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2. Chapter Menu Chapter Introduction Studying Genomes 15.1 The Genome Projects 15.2 Functional Genomics 15.3 Technologies Applications and Issues in Molecular Genetics 15.4 Mutations and DNA Repair 15.5 Genetic Disorders and Gene Therapy 15.6 Ethical, Legal, and Social Issues Chapter Highlights Chapter Animations Contents

3. Learning Outcomes By the end of this chapter you will be able to: A Describe the Human Genome Project. BExplain how functional genomics helps scientists use sequence data. C Describe new technologies that have contributed to the genome project. D Describe mechanisms of mutation and relate them to disease. E Distinguish between gene therapy and traditional treatments for genetic disease. F Discuss ethical, legal, and social implications of genetic technology. Learning Outcomes

4. This photo shows a robot picker selecting petri dishes containing bacterial colonies with human DNA for the Human Genome Project. Advances in Molecular Genetics • Why is human DNA placed in bacterial colonies? • What practical uses can you think of for this technology? Chapter Introduction 1

5. This photo shows a robot picker selecting petri dishes containing bacterial colonies with human DNA for the Human Genome Project. Advances in Molecular Genetics • The Human Genome Project was completed in 2003 and established that human DNA consists of over 3 billion base pairs and contains information for approximately 35,000 genes. • This data is being analyzed to help find the causes of genetic diseases and to aid in their treatment and prevention. Chapter Introduction 2

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7. Studying Genomes 15.1 The Genome Projects • An organism’s genome consists of all of its genetic information. • An international organization called the Human Genome Organization was formed to coordinate human genome research around the world. • Pharmaceutical companies are trying to apply this knowledge to the development of better treatments for genetic diseases. 15.1 The Genome Projects 1

8. Studying Genomes 15.1 The Genome Projects (cont.) • The Human Genome Project (HGP) was an international effort to study the genomes of humans and other organisms. • The HGP’s central goal was to determine the sequence of the approximately 3 billion base pairs of DNA that make up the 24 different human chromosomes. • Other important goals include the study of how the genes function and the ethical, legal, and social issues related to genetic research 15.1 The Genome Projects 2

9. The dark and light bands shown on each human chromosome represent banding patterns seen with specific stains. These bands do not identify actual genes but serve as landmarks to help researchers label large regions of chromosomes that may contain genes. These diagrams are highly simplified representations of chromosomes that include many, many more genes than are being shown here. 15.1 The Genome Projects 3

10. Studying Genomes 15.1 The Genome Projects (cont.) • The HGP began with efforts to map genes to specific chromosomes. • Genome scientists are studying many different organisms besides humans, including viruses and bacteria that cause human infections. • This information being discovered is being used to help design new and more effective antibiotics. 15.1 The Genome Projects 4

11. Studying Genomes 15.1 The Genome Projects (cont.) • One of the first bacterial genomes sequenced was that of Escherichia coli, which lives symbiotically in human intestines, where it helps digest our food. • Much of our knowledge of cell biology and the functions of DNA are based on studies of E. coli. 15.1 The Genome Projects 5

12. Studying Genomes 15.1 The Genome Projects (cont.) • Saccharomyces cerevisiae, or brewer’s yeast, was the first eukaryotic genome to be sequenced, providing a model of more complex eukaryotic cells • A tiny worm, Caenorhabditis elegans, was the first multicellular organism sequenced. • This greatly aided the understanding of how genetic programs direct the development of more complex organisms such as humans. 15.1 The Genome Projects 6

13. The embryonic origin of each cell of Caenorhabditis elegans(a), a nematode worm, has been identified,x200. A fate map (b) of its cells shows how the worm normally develops. The effects of mutations in various genes on the development of specific cells reveal the genetic basis of development in this simple animal. 15.1 The Genome Projects 7

14. Studying Genomes 15.1 The Genome Projects (cont.) • Other genomes now under study include those of organisms with substantial commercial value, such as corn, rice, cotton, pigs, and cattle. • Breeders have begun to use the new genetic knowledge to modify the genomes of crops and livestock. • While these changes have increased crop yields and created more effective disease resistance, there is concern about whether people might be harmed by eating products made from genetically modified organisms. 15.1 The Genome Projects 8

15. Studying Genomes 15.2 Functional Genomics • Functional genomics is the study of DNA sequence information to help explain cell functions. • An important tool in functional genomics is the computer analysis of DNA sequences to predict the structures and functions of the proteins they encode. 15.2 Functional Genomics 1

16. Studying Genomes 15.2 Functional Genomics (cont.) In this computer image of DNA sequence data, each band contains DNA that is one base longer than the band below it. The color of each band indicates the terminal nucleotide in that band’s sequence. 15.2 Functional Genomics 2

17. Studying Genomes 15.2 Functional Genomics (cont.) • For diseases caused by mutations, specialists can use the DNA sequence of a mutant protein allele to predict how the mutation alters the shape and function of the protein. 15.2 Functional Genomics 3

18. Once the DNA sequence of a gene has been determined (a), it is stored in a computer database. A computer program identifies start and stop codons and introns and converts the DNA sequence into a predicted amino acid sequence (b) for the encoded protein. In a different example (c), a complete amino acid sequence is analyzed by a sophisticated program that predicts the three-dimensional structure of the protein. Comparison of the structures of normal and mutant proteins can reveal the nature of a genetic defect. 15.2 Functional Genomics 4

19. Studying Genomes 15.2 Functional Genomics (cont.) • In humans, the cytochrome P450 gene encodes the enzyme Cytochrome P450, which breaks down medicines and toxins that enter our bodies. • Scientists can use computer models of these variations to determine whether some people may have difficulty in metabolizing a new medicine. 15.2 Functional Genomics 5

20. Studying Genomes 15.2 Functional Genomics (cont.) Interactions of normal (a) and mutant (b) enzymes with a substrate molecule. If the altered amino acid sequence of the mutant enzyme interferes with binding of the substrate, the enzyme’s activity will decrease, and the substrate will not be metabolized as quickly or efficiently. 15.2 Functional Genomics 6

21. Studying Genomes 15.2 Functional Genomics (cont.) • Most genes interact in conjunction with many other genes. • DNA microarrays are used to look at the expression of thousands of genes at once. • Computer analysis of microarray binding can identify groups of genes that are turned on and off together. 15.2 Functional Genomics 7

22. Studying Genomes 15.3 Technologies • Humans have a long history practicing the selective breeding which manipulates traits that are part of an organism’s normal genetic potential. • Current methods enable the manipulation of individual genes that can be isolated and enable the alteration of their sequences. 15.3 Technologies 1

23. Studying Genomes 15.3 Technologies (cont.) • Using genetic engineering, biologists can prepare recombinant DNA–DNA whose segments are from different sources. • Recombinant DNA, once inserted into host cells, can be replicated, or cloned, to provide multiple copies of the gene for study. 15.3 Technologies 2

24. Studying Genomes 15.3 Technologies (cont.) • Creating a recombinant organism requires: • identifying a specific gene and excising it from a chromosome, usually with a restriction enzyme • finding a DNA carrier, or vector, that can be introduced into living host cells and can replicate there • finding a method of joining the gene to the vector DNA • finding a method to detect cells that have replicated the recombinant DNA 15.3 Technologies 3

25. Genetic engineering 15.3 Technologies 4

26. Studying Genomes 15.3 Technologies (cont.) • Advances in the genome project have also benefited greatly from improved technologies. • An efficient machine can sequence almost 100 different fragments of up to 1,000 base pairs of DNA at a time in a few hours. 15.3 Technologies 5

27. DNA is being loaded into plastic plates via this robotic arm for lab research to map the human genome. Studying Genomes 15.3 Technologies (cont.) • Preparing samples for DNA sequencing requires isolating pure DNA and mixing it with the appropriate chemicals to carry out the sequencing reaction. • Many of these reactions also use the polymerase chain reaction (PCR), a method of producing many copies of a tiny sample of DNA. 15.3 Technologies 6

28. Studying Genomes 15.3 Technologies (cont.) • Before DNA can be sequenced, restriction enzymes must be used to cut it into more manageable pieces. • Once the sequence of each piece has been determined, they must be assembled in the correct order to determine whether the sequences include any errors. 15.3 Technologies 7

29. Restriction enzymes cut DNA into pieces that are short enough to be sequenced individually. The sequences of the pieces must then be arranged in the correct order. Treating a second sample of the same DNA with a different restriction enzyme results in a different series of cuts. Overlaps between the two sets of pieces reveal their correct order. 15.3 Technologies 8

30. Studying Genomes 15.3 Technologies (cont.) • RFLP (restriction fragment length polymorphism) analysis is another tool for DNA analysis. • RFLP compares the lengths of DNA fragments to identify individuals or mutations that create or eliminate sites where a restriction enzyme binds to and cuts the DNA. • A problem in RFLP analysis comes from the fact that most restriction enzymes recognize DNA sequences that are only six bases long. 15.3 Technologies 9

31. Studying Genomes 15.3 Technologies (cont.) • When RFLP analysis is used to identify individuals, a short piece of synthetic DNA, called a probe, is added to the DNA after electrophoresis. • A radioactive atom that will expose X-ray film is attached to the probe. • Because of variation among individuals in the size of the DNA fragments, the positions of these bands can be used to identify the source of the DNA. 15.3 Technologies 10

32. Studying Genomes 15.3 Technologies (cont.) • A variation of RFLP can be used to determine whether a person carries a disease-causing allele. • A probe that binds to the globin gene can reveal the presence of the sickle-cell allele in the DNA of a healthy heterozygote. 15.3 Technologies 11

33. The restriction enzyme Mst II cuts the globin gene at three sites. The sickle-cell mutation eliminates one of these sites. The gene fragment that binds to the radioactive probe is longer than normal in the case of sickle-cell DNA. 15.3 Technologies 12

34. Studying Genomes 15.3 Technologies (cont.) • Another method, called PCR-RFLP, avoids the use of radioactively labeled probes. • Alternate alleles can be detected by first amplifying the chromosomal region containing the gene, then trying to cut these DNA fragments with a useful restriction enzyme. 15.3 Technologies 13

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36. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair • Mutations are changes in the DNA sequence that have many causes, including exposure to certain chemicals and radiation. • Most mutations probably result from failure of the DNA copying and repair mechanisms to work with 100% accuracy. 15.4 Mutations and DNA Repair 1

37. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • The most common type of mutation is the point mutation, in which one base pair changes into a different one. • Missense mutations are point mutations that change an amino acid that is important to protein structure or function. • Nonsense mutations change the codon for an amino acid into a stop codon, resulting in an abnormally short protein. 15.4 Mutations and DNA Repair 2

38. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • Frameshift mutations occur when one or two base pairs are inserted or deleted from the DNA, thereby disrupting the pattern of three-base codons. • Frameshift mutations change every subsequent codon resulting in a shortened, nonfunctional protein in which all amino acids after a certain point are changed. 15.4 Mutations and DNA Repair 3

39. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) Mutations in DNA can be illustrated in an English sentence. The effects of point mutations vary widely. Some missense mutations have little or no effect on phenotype; others have significant effects. Most nonsense and frameshift mutations eliminate normal gene function. 15.4 Mutations and DNA Repair 4

40. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • Different mutations in a gene can produce the same phenotype. • Many different mutations have been found in the human gene that encodes the enzyme phenylalanine hydroxylase. • Any mutation that reduces the activity of this enzyme results in a disorder called phenylketonuria (PKU). 15.4 Mutations and DNA Repair 5

41. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • Some genetic disorders, such as sickle-cell anemia and a form of dwarfism called achondroplasia, are caused by one specific point mutation. • When only one mutation causes all or most cases of a disorder, detection of the condition is easier because a single genetic test can be used. 15.4 Mutations and DNA Repair 6

42. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • Like the DNA in individual genes, large regions of chromosomes can be mutated by the deletion or movement of genetic material. • Chromosomal mutations are especially common in cancer cells. 15.4 Mutations and DNA Repair 7

43. Chromosome rearrangements 15.4 Mutations and DNA Repair 8

44. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • The study of a form of leukemia (cancerous growth of white blood cells) revealed that about 70% of people had cancer cells with a mutation called the Philadelphia chromosome. • The resulting protein has been found to activate the ras pathway that regulates cell division 15.4 Mutations and DNA Repair 9

45. A translocation transfers a small piece of chromosome 9 to chromosome 22, which is now referred to as the Philadelphia chromosome. A fluorescent probe reveals the presence of this out-of-place DNA. Look closely to notice that the c-abl gene is pink, the bcr gene is green, and the fusion gene (c-abl/bcr) shows up as yellow. The translocation joins parts of two genes, resulting in a fused protein. 15.4 Mutations and DNA Repair 10

46. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • In multicellular organisms, mutations that are not inherited can occur in somatic cells. • Some somatic-cell mutations are not important; at worst, they result in the death of one mutant cell. • Others cause uncontrolled cell growth, eventually leading to cancer. 15.4 Mutations and DNA Repair 11

47. ApplicationsandIssuesinMolecularGenetics 15.4 Mutations and DNA Repair (cont.) • Gene amplification, a somatic-cell mutation that is a normal part of development in many animal species, creates extra copies of specific genes. • During differentiation, some types of cells produce extra copies of chromosomal regions that contain the genes for specific proteins such as insulin. • Oncogenes are often amplified in cancer cells, contributing to the development of cancer. 15.4 Mutations and DNA Repair 12

48. ApplicationsandIssuesinMolecularGenetics 15.5 Genetic Disorders and Gene Therapy • The most important result of the HGP will be new ways of treating or preventing inherited disease. • Current treatments deal with the symptoms of the disease and not its genetic cause. 15.5 Genetic Disorders and Gene Therapy 1

49. ApplicationsandIssuesinMolecularGenetics 15.5 Genetic Disorders and Gene Therapy (cont.) • Gene therapy attempts to treat the genetic defect itself rather than its results. • It can be divided into two general categories: • In germ-line therapy, the DNA of an affected individual’s gametes would be changed so that the abnormal allele would not pass on to any future offspring. • Other gene therapy treats somatic cells, correcting the disease-causing allele in the cells that express the gene. 15.5 Genetic Disorders and Gene Therapy 2

50. ApplicationsandIssuesinMolecularGenetics 15.5 Genetic Disorders and Gene Therapy (cont.) • Gene therapy requires that genes be brought into cells and then integrated, or moved, into the cells’ DNA. • There are several methods of doing this: • placing the DNA in a lipid vesicle that will allow it to move through the plasma membrane • inserting the gene in the DNA of a virus that is adapted to bring its own DNA into the cell 15.5 Genetic Disorders and Gene Therapy 3