Recombinant DNA and Genetic Engineering Chapter 16

Familial Hypercholesterolemia Gene encodes protein that serves as cell’s LDL receptor Gene encodes protein that serves as cell’s LDL receptor Two normal alleles for the gene keep blood level of LDLs low Two normal alleles for the gene keep blood level of LDLs low Two mutated alleles lead to abnormally high cholesterol levels & heart disease Two mutated alleles lead to abnormally high cholesterol levels & heart disease

Example of Gene Therapy Woman with familial hypercholesterolemia Woman with familial hypercholesterolemia Part of her liver was removed Part of her liver was removed Virus used to insert normal gene for LDL receptor into cultured liver cells Virus used to insert normal gene for LDL receptor into cultured liver cells Modified liver cells placed back in patient Modified liver cells placed back in patient

Results of Gene Therapy Modified cells alive in woman’s liver Modified cells alive in woman’s liver Blood levels of LDLs down 20 percent Blood levels of LDLs down 20 percent No evidence of atherosclerosis No evidence of atherosclerosis Cholesterol levels remain high Cholesterol levels remain high Remains to be seen whether procedure will prolong her life 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 Humans have been changing the genetics of other species for thousands of years Artificial selection of plants and animals Artificial selection of plants and animals Natural processes also at work Natural processes also at work Mutation, crossing over Mutation, crossing over

Genetic Engineering Genes are isolated, modified, and inserted into an organism Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology Made possible by recombinant technology Cut DNA up and recombine pieces Cut DNA up and recombine pieces Amplify modified pieces Amplify modified pieces

Discovery of Restriction Enzymes Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Discovered bacteria have an enzyme that chops up viral DNA Discovered bacteria have an enzyme that chops up viral DNA

Specificity of Cuts Restriction enzymes cut DNA at a specific sequence Restriction enzymes cut DNA at a specific sequence Number of cuts made in DNA will depend on number of times the “target” sequence occurs Number of cuts made in DNA will depend on number of times the “target” sequence occurs

Making Recombinant DNA 5’ 3’ G C T T A A A A T T C G G C T T A AG 3’ 5’ one DNA fragmentanother DNA fragment 3’ 5’ In-text figure Page 254

Making Recombinant DNA nick 5’ 3’ 5’ GA A T T C C T T A AG nick GA A T T C C T T A AG DNA ligase action In-text figure Page 254

Using Plasmids Plasmid is small circle of bacterial DNA Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid Foreign DNA can be inserted into plasmid Forms recombinant plasmids Forms recombinant plasmids Plasmid is a cloning vector Plasmid is a cloning vector Can deliver DNA into another cell Can deliver DNA into another cell

Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids Figure 16.4 Page 255

Amplifying DNA Fragments can be inserted into fast-growing microorganisms Fragments can be inserted into fast-growing microorganisms Polymerase chain reaction (PCR) Polymerase chain reaction (PCR)

Polymerase Chain Reaction Sequence to be copied is heated Sequence to be copied is heated Primers are added and bind to ends of single strands Primers are added and bind to ends of single strands DNA polymerase uses free nucleotides to create complementary strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA Doubles number of copies of DNA

Polymerase Chain Reaction Double-stranded DNA to copy 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 Figure 16.6 Page 256 Stepped Art

Polymerase Chain Reaction Figure 16.6 Page 256 Stepped Art Mixture heated again; makes all DNA fragments unwind Mixture cooled; base- pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments

DNA Fingerprints Unique array of DNA fragments Unique array of DNA fragments Inherited from parents in Mendelian fashion Inherited from parents in Mendelian fashion Even full siblings can be distinguished from one another by this technique Even full siblings can be distinguished from one another by this technique

Tandem Repeats Short regions of DNA that differ substantially among people Short regions of DNA that differ substantially among people Many sites in genome where tandem repeats occur Many sites in genome where tandem repeats occur Each person carries a unique combination of repeat numbers Each person carries a unique combination of repeat numbers

Gel Electrophoresis DNA is placed at one end of a gel DNA is placed at one end of a gel A current is applied to the gel A current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones Smaller molecules move faster than larger ones

Analyzing DNA Fingerprints DNA is stained or made visible by use of a radioactive probe DNA is stained or made visible by use of a radioactive probe Pattern of bands is used to: Pattern of bands is used to: Identify or rule out criminal suspects Identify or rule out criminal suspects Identify bodies Identify bodies Determine paternity Determine paternity

Genome Sequencing Sequence of bacterium Haemophilus influenzae determined Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method Automated DNA sequencing now main method Draft sequence of entire human genome determined in this way Draft sequence of entire human genome determined in this way

Gene Libraries Bacteria that contain different cloned DNA fragments Bacteria that contain different cloned DNA fragments Genomic library Genomic library cDNA library cDNA library

Engineered Proteins Bacteria can be used to grow medically valuable proteins Bacteria can be used to grow medically valuable proteins Insulin, interferon, blood-clotting factors Insulin, interferon, blood-clotting factors Vaccines Vaccines

Cleaning Up the Environment Microorganisms normally break down organic wastes and cycle materials 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 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 Researchers replace tumor- causing genes with beneficial genes Plasmid transfers these genes to cultured plant cells Plasmid transfers these genes to cultured plant cells foreign gene in plasmid plant cell Figure Page 261

Engineered Plants Cotton plants that display resistance to herbicide Cotton plants that display resistance to herbicide Aspen plants that produce less lignin and more cellulose Aspen plants that produce less lignin and more cellulose Tobacco plants that produce human proteins Tobacco plants that produce human proteins Mustard plant cells that produce biodegradable plastic Mustard plant cells that produce biodegradable plastic

First Engineered Mammals Experimenters used mice with hormone deficiency that leads to dwarfism Experimenters used mice with hormone deficiency that leads to dwarfism Fertilized mouse eggs were injected with gene for rat growth hormone Fertilized mouse eggs were injected with gene for rat growth hormone Gene was integrated into mouse DNA Gene was integrated into mouse DNA Engineered mice were 1-1/2 times larger than unmodified littermates Engineered mice were 1-1/2 times larger than unmodified littermates

Cloning Dolly A sheep cloned from an adult cell Nucleus from mammary gland cell was inserted into enucleated egg Nucleus from mammary gland cell was inserted into enucleated egg Embryo implanted into surrogate mother Embryo implanted into surrogate mother Sheep is genetic replica of animal from which mammary cell was taken Sheep is genetic replica of animal from which mammary cell was taken

Designer Cattle Genetically identical cattle embryos can be grown in culture Genetically identical cattle embryos can be grown in culture Embryos can be genetically modified Embryos can be genetically modified create resistance to mad cow disease create resistance to mad cow disease engineer cattle to produce human serum albumin for medical use 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 Initially thought by many to be a waste of resources Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Sequencing was completed ahead of schedule in early 2001 Sequencing was completed ahead of schedule in early 2001

Genomics Structural genomics: actual mapping and sequencing of genomes of individuals Structural genomics: actual mapping and sequencing of genomes of individuals Comparative genomics: concerned with possible evolutionary relationships of groups of organisms 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 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 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 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 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 Genes are included that will be turned on in outside environment, triggering death

Ethical Issues Who decides what should be “corrected” through genetic engineering? Who decides what should be “corrected” through genetic engineering? Should animals be modified to provide organs for human transplants? Should animals be modified to provide organs for human transplants? Should humans be cloned? Should humans be cloned?