1 Recombinant DNA and Genetic Engineering Chapter 15

2 Lipoproteins Form when certain blood proteins combine with cholesterol High-density lipoproteins (HDLs) Low-density lipoproteins (LDLs)

3 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

4 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

5 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

6 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

7 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

8 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

9 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

10 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’

11 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

12 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 be used to deliver DNA into another cell

13 Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids

14 Making cDNA mRNA transcript mRNA–cDNA hybrid single-stranded cDNA double-stranded cDNA

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

16 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

17 Polymerase Chain Reaction DNA is heated Primers are added DNA to be amplified

18 Polymerase Chain Reaction Mixture cools Base pairing occurs Complementary strand synthesized

19 Primers Short sequences that DNA polymerase recognizes as start tags To carry out PCR, must first determine nucleotide sequences just before and after the gene to be copied Complementary primers are then created

20 The DNA Polymerase Most DNA polymerase is denatured at high temperature Polymerase used in PCR is from bacteria that live in hot springs

21 Temperature Cycles DNA is heated to unwind strands Cooled to allow base-pairing with primers and complementary strand synthesis DNA is heated again to unwind strands Cycle is repeated over and over again

22 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

23 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

24 RFLPs Restriction fragment length polymorphisms DNA from areas with tandem repeats is cut with restriction enzymes Because of the variation in the amount of repeated DNA, the restriction fragments vary in size Variation is detected by gel electrophoresis

25 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

26 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 –Determine paternity

27 Genome Sequencing Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method 3.2 billion nucleotides in human genome determined in this way

28 Nucleotides for Sequencing Standard nucleotides (A,T,C, G) Modified versions of these nucleotides –Labeled so they fluoresce –Structurally different so that they stop DNA synthesis when they are added to a strand

29 Reaction Mixture Copies of DNA to be sequenced Primer DNA polymerase Standard nucleotides Modified nucleotides

30 Reactions Proceed Nucleotides are assembled to create complementary strands When a modified nucleotide is included, synthesis stops Result is millions of tagged copies of varying length

31 Recording the Sequence T C C A T G G A C C T C C A T G G A C T C C A T G G A T C C A T G G T C C A T G T C C A T T C C A T C C T C T electrophoresis gel one of the many fragments of DNA migrating through the gel one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A DNA is placed on gel Fragments move off gel in size order; pass through laser beam Color each fragment fluoresces is recorded on printout

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

33 Using a Probe to Find a Gene You want to find which bacteria in a library contain a specific gene Need a probe for that gene –A radioisotope-labeled piece of DNA –It will base-pair with the gene of interest

34 Use of a Probe Colonies on plate Cells adhere to filter Cells are lysed; DNA sticks to filter Probe is added Location where probe binds forms dark spot on film, indicates colony with gene

35 Engineered Proteins Bacteria can be used to grow medically valuable proteins –Insulin, interferon, blood-clotting factors –Vaccines Human gene is inserted into bacteria, which are then grown in huge vats

36 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 –Break down oil, sponge up heavy metals

37 Basic Research Recombinant DNA technology allows researchers to: –Investigate basic genetic processes –Reconstruct life’s evolutionary history –Devise counterattacks against rapidly mutating pathogens

38 The Ti plasmid Researchers replace tumor- causing genes with beneficial genes Plasmid transfers these genes to cultured plant cells foreign gene in plasmid plant cell

39 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

40 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

41 More Mouse Modifications Experiments showed that human growth hormone genes can be expressed in mice Human genes are inserted into mice to study molecular basis of genetic disorders, such as Alzheimer’s disease Variety of methods used to introduce genes

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

43 Designer Cattle Genetically identical cattle embryos can be grown in culture Embryos can be genetically modified –Experimenters are attempting to create resistance to mad cow disease –Others are attempting to engineer cattle to produce human serum albumin for medical use

44 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

45 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

46 Eugenic Engineering Selecting “desirable” human traits Who decides what is desirable? 40 percent of Americans say gene therapy to make a child smarter or better looking would be OK

47 Where Do We Go Now? Can we bring about beneficial changes without harming ourselves or the environment? Gene therapy is not harmless –A young man died after gene therapy that used an adenovirus Gene therapy can save lives –Infants with disabled immune systems are now healthy

48 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

49 Effects of Engineered Organisms Opposition to any modified organisms What if engineered genes escape into other species?