1. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174

2. Biotechnology - use of living organisms to create products or help processes Ex. HGH, insulin Recombinant DNA - segment of DNA containing sequences from different organisms How is DNA manipulated?

3. A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C G C G C G C G C G C G C G C G C G C G C G G C C G C G C G C G C G C G C G G G C C C C G G C G C G G C G C G C G G GG G G G C C C C C Restriction enzymes cut DNA at specific sites and create sticky ends Complementary ends will fuse to produce a long strand of DNA

4. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The DNA is then integrated into the recipient cell’s chromosome Figure 12.1D Donated DNA Recipient cell’s chromosome Crossovers Degraded DNA Recombinant chromosome

5. bacterium bacterial chromosome plasmid Plasmids are extra rings of DNA that replicate in bacteria. DNA can be inserted into plasmids.

6. Cloning Vectors

7. 1. Use restriction enzymes. 2. Insert gene into plasmid. 3. Transfer the plasmid back into bacterial cell. 4. Let bacterial cells replicate. bacterial clones replication transformation recombinant DNA Plasmid Bacterium Human cell DNA Human protein Bacterial chromosome

8. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant DNA products “seed” protein for artificial snow Insulin for diabetes treatment Enzymes that clean up toxic waste spills Growth Hormones (Human, Bovine) TPA: Tissue Plasminogen Activator for treatment of heart attacks

9. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube Selection of specific sequence Figure 12.12 Initial DNA segment 1248 Number of DNA molecules

10. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings

11. Restriction fragments of DNA are compared by size Gel electrophoresis sorts DNA molecules by size Figure 12.10 Mixture of DNA molecules of different sizes Power source Gel Glass plates Longer molecules Shorter molecules Completed gel

12. DNA forensics

13. Egg manipulation via microinjection. Credit: © Science VU/Visuals Unlimited Egg microinjection to produce transgenic animal

14. Grow bigger fish faster. Salmon with gene from another fish species

15. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Uses of transformed animals: Produce medicines more easily Ex. sheep and gene to treat cystic fibrosis Goats and AT3 gene to prevent blood clots Figure 12.16

16. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Fig. 11-14, p.173

17. An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary tumors on Kalanchoe stem. Credit: © Brad Mogen Genetic engineering of plants Methods to insert DNA: 1.Ballistics 2.Protoplasts 3.Agrobacterium as vector - Ti plasmid

18. a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue). b The bacterium infects a plant and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the plant’s chromosomes. c The plant cell divides. Its descendant cells form an embryo, which may develop into a mature plant that can express the foreign gene. A young plant expressing a fluorescent gene product Fig. 11-12, p.171

19. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Genetically modified crops Golden rice with Vitamin A Cotton resistant to boll weevil Soybeans resistant to herbicide (Roundup) Corn resistant to European corn borer Rapeseed with healthier vegetable oil Benefits and risks

21. white sheep black sheep egg cell udder cells DNA embryo Dolly surrogate mother How Dolly was cloned

22. Cloning of human cells Regenerative medicine –Bone, pancreas cells, skin Stem cells - the $6 billion promise?

23. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings treat disease by altering an afflicted individual’s genes –Ex vivo –In vivo –Stem cells Gene therapy may someday help treat a variety of diseases Figure 12.19 Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells into patient

24. Human Genome Project 3.2 billion bases in 22 autosomes + X, Y Draft sequence completed in 2003 Available at www.ornl.gov/sci/techresources/Human_Genome/ home.shtml www.ucsc.genome.edu

25. What does the human genome sequence tell us? 20 K to 25 K genes 99.9% alike, across all races 97% of DNA is not transcribed - Spacers between genes - Structural (centromeres, telomeres) - Regulatory (enhancers, promoters) - Leftovers of evolution?

26. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings How are specific genes identified? 1. Isolate it from a genomic library by homology with a gene from another organism. 2.Find mRNA for the gene, make cDNA from it. 3.Make DNA sequence based on protein sequence.

27. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 1. Nucleic acid probes identify clones carrying specific genes Figure 12.8A Radioactive probe (DNA) Mix with single- stranded DNA from various bacterial (or phage) clones Single-stranded DNA Base pairing indicates the gene of interest A nucleic acid probe can tag a desired gene in a library

28. Fig. 11-4, p.164 mRNA cDNA DNA reverse transcriptase DNA polymerase cDNA Complementary DNA Using reverse transcriptase Assembles DNA on mRNA template

29. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings A labeled probe can reveal patterns of gene expression in different kinds of cells DNA microarrays test for the expression of many genes at once Figure 12.9 cDNA DNA of gene DNA microarray, actual size (6,400 genes)

30. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene Therapy What is it? How is it done? Does it work?

31. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene therapy Goal - Treat diseases caused by mutated genes Method - Add a normal gene or block an abnormal gene in enough cells to restore normal function Target - somatic cells

32. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Which disorders are candidates for gene therapy treatment? Disorders due to mutations in one or more genes The responsible gene is known The affected tissues are known and accessible

33. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Knockout gene therapy Goal: turn off a gene that is causing a disorder Strategies: –Antisense –Triple helix oligos –Spliceosome –Ribozyme

34. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings How is gene therapy done? 1.Identify the gene(s) responsible for the disorder 2.Make copies of the normal gene 3.Insert the copies into vectors (i.e., viruses) 4.Infect the affected cells with the vectors 5.Activate the gene –Transcription and translation take place

35. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Critical factors in choosing a vector Gene size –Limited room in vector genome Target tissue –What cells can the vector infect? Integration into the genome –Without integration, only short-term effect –Random integration may disrupt other genes Cell cycle stage (dividing vs. non-dividing)

36. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene Therapy Successes Ashanti de Silva successfully treated for ADA deficiency - 1990 Ryes Evans successfully treated for SCID - 2001 Photo courtesy of Van de Silva

37. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Gene Therapy Problems Jesse Gelsinger died of complications due to an immune system response while participating in a clinical trial Three children treated for SCID developed leukemia due to disruption of a gene that regulates cell division

38. Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Ethical and Social Issues Patient safety while participating in clinical trials Which applications are therapies and which are enhancements? –“ Designer ” babies Access to gene therapies