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Recombinant DNA and Genetic Engineering. Chapter 16. Familial Hypercholesterolemia. Gene encodes protein that serves as cell’s LDL receptor Two normal alleles for the gene keep blood level of LDLs low Two mutated alleles lead to abnormally high cholesterol levels & heart disease.
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Recombinant DNA and Genetic Engineering Chapter 16
Familial Hypercholesterolemia • Gene encodes protein that serves as cell’s LDL receptor • Two normal alleles for the gene keep blood level of LDLs low • Two mutated alleles lead to abnormally high cholesterol levels & heart disease
Example of Gene Therapy • Woman with familial hypercholesterolemia • Part of her liver was removed • Virus used to insert normal gene for LDL receptor into cultured liver cells • Modified liver cells placed back in patient
Results of Gene Therapy • Modified cells alive in woman’s liver • Blood levels of LDLs down 20 percent • No evidence of atherosclerosis • Cholesterol levels remain high • Remains to be seen whether procedure will prolong her life
Genetic Changes • Humans have been changing the genetics of other species for thousands of years • Artificial selection of plants and animals • Natural processes also at work • Mutation, crossing over
Genetic Engineering • Genes are isolated, modified, and inserted into an organism • Made possible by recombinant technology • Cut DNA up and recombine pieces • Amplify modified pieces
Discovery of Restriction Enzymes • Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack • Discovered bacteria have an enzyme that chops up viral DNA
Specificity of Cuts • Restriction enzymes cut DNA at a specific sequence • Number of cuts made in DNA will depend on number of times the “target” sequence occurs
Making Recombinant DNA 5’ G A A T T C 3’ C T T A A G one DNA fragment another DNA fragment 5’ G A A T T C 3’ 5’ C T T A A G 3’ In-text figurePage 254
Making Recombinant DNA nick 5’ G A A T T C 3’ 3’ C T T A A G 5’ nick DNA ligase action G A A T T C C T T A A G In-text figurePage 254
Using Plasmids • Plasmid is small circle of bacterial DNA • Foreign DNA can be inserted into plasmid • Forms recombinant plasmids • Plasmid is a cloning vector • Can deliver DNA into another cell
Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids Figure 16.4Page 255
Amplifying DNA • Fragments can be inserted into fast-growing microorganisms • Polymerase chain reaction (PCR)
Polymerase Chain Reaction • Sequence to be copied is heated • Primers are added and bind to ends of single strands • DNA polymerase uses free nucleotides to create complementary strands • Doubles number of copies of DNA
DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands Polymerase Chain Reaction Double-stranded DNA to copy Stepped Art Figure 16.6Page 256
Mixture cooled; base-pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments Polymerase Chain Reaction Mixture heated again; makes all DNA fragments unwind Stepped Art Figure 16.6Page 256
DNA Fingerprints • Unique array of DNA fragments • Inherited from parents in Mendelian fashion • Even full siblings can be distinguished from one another by this technique
Tandem Repeats • Short regions of DNA that differ substantially among people • Many sites in genome where tandem repeats occur • Each person carries a unique combination of repeat numbers
Gel Electrophoresis • DNA is placed at one end of a gel • A current is applied to the gel • DNA molecules are negatively charged and move toward positive end of gel • Smaller molecules move faster than larger ones
Analyzing DNA Fingerprints • DNA is stained or made visible by use of a radioactive probe • Pattern of bands is used to: • Identify or rule out criminal suspects • Identify bodies • Determine paternity
Genome Sequencing • 1995 - Sequence of bacterium Haemophilus influenzae determined • Automated DNA sequencing now main method • Draft sequence of entire human genome determined in this way
Gene Libraries • Bacteria that contain different cloned DNA fragments • Genomic library • cDNA library
Engineered Proteins • Bacteria can be used to grow medically valuable proteins • Insulin, interferon, blood-clotting factors • Vaccines
Cleaning Up the Environment • Microorganisms normally break down organic wastes and cycle materials • Some can be engineered to break down pollutants or to take up larger amounts of harmful materials
The Ti plasmid • Researchers replace tumor-causing genes with beneficial genes • Plasmid transfers these genes to cultured plant cells plant cell foreign gene in plasmid Figure 16.11Page 261
Engineered Plants • Cotton plants that display resistance to herbicide • Aspen plants that produce less lignin and more cellulose • Tobacco plants that produce human proteins • Mustard plant cells that produce biodegradable plastic
First Engineered Mammals • Experimenters used mice with hormone deficiency that leads to dwarfism • Fertilized mouse eggs were injected with gene for rat growth hormone • Gene was integrated into mouse DNA • Engineered mice were 1-1/2 times larger than unmodified littermates
Cloning Dolly 1997 - A sheep cloned from an adult cell • Nucleus from mammary gland cell was inserted into enucleated egg • Embryo implanted into surrogate mother • Sheep is genetic replica of animal from which mammary cell was taken
Designer Cattle • Genetically identical cattle embryos can be grown in culture • Embryos can be genetically modified • create resistance to mad cow disease • engineer cattle to produce human serum albumin for medical use
The Human Genome Initiative Goal - Map the entire human genome • Initially thought by many to be a waste of resources • Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes • Sequencing was completed ahead of schedule in early 2001
Genomics • Structural genomics: actual mapping and sequencing of genomes of individuals • Comparative genomics: concerned with possible evolutionary relationships of groups of organisms
Using Human Genes • Even with gene in hand it is difficult to manipulate it to advantage • Viruses usually used to insert genes into cultured human cells but procedure has problems • Very difficult to get modified genes to work where they should
Can Genetically Engineered Bacteria “Escape”? • Genetically engineered bacteria are designed so that they cannot survive outside lab • Genes are included that will be turned on in outside environment, triggering death
Ethical Issues • Who decides what should be “corrected” through genetic engineering? • Should animals be modified to provide organs for human transplants? • Should humans be cloned?