6. Fig. 20-2a DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2

7. Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 3

11. Fig. 20-9 Mixture of DNA mol- ecules of different sizes Power source Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE RESULTS 1 2 + + – –

13. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment Large fragment 376 bp 201 bp 175 bp Dde I Dde I Dde I Dde I Dde I Dde I

16. Fig. 20-17 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage

18. Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2

21. Fig. 20-20 Cultured stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) Different culture conditions Different types of differentiated cells Blood cells Nerve cells Liver cells Cells generating all embryonic cell types Adult stem cells Cells generating some cell types Embryonic stem cells From bone marrow in this example

24. Fig. 20-22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4

26. Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. (a) Semen on victim Earl Washington Source of sample Kenneth Tinsley STR marker 1 STR marker 2 STR marker 3 (b) 17, 19 16, 18 17, 19 13, 16 12, 12 14, 15 11, 12 13, 16 12, 12

29. Fig. 20-25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS

31. Genetic Engineering of Microorganisms Many medicines & useful chemicals are now produced by genetically engineered bacteria. For instance, all insulin is synthesized by transgenic E. coli that have the genes to produce human insulin . This has greatly reduced the cost and made insulin available to all diabetics who need it. Before the 1980s, all insulin was derived from the pancreases of animals (insulin from pigs differs from human insulin by only one amino acid).

32. The leading artificial sweetener, aspartame , is made from two amino acids, aspartic acid and phenylalanine . Both amino acids are synthesized by bacteria, and the strain that produces the phenylalanine has been genetically engineered. Genetic Engineering of Microorganisms

33. Vitamin A deficiency - 1 million children under 5 die per year; many go blind . Modifications to improve the nutritional value of food. Iron deficiency - 2 billion affected; 100,000 maternal deaths /yr in Africa & Asia. Severe mental impairment in women & children. Genetic engineering of rice to contain vitamin A & increased levels of iron and zinc . “Golden rice” How important is this? Fig. 38.18 Genetic engineering of corn to reduce the level of omega-6 fatty acids . http://www.nysaes.cornell.edu/comm/gmo/corn.jpg

34. Some crop plants have been genetically engineered to extract limiting nutrients more efficiently from the soil. Other plants have been engineered to increase the bioavailability of their essential nutrients for digestion and uptake.

35. Genetic engineering of crop plants to increase the quantity & quality of food that is harvested and reaches the consumer. • Shift in emphasis from vegetative body to seed/fruit. The Green Revolution was driven in part by the introduction of genes for dwarfism into cereal grains like wheat. The same genes are now engineered into other crops such as basmati rice. http://www.lsuagcenter.com/en/crops_livestock/crops/WheatOats/Diseases/Wheat+Diseases.htm

36. - Salty soils (77 million hectares). Plants also remove the salt from the soil! http://www.environment.gov.au/soe/2001/publications/theme-reports/water/water02-1c.html • Decreased losses due to harsh environmental conditions :

37. - Extreme heat or cold http://cropwatch.unl.edu/photos/cwphoto/crop07-6wheat-3.jpg http://web1.msue.msu.edu/vanburen/frstapp.htm http://boxer.senate.gov/news/photos/events/2007/01/bigfreeze/16.html • Decreased losses due to harsh environmental conditions :

38. Luciferase engineered in tobacco There is interest in engineering plants to signal when they are water stressed or require more nutrients through the use of reporter genes (luciferase or one of the many fluorescent proteins that can be engineered to upregulate during a particular stress). Luciferase in tobacco Fig. 17.6

40. Fungi (powdery mildews, potato blight, ergot, etc.) http://www.potatomuseum.com/images/exblightfieldwithinsert.jpg Fig. 31.25 - Pathogenic microorganisms • Decreased losses due to pests & pathogens :

41. • Increased shelf life for fruits and vegetables. http://www.ces.ncsu.edu/depts/cs/biotech/pics/c2h.jpg Normal Flavor saver

42. Other genetic improvements to the foods and beverages that humans and animals consume. To decrease the allergenicity of food . A transgenic soybean was recently engineered that does not produce a protein to which some individuals are allergic. Peanuts are currently a major target. Decaffeinated coffee - “naturally”. A Japanese group recently engineered coffee that does not synthesize caffeine. Several forage crops have been engineered to have reduced lignin content so that they are more easily digested (by sheep, cows, etc.). Efforts are also underway to engineer grains to release more carbo-hydrates, or more essential nutrients, for animal feed .

43. http://arabidopsis.info/students/dom/mainpage.html A number of species have been genetically engineered to more effectively clean up pollution or to detoxify specific toxins. One example is the transfer of genes from bacteria into plants, resulting in plants that can take up highly toxic methylmercury and convert it to elemental mercury. Phytoremediation