1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 19 BIOTECHNOLOGY

2. INTRODUCTION Biotechnology is broadly defined as technologies that involve the use of living organisms, or their products, to benefit humans It is not a new topic It began about 12,000 years ago when humans began to domesticate animal and plants for the production of food Since the 1970s, molecular genetics has provided new, improved ways to make use of organisms to benefit humans An organism that has integrated recombinant DNA into its genome is called transgenic 19-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. Microorganisms are used to benefit humans in various ways Refer to Table 19.1 Molecular genetic tools are very important in influencing and improving our use of microorganisms Overall, the use of recombinant microorganisms is an area of great research interest and potential However, there are problems such as safety concerns and negative public perception Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19.1 USES OF MICROORGANISMS IN BIOTECHNOLGY 19-3

4. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-4

5. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many Important Medicines Are Produced by Recombinant Microorganisms 19-16

6. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells It is produced by the  cells of the pancreas Persons with insulin-dependent diabetes have a defect in their  cells Therefore, they cannot synthesize enough insulin Sources of insulin included Cows Human cadavers! But now, patients can use insulin made by recombinant bacteria Refer to Figure 19.2 19-17 Insulin

7. Figure 19.2 The use of bacteria to make human insulin 19-18 Insulin is a hormone composed of two polypeptide chains, called the A and B chains

8. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transgenic animals Gene replacemen t Gene addition

9. Fig. 19.4

10. Cloned genes can be introduced into plant and animal cells However, the gene will not be inherited stably if it does not become integrated into the host cell’s genome This integration occurs by recombination The introduction of a cloned gene into a cell can lead to one of two outcomes Gene replacement Gene addition Refer to Figure 19.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Gene Addition versus Gene Replacement 19-26

11. 19-27 Figure 19.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display If rendered inactive by mutation => gene knockout

12. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

13. In bacteria and yeast, gene replacement is the common outcome These have relatively small genomes, so homologous recombination occurs at a relatively high rate In complex eukaryotes, gene addition is the norm These have very large genomes, so homologous recombination is rare Only 0.1% of the time Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Production of Mice That Contain Gene Replacements 19-28

14. To produce mice with gene replacements, molecular biologists have resorted to trickery Cells in which homologous recombination has occurred are preferentially selected This approach is shown in Figure 19.6 The cloned gene is altered using two selectable markers A neomycin-resistant gene (Neo R ) is inserted into the center of the coding sequence of the target gene A thymidine-kinase gene (TK) is inserted adjacent (not within) the target gene TK renders cells sensitive to killing by a drug called gancyclovir The modified target gene is then introduced into mouse embryonic cells which can be grown in the lab Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-29

15. 19-30 Figure 19.6 Sensitive to gancyclovir Resistant to both drugs

16. 19-30 Figure 19.6 Blastocyst Chimeric offspring A chimera is an organism that contains cells from two different individuals

20. Gene replacements were discussed in Figure 19.6 When a mouse is homozygous for an inactivated gene, this is called a gene knockout The inactive mutant gene has replaced both copies of the normal gene Gene replacements and gene knockouts have become powerful tools for understanding gene function Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Use of Gene Replacements in Mice 19-53

21. In some cases, gene knockouts produce phenotypic effect This shows that a gene is critical in a certain tissue or during a specific stage of development In other cases, gene knockouts produce no detectable phenotypic effect at all This led to the conclusion that mammalian genomes have a fair amount of gene redundancy A particularly exciting avenue of gene replacement research is its application in the study of human disease Cystic fibrosis (CF) Researchers have produced mice that are homozygous for the same type of mutation that is found in humans with CF These can be used as models organisms to study this human disease Furthermore, these mice models have been used to test various treatments for the disease Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-54

22. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-56 Transgenic Livestock

23. The production of proteins from mammals is more advantageous than the from bacteria 1. Certain proteins are more likely to function properly when expressed in mammals Post-translational modifications occur in eukaryotes Degradation and misfolding occur in bacteria The strategy for expressing human genes in animals is shown in Figure 19.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-57

24. 19-58 Figure 19.13 And use it to treat humans

25. DNA fingerprinting is a technology that identifies particular individuals using properties of their DNA It is also termed DNA profiling The application of DNA fingerprinting to forensics has captured the most public attention In addition, DNA fingerprinting can also be used to determine if two individuals are genetically related For example, it is used routinely in paternity testing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19.4 DNA FINGERPRINTING 19-62

26. When subjected to DNA fingerprinting, chromosomal DNA gives rise to a series of bands on a gel Refer to Figure 19.16 The order of bands is an individual’s DNA fingerprint It is the unique pattern of these bands that makes it possible to distinguish individuals In the 1980s, Alec Jeffries found that certain loci in human chromosomes are variable in length These loci contain tandemly repeated sequences called minisatellites In humans, the number of tandem repeats varies substantially Variable Number of Tandem Repeats (VNTRs) Refer to Figure 19.17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-63

27. 19-64 Figure 19.17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display VNTRs Restriction enzyme sites DNA probes are used to hybridize specifically to the repeat sequence located within VNTRs

28. Figure 19.18 Protocol for DNA fingerprinting 19-65

29. 19-66 Figure 19.18 Protocol for DNA fingerprinting The probe is called a multilocus probe (MLS) It binds to ~ 20 to 40 fragments of DNA that contain the sequence

31. RFLPs as markers for disease- causing alleles RFLP marker DNA Disease-causing allele Normal allele

32. In the past decade, the technique of DNA fingerprinting has become automated It is now done using PCR, which amplifies short tandem repeat sequences (STRs) Like VNTRs, STRs are found in multiple sites in human genomes and are variable among different individuals The main difference between a VNTR and STR is size STRs are much shorter, usually 100–450 bp STRs are called microsatellites, and VNTRs minisatellites The amplified STRs are fluorescently labeled They are separated by gel electrophoresis A laser excites the fluorescent molecule within the STR A detector records the amount of emission for each STR Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-67

34. 19-68 Figure 19.18 Protocol for DNA fingerprinting Each peak has a characteristic mass The pattern of peaks is an individual’s DNA fingerprint Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

35. Within the past decade, the uses of DNA fingerprinting have expanded in many ways In medicine, it is used identify different species of bacteria and fungi and also different strains of the same species This is useful for appropriate antibiotic treatment DNA fingerprinting is also used in forensics and relationship testing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Uses of DNA Fingerprinting 19-69

36. 19-71 Figure 19.19 The use of DNA fingerprinting to establish paternity Male 2 does not have many of the child’s paternal bands Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Therefore he cannot be the biological father Male 1 has all the child’s paternal bands The probability of this occurring by chance alone is very small Therefore he is the biological father

37. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA fingerprints from a murder case Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) D Jeans shirt V 4  g 8  g