificial Bacterial Plasmid Cut plasmid containing resistance transfer factor with EcoRI Contained replication origin and tetracycline resistance gene Complementary ends joined forming pSC101 plasmid fig 19.4 Same restriction enzymes used to cut frog genome Frog DNA pieces added to open pSC101 circles Added to bacteria, select for tetracycline resistance fig 19.5 Isolated cells with plasmids containing frog genes Recombinant DNA: a molecule created in laboratory¨ A CLOSER LOOK AT GENETIC ENGINEERING Experiments Generally Consist of Four Stages Stage 1: Cleavage Via restriction endonucleases Large number of specific fragments called library Different library for each specific sequence Fragments compared by electrophoresis fig 19.6 Stage 2: Producing recombinant DNA Fragments put into plasmids or virus vehicles Fragment replicated with vehicle genome Stage 3: Cloning Fragment-containing vehicles introduced into bacteria Bacteria reproduce making identical replicas Each cell line maintained separately Whole set constitutes clone library of original DNA Stage 4: Screening Identify clone line containing fragment of interest Among most difficult and critical steps Preliminary Screening of Clones Eliminate bacteria not containing proper DNA fragment Use genes conferring antibiotic resistance fig 19.7a Eliminate bacteria without vehicle Culture clones on medium containing antibiotic Only bacteria resistant to antibiotic will grow on it Eliminate bacteria with vehicle, but lacking fragment Use vector with gene that enables cell to metabolize X-gal sugar Metabolism of X-gal produces blue product Cells with vector and functional gene will turn blue fig 19.7b Test clones for presence of X-gal metabolism Clones with fragment lose ability to metabolize sugar DNA fragment within gene makes it inoperative Cells remain colorless in presence of X-gal Finding the Gene of Interest fig 19.8 Clone library may contain thousands of DNA fragments Southern blot technique Fragments spread apart by electrophoresis Gel blotted with nitrocellulose, DNA transfers to sheet Probe poured onto nitrocellulose sheet Only fragments with proper gene hybridize with probe Probe may be radioactive chemical Analysis of restriction fragment length polymorphisms (RFLP's) fig 19.9 Cut DNA samples with particular restriction Separate fragments according to length with electrophoresis Use radioactive probe to identify fragments Obtain unique pattern of bands in gel Called "DNA fingerprinting" Used in criminal forensic investigations Used as markers to identify carriers of certain genetic disorders Getting Enough DNA to Work With: The Polymerase Chain Reaction Produce multiple identical copies of DNA fig 19.10 PCR used to amplify sequences or add sequences as primers to cleaved DNA Five steps in PCR process Tagging Primer of synthetic nucleotides mixed with DNA fragment template Increase size of fragment and give it a unique tag Heating Temperature of mixture increased to 98% C Both primed fragment and oligonucleotide dissociate into single strands Priming Solution cooled to 60% C Single strands of DNA reassociate into double strands Fragment base-pairs with complementary primer nucleotide Part of fragment still single stranded Copying Heat stable DNA polymerase added along with supply of all four nucleotides Polymerase copies rest of fragment as in DNA replication Oligonucleotide primer lengthened into complementary copy of single-stranded fragment Two copies of original now exist Repeating the cycle Repeat heating and cooling in short cycles Each cycle doubles amount of DNA After twenty cycles one fragment can become more than one million PCR allows investigation of minute samples of DNA Has had enormous impact on all aspects of biology fig 19.11 BIO TECHNOLOGY: A SCIENTIFIC REVOLUTION Pharmaceuticals Most obvious commercial application of gene technology Bacteria can produce gene products in bulk Several forms of interferon, human insulin Manufacture valuable nonhuman enzymes Produce medically important proteins Atrial peptides: regulate blood pressure, kidney function Tissue plasminogen activator: dissolves blood clots Must separate desired protein from bacterial proteins Time-consuming and expensive Produce RNA transcripts of genes Make proteins directly in cell-free culture Probing the Human Genome Localize cloned gene location via radioactive probe Construction of clonal libraries Use large-size restriction fragments Associate disease genes with restriction fragments Identify presence of fragments with electrophoresis Do genetic screening for potential birth defects Attempt treatment or cure with gene therapy Example: cystic fibrosis Propose sequencing of entire human genome fig 19.12 Construct detailed map of human genome Controversial as it requires significant resources Piggyback Vaccines Subunit vaccines for herpes virus and hepatitis viruses fig 19.13 Protein-polysaccharide coat genes isolated Spliced to vaccinia virus DNA Live vaccinia added to cell culture with fragments Recombinant virus carries coat genes of other virus Infected animal produces antibodies to outer surface of virus Make antibodies against virus without exposure to it Agriculture Initial difficulty in identifying suitable plant vector Currently use Ti plasmid of Agrobacterium Infects broad leaf plants but not cereal plants Attach other genes to this plasmid fig 19.14 Development of Flavr Savr tomatoes Contain fish antifreeze gene Produce ethylene glycol from ethylene Lack of ethylene delays ripening of fruit Herbicide Resistance Broadleaf plants engineered to be resistant to glyphosate Glyophosate is the active ingredient in Roundup herbicide fig 19.15 Extra copies of EPSP synthetase gene via Ti plasmid Plants overproduce enzyme Overcome glyphosate suppression Advantages Crops would not need to be weeded Wide variety of weeds killed and desired crop spared Glyphosate readily degradable Virus Resistance Ti plasmids introduce genes into broadleaf plants TMV protein coat genes placed into tobacco chromosomes fig 19.16 Grow plant via tissue culture All progeny cells contain TMV coat genes Transgenic plants do not develop disease as if infected with whole TMV Insect Resistance Insects presently controlled via chemical insecticides Engineer plants for resistance to insects Bacillus thuringiensis insecticidal protein genes fig 19.17 Ingested by tomato hornworm, converted to poison Harmless to animals with different stomach enzymes Genes introduced into plants via Ti plasmid Plants safe from attack by insects that eat them fig 19.18 Examples: Genetically altered potato kills Colorado potato beetle Cotton resistant to bollworms Corn resists European corn borer Isolation insect-killing enzyme from a fungus Cholesterol oxidase disrupts insect gut membranes Fungal gene inserted into a variety of crops Kills variety of insects including cotton boll weevil and Colorado potato beetle Introduce insecticidal protein into root bacteria B. thuringiensis does not normally inhabit roots Protect roots from various pests, including Pseudomonas Nitrogen Fixation Insert proper genes into non-leguminous plants Provide plants with own fertilizer Farm Animals Somatotropin growth hormone (BST) synthetically produced Added to diary cow`s diet to increase milk yield fig 19.19 Potential to increase weight of cattle and pigs fig 19.20 Human tests to increase size of hormonal dwarfs Public resistance to BST in milk Generalized fears of gene technology BST is a proteins, digested in stomach Development of transgenic animals Other Applications Create strains of bacteria to eat oil spills Grow "synthetic cotton" Forensic use Identification of individuals Ethics and Regulation Concerns regarding tampering with genetic material Accidental production of a cancer-transmitting bacterium Intentional development of a killer virus Dangerous complications of genetically engineered products administered to plants or animals in future generations Ecological impact of "improved" crops Potential of creating "genetically superior" organisms, including humans Most of public's concerns not well-founded Most organisms used in genetic engineering incompatible with human hosts Recombinant technology like natural crossing, only faster Genetic "dabbling" by humans minuscule compared to natural mutations Genetic engineering research under close scrutiny Appropriate experimental safeguards established Scientists well-trained Products tested for years prior to marketing Risk to humans, organisms and environment rigorously assessed Benefits far outweigh the risks