10.1 A scientific Revolution

2. Fig. 10.1 Producing insulin Curing disease Increasing yields 10.1 A scientific Revolution • Genetic engineering is the process of moving genes from one organism to another • Having a major impact on agriculture & medicine

3. 10.2 Restriction Enzymes • Restriction enzymes bind to specific short sequences (usually 4- to 6- bases long) on the DNA • The nucleotide sequence on both DNA strands is identical when read in opposite directions GAATTC CTTAAG • Most restriction enzymes cut the DNA in a staggered fashion • This generates “sticky” ends • These ends can pair with any other DNA fragment generated by the same enzyme • The pairing is aided by DNA ligase

4. Fig. 10.2

5. All gene transfer experiments share four distinct stages 1. Cleaving DNA 2. Producing recombinant DNA 3. Cloning 4. Screening 10.3 The Four Stages of a Genetic Engineering Experiment

6. Fig. 10.3

7. Fragments appear as bands under fluorescent light Fig. 10.4 1. Cleaving the DNA • The large number of fragments produced are separated by electrophoresis

8. 2. Producing Recombinant DNA • Fragments of source DNA are inserted into vectors • Vectors are plasmids or viruses that carry foreign DNA into the host cell • Vector DNA is cut with the same enzyme as the source DNA, thus allowing the joining of the two 3. Cloning • Host cells are usually bacteria • As each bacterial cell reproduces, it forms a clone of cells containing the fragment-bearing vector • Together all clones constitute a clone library

9. 4. Screening • A preliminary screen of the clone library eliminates • 1. Clones without vectors • 2. Clones with vectors that do not contain DNA • The vector employed usually has genes for • a. Antibiotic resistance • This eliminates the first type of clones because they are sensitive to antibiotics • b. b-galactosidase • This eliminates the second type of clones based on X-gal metabolism and color changes

10. Fig. 10.5

11. 4. Screening • To find the gene of interest, the clone library is screened by a process termed hybridization • The cloned genes form base pairs with complementary sequences on another nucleic acid, termed the probe • The bacterial colonies are first grown on agar • They are then transferred to a filter • The filter is treated with a radioactive probe • The filter is then subjected to autoradiography

12. Fig. 10.6

13. 10.4 Working with DNA • Key techniques used by today’s genetic engineers include • PCR amplification • Used to increase the amounts of DNA • cDNA formation • Used to build genes from their mRNA • DNA fingerprinting • Used to identify particular individuals

14. PCR Amplification • The polymerase chain reaction (PCR) requires primers • Short single-stranded sequences complementary to regions on either side of the DNA of interest • PCR consists of three basic steps • 1. Denaturation • 2. Primer annealing • 3. Primer extension

15. Target sequence Heat Primers Denaturation 1 Cool Cycle 1 Annealing of primers 2 DNA polymerase Free nucleotides 2 copies Primer extension 3 Heat Cycle 2 Cool 4 copies Heat Cool 8 copies Cycle 3 Fig. 10.7

16. cDNA Formation • The primary mRNA transcript contains exons and introns • The processed mRNA contains only exons • It is used as a template to create a single strand of DNA termed complementary DNA (cDNA) • cDNA is then converted to a double-stranded molecule

17. Fig. 10.8

18. DNA Fingerprinting • This is a process that is used to determine if two DNA samples are from the same source • TheDNA from the two sources is fragmented using restriction enzymes • The fragments are separated using gel electrophoresis • They are transferred to a filter • The filters are screened with radioactive probes • Then subjected to autoradiography

19. Fig. 10.9 Two of the DNA profiles that lead to conviction First time DNA profiles were used in court of law

20. Genetic engineering has been used in many medical applications 1. Production of proteins to treat illnesses 2. Creation of vaccines to combat infections 3. Replacement of defective genes Gene therapy is discussed in Chapter 12 10.5 Genetic Engineeringand Medicine

21. Fig. 10.1 Making “Magic Bullets” • In diabetes, the body is unable to control levels of sugar in the blood because of lack of insulin • Diabetes can be cured if the body is supplied with insulin • The gene encoding insulin has been introduced into bacteria

22. Fig. 10.10 Making “Magic Bullets” • Other genetically engineered drugs include Has only one extra gene: HGH • Anticoagulants • Used to treat heart attack patients • Factor VIII • Used to treat hemophilia • Human growth hormone (HGH) • Used to treat dwarfism

23. Piggyback Vaccines • Genetic engineering has also been used to create subunit vaccines against viruses • A gene encoding a viral protein is put into the DNA of a harmless virus and injected into the body • The viral protein will elicit antibody production in the animal A novel kind of vaccine was introduced in 1995 • The DNA vaccine uses plasmid vectors • It elicits a cellular immune response, rather than antibody production

24. Fig. 10.11 Constructing a piggyback vaccine for the herpes simplex virus

25. In 1994, the recombinant hormone bovine somatotropin (BST) became commercially available Dairy farmers used BST as a supplement to enhance milk production in cows Consumers are concerned about the presence of the hormone in milk served to children This fear is unfounded 10.6 Genetic Engineeringof Farm Animals

26. Fig. 10.12 The production of BST through genetic engineering

27. Successful manipulation of the genes of crop plants has improved the quality of these plants Pest resistance Leads to a reduction in the use of pesticides Bt, a protein produced by soil bacteria, is harmful to pests but not to humans The Bt gene has been introduced into tomato plants, among others 10.7 Genetic Engineeringof Crop Plants

28. Herbicide resistance Crop plants have been created that are resistant to glyphosate Fig. 10.14 Petunias 10.7 Genetic Engineeringof Crop Plants Glyphosate-resistant plants Glyphosate-sensitive plants • Herbicide resistance offers two main advantages • 1. Lowers the cost of producing crops • 2. Reduces plowing and conserves the top soil

29. More Nutritious Crops Worldwide, two major deficiencies are iron and vitamin A Fig. 10.15 10.7 Genetic Engineeringof Crop Plants • Deficiencies are especially severe in developing countries where the major staple food is rice • Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden” rice to solve this problem

30. Fig. 10.15 Transgenic “golden” rice

33. Potential Risks of Genetically Modified (GM) Crops • The promise of genetic engineering is very much in evidence • However, it has generated considerable controversy and protest • Are genetic engineers “playing God” by tampering with the genetic material? • Two sets of risks need to be considered • 1. Are GM foods safe to eat? • 2. Are GM foods safe for the environment?

34. Potential Risks of Genetically Modified (GM) Crops • 1. Are GM foods safe to eat? • The herbicide glyphosate blocks the synthesis of aromatic amino acids • Humans don’t make any aromatic amino acids, so glyphosate doesn’t hurt us • However, gene modifications that render plants resistant to glyphosate may introduce novel proteins • Moreover, introduced proteins may cause allergies in humans

35. Potential Risks of Genetically Modified (GM) Crops • 2. Are GM foods safe for the environment? • Three legitimate concerns are raised • 1. Harm to other organisms • Will other organisms be harmed unintentionally? • 2. Resistance • Will pests become resistant to pesticides? • 3. Gene flow • What if introduced genes will pass from GM crops to their wild or weedy relatives?

36. Fig. 10.16 Potential Risks of Genetically Modified (GM) Crops • Should GM foods be labeled? • Every serious scientific investigation has concluded that GM foods are safe • So there is no health need for a GM label • However, people have a right to know what is in their food • So there may be a need for label after all