Chapter 14 Gene Mutation, DNA Repair and Transposable Element

1. Chapter 14 Gene Mutation, DNA Repair and Transposable Element Essentials of genetics pp278-301 遗传学 王亚馥 pp440-460

2. Aside from chromosomal mutation (ch7), • major basis of diversity among organisms, from genetic variation at the level of the gene. • The origin of such variation are gene mutations,whereby coding sequences are altered as a result of substitution ,addition,or deletion of one or more bases within those sequences. • Such mutations are subject to elaborate repair mechanisms, make the study of genetics possible, and the raw material on which evolution relies.

3. Contents in this Ch. 14.1 Classification of Mutations 14.2 Detection of Mutation 14.3 Spontaneous Mutation Rate 14.4 The Molecular Basis of Mutation 14.5 Ultraviolet and High Energy Radiation 14.6 Mutations in Humans: case Studies 14.7 Detection of Mutagenicity:The Ames Test 14.8 Counteracting DNA Damage:Repair Systems 14.9 Site-Directed Mutagenesis 14.10 Knockout Genes and Transgenes 14.11 Transposable Genetic Elements

4. GENETIC CHANGE • 5 ways to change & reorganize genotypes • chromosome structure – CH7 • chromosome number – CH7 • recombination – CH 9 and 8 • Mutation genes • transposable elements

5. Mutation  phenotype variabilities Form the basis for genetic study Identify and study the gene that control the modified trait

8. 14.1 Classification of Mutations • Mutations can be classified by various schemes. • Spontaneous Versus Induced Mutations • All mutations are described as either spontaneous or induced. Although these two categories overlap to some degree.

9. spontaneous mutations are those that just happen in nature. No specific agents are associated with their occurrence, and they are generally assumed to be random changes in the nucleotide sequences of genes. • Most such mutations are linked to normal chemical processes in the organism that alter the structure of the nitrogenous bases that are part of the existing genes. • Most spontaneous mutations are thought to occur during the enzymatic process of DNA replication, an idea that we discuss later in this chapter. • Once an error is present in the genetic code, it may be reflected in the amino acid composition of the specified protein. If the changed amino acid is present in a part of the molecule critical to the structure or biochemical activity, a functional alteration can result.

10. Induced mutations; In contrast to such spontaneous events, those that result from the influence of any artificial factor are considered to be. • It is generally agreed that any natural phenomenon that heightens chemical reactivity in cells will induce mutations. • For example, radiation from cosmic and mineral sources and ultraviolet radiation from the sun are energy sources that most organisms are exposed to and, as such, may be factors that cause spontaneous mutations.

11. The earliest demonstration of the artificial induction of mutation occurred in 1927, when Hermann J. Muller reported that X rays could cause mutations in Drosophila, In 1928, Lewis J. Stadler reported that X rays had the same effect on barley. In addition to various forms of radiation, a wide spectrum of chemical agents is also known to be mutagenic, as we shall see later in this chapter.

12. mutagens = agents that  mutation rates • radiation • chemicals, e.g... • preservatives • pesticides • herbicides • smoke • natural substances in food... • mechanisms  generate / study mutation

13. Gametic Versus Somatic Mutations • gene mutation ~ random • tissue • development • mutations are rare... selection systems  recovery • only germ-line mutations  transmitted to progeny • mutations  genetic dissection of biological processes

14. clone = cell population  (asexual) 1 progenitor • mutant sector = patch of mutant cells • only germinal mutations inherited

15. clone = cell population  (asexual) 1 progenitor • mutant sector = patch of mutant cells

16. SOMATIC vs GERMINAL MUTATIONS • clone = cell population  (asexual) 1 progenitor • mutant sector = patch of mutant cells

17. mutation = process of genetic change • 1 allelic form  another • forward: +  – • reverse: –  + • mutations  phenotype • loss-of-function  recessive (usually) • gain-of-function  dominant (usually)

18. Other Categories of Mutation • Various types of mutations are classified on the basis of their effect on the organism. Note that a single mutation may well fall into more than one category. • morphological Trait • The most easily observed mutations are those affecting a morphological Trait. For example, all of Mendel's pea characters and many genetic variations encountered in the study of Drosophila fit this designation. They cause obvious changes in morphology.

19. morphological mutations  visible phenotype

20. nutritional or biochemical variations : A second broad category of mutations includes those that exhibit nutritional or biochemical variations in phenotype. In bacteria and fungi, the inability to synthesize a particular amino acid or vitamin is an example of a typical nutritional mutation. In humans, sickle-cell anemia and hemophilia are examples of biochemical mutations. While such mutations in these organisms are not visible and do not always affect specific morphological characters, they can have a more general effect on the well-being and survival of the affected individual.

21. biochemical mutations  auxotrophs

22. loss-of-function function • usually + > m null • m null  lethal ? • m / m + > m • + / m m > + • usually + > m' leaky • gain-of-function • usually M > + • M / M lethal ?

23. behavior mutations :A third category consists of mutations that affect behavior patterns of an organism. For example, mating behavior or circadian rhythms of animals may be altered. The primary effect of behavior mutations is often difficult to discern. For example, the mating behavior of a fruit fly can be impaired if it cannot beat its wings. However, the defect may be in (1) the flight muscles, (2) the nerves leading to them, or (3) the brain, where the nerve impulses that initiate wing movements originate. The study of behavior and the genetic factors influencing it has benefited immensely from investigations of behavior mutations.

24. regulatory mutations :Another type of mutation can affect the regulation of genes. A regulatory gene can produce a product that controls the transcription of another gene. In other instances, a region of DNA either close to, or far from a gene can modulate its activity. In either case, regulatory mutations can disrupt normal regulatory processes and permanently activate or inactivate a gene. Our knowledge of genetic regulation depends on the study of mutations that disrupt this process.

25. Still another group consists of lethal mutations. Nutritional and biochemical mutations can also fall into this category. A mutant bacterium that cannot synthesize a specific amino acid it needs will be unable to grow and divide if plated on a medium lacking that amino acid. Various human bio­chemical disorders, such as Tay-Sachs disease and Huntington disease, are lethal at different points in the life cycle of humans.

26. P gametes F1 A/a♀A/a♂ ½ A + ½ a ½ A + ½ a  ¾ viable ¼ lethal ¼ A/A + ½ A/a + ¼ a/a • lethal mutations  death (recessive or dominant)

27. Finally, any of these categories can exist as conditional mutations. Even though a mutation is present in the genome of an organism, it may not be evident under certain conditions. Among the best examples are temperature-sensitive mutations, found in a variety of organisms. At certain "permissive" temperatures, a mutant gene product functions normally, only to lose its functional capability at a different, "restrictive" temperature. When shifted to a restrictive temperature, the impact of the mutation becomes apparent, even lethal, and is amenable to investigation. The study of conditional mutations has been extremely important in experimental genetics, particularly in understanding the function of genes essential to the viability of organisms.

28. conditional mutations • permissive conditions • restrictive conditions • host • temperature • nutrition • developmental stage-specific suppressor mutation

29. conditional mutations  (e.g., T4 rII phage E. coli ) • permissive conditions (E. coli B) • restrictive conditions (E. coli K) JAPANESE QUAIL

30. 14.2 Detection of Mutation • Detection in Bacteria and Fungi • Detection of mutations is most efficient in haploid microorganisms such as bacteria and fungi. Detection depends on a selection system to isolate mutant cells easily from nonmutant cells. Neuwspora is a pink mold that normally grows on bread. It can also be cultured in the laboratory. • selective system = separate mutants from + • minimal culture medium. • supplemented or complete medium

31. selective system for auxotroph mutation

32. SELECTIVE SYSTEMS for • filtration enrichment of fungi  auxotrophs (hyphae) • e.g., leu+  leu • prototrophs held in filter • auxotrophs pass filter • leu auxotrophs grow on MM + leucine... all others do not

33. SELECTIVE SYSTEMS for • reversion of auxotroph bacteria  prototrophs • e.g., ad  ad+ • auxotrophs grow in MM + adenine • some revertants • only prototrophs on MM plate • true revertant vs suppressor ? ...

34. SELECTIVE SYSTEMS for • true revertant vs suppressor ... revertant  wild type • true revertant... ad  ad+ad+  all ad+ prototrophs • suppressor... ad• su ad+  ad • su adad+ • su ad+  somead • su ad+ auxotrophs

35. SELECTIVE SYSTEMS leu auxotrophs other auxotrophs form colonies form no colonies leu+prototrophs grow & die auxotrophs do not grow & die MM + leucine without penicillin MM + penicillin • antibiotic enrichment  kill dividing prototrophs only • e.g., penicillin & leu+  leu • penicillin kills dividing cells • auxotrophs do not divide &  not killed • leu auxotrophs grow in MM + leucine... all others do not

36. SELECTIVE SYSTEMS T1-resistant T1-sensitive form colonies form no colonies T1 phage sensitive cells T1 phage resistant cells MM + T1 bacteriophage MM • resistance  grow in presence of selective agent • e.g., T1 phage-resistant E. coli • all cells grow in absence of T1 • only T1-resistant grow in presence of T1 phage • system to study the nature of mutation... adaptation or pre-existing ?

37. SELECTIVE SYSTEMS • fluctuation test (Luria & Delbrück, 1943)... Nobel • T1-resistant cells  Tonr ... grow in presence of T1 • T1-sensitive cells  Tons ... do not grow

38. SELECTIVE SYSTEMS • 2 hypotheses about origin of mutations 1. adaptation & constant • expect ~ identical sampled individual culture • dependent on exposure to selective agent only 2. pre-existing & random • expect variable sampled individual cultures • early mutations make larger populations • later mutations make smaller populations

39. SELECTIVE SYSTEMS • 2 hypotheses about origin of mutations (bulk  = 10) 1. adaptation & constant • expect ~ identical sampled individual culture • dependent on exposure to selective agent only

40. SELECTIVE SYSTEMS • 2 hypotheses about origin of mutations (bulk  = 10) 2. pre-existing & random • expect individual cultures to vary or fluctuate... • early mutations make larger populations • later mutations make smaller populations

41. SELECTIVE SYSTEMS • mutation random & not adaptive... direct evidence ? • replica plating...

42. SELECTIVE SYSTEMS • mutation random & not adaptive... direct evidence... • replica plating... • on T1 plates • all replicas = master •  resistance from mutations occur prior to exposure

43. SELECTIVE SYSTEMS        • measuring mutation rate • described by Poisson distribution • 11 individual cultures  0 mutations • # cell divisions...  n – initial cell # (e.g., 8 – 1  7) n in normal bacterial cultures

44. Detection in Drosophila • Müller, in his studies demonstrating that X-rays are mutagenic, developed a number of detection systems in Drosophila melanogaster. • These systems are used to estimate both spontaneous and induced rates of X-linked and autosomal recessive lethal mutations.

45. Detection in Drosophila attached-X procedure (并联) to test the X-linked mutation

46. Detection in Drosophila • ClB chromosome • Müller, 1920s (Nobel) • e.g.recessive X-linked lethals • C = large inversion (C/C+, C suppresses recombination in C+ homologue) • l = recessive lethal allele (l/l & l/Y= lethal) • B = Bar, dominant visible marker (B/B+ = Bar)

47. Detection in Drosophila       • mutation induction... e.g.recessive X-linked lethals • C = large inversion, suppresses recombination • l = recessive lethal allele (l/l & l/Y= lethal) • B = Bar, dominant visible marker (B/B+ = Bar) C+l+B+/Y♂ClB/C+l+B+♀ [ClB/C+l+B+ ♀ + ♂] or [ClB/C+l+B+m ♀ + ♂] ClB/Y C+l+B+/Y ClB/Y C+l+B+m/Y lethal viable lethal lethal

49. Muller－ 5（=“Base”） Bar棒眼和wa(apricot，杏色眼）、sc(scute,小盾片少刚毛 inversion

50. Detection in Plants • visual technique -observation • analysis of a plant's biochemical composition. For example, the isolation of proteins from maize endosperm, hydrolysis of the proteins, and determination of the amino acid composition have revealed that the opaque-2 mutant strain contains significantly more lysine than do other, non mutant lines.