Chapter 16

1. Chapter 16 Studying and Manipulating Genomes

2. Impacts, Issues: Golden Rice, or Frankenfood? • 124 million children around the world have vitamin A deficiencies • Golden rice –Rice plants engineered with genes from daffodils allowing it to produce beta-carotine in its seeds (rice) –Beta carotine is the precursor to Vitamin A • Rice is the main food for 3 billion people

3. 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

4. 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

5. 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

6. Making Recombinant DNA 5’ G A A T T C 3’ C T T A A G another DNA fragment one DNA fragment 5’ G A A T T C 3’ 5’ C T T A A G 3’

7. 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

8. cut fragments enzyme recognition site A A T T C G 3’ G 5’ A A T T C 5’ 3’ 5’ 3’ G 5’ 5’ 3’ 3’ G C C T T T T A A A A one DNA fragment another DNA fragment nick 3’ 5’ 5’ 5’ 3’ 3’ nick DNA ligase action 3’ 5’ G G G A A A A A A T T T T T T C C C 5’ C C C T T T T T T A A A A A A G G G 3’ Stepped Art Fig. 16-2, p.244

9. 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

10. Plasmids Fig. 16-3b, p.244

11. Restriction enzyme cuts molecule of chromosomal DNA or cDNA DNA or cDNA fragments with sticky ends Foreign DNA, plasmid DNA, and modification enzymes are mixed recombinant plasmids containing foreign DNA Same enzyme cuts same sequence in plasmid DNA plasmid DNA with sticky ends host cells containing recombinant plasmids Stepped Art Fig. 16-4, p.245

12. Using Plasmids e The DNA fragments and the plasmid DNA are mixed with DNA ligase. a A restriction enzyme cuts a specific base sequence everywhere it occurs in DNA. b The DNA fragments have sticky ends. f The result? A collection of recombinant plasmids that incorporate foreign DNA fragments. c The same enzyme cuts the same sequnece in plasmid DNA. d The plasmid DNA also has sticky ends gHost cells that can divide rapidly take up the recombinant plasmids. Fig. 16-4, p.245

13. Making cDNA Fig. 16-5, p.245

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

15. 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 • Will base-pair with gene of interest

16. Use of a Probe aBacterial colonies, each derived from a single cell, grow on a culture plate. Each colony is about 1 millimeter across. bA nitrocellulose or nylon filter is placed on the plate. Some cells of each colony adhere to it. The filter mirrors how the colonies are distributed on the culture plate. cThe filter is lifted off and put into a solution. Cells stuck to it rupture; the cellular DNA sticks to the filter. dThe DNA is denatured to single strands at each site. A radioactively labeled probe is added to the filter. The probe binds to DNA with a complementary base sequence. e The probe’s location is identified by exposing the filter to x-ray film. The image that forms on the film reveals the colony that has the gene of interest. Fig. 16-6, p.246

17. 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

18. 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

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

20. 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

21. 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 Fig. 16-6, p. 256

22. 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 Fig. 16-6, p. 256

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

24. 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

25. 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

26. 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

27. 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

28. Gel Electrophoresis Fig. 16-9b, p.249

29. 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

30. Bozeman video—dna fingerprinting http://www.youtube.com/watch?v=DbR9xMXuK7c

31. 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

32. Genomics • Structural genomics: actual mapping and sequencing of genomes of individuals • Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

33. 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

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

35. 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

36. 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

37. 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

38. 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

39. The Ti plasmid bThe bacterium infects a plant and transfers the Ti plasmid into it. aA bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue). eYoung plants with a fluorescent gene product. cThe plant cell divides. dTransgenic plants. Fig. 16-13, p.253

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. Transgenic Mice Fig. 16-15, p.254

42. 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

43. 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

44. Xenotransplantation • Researchers knockout the Ggta1genes in transgenic piglets • Ggta1 gene produces proteins that human antibodies recognize • Pig’s organs are less prone to rejection by a human

45. Genetically Modified Animals Fig. 16-14c, p.254

46. 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

47. 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

48. 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?