Geneticists  Mutants

1. Geneticists Mutants Mutations are essential for: Genetic analysis and gene mapping Identifying and isolating disease genes Understanding gene function Discovering biochemical pathways Evolution: Most new mutations - deleterious Some provide selective advantage 1

2. Types of Mutations Wild type Mutant Wild type Forward Reverse (Backward) 2

3. Types of Mutations Point Mutations - Base-pair substitutions transition transversion purpur; pyrpyr purpyr; pyrpur 3

4. Types of Mutations Point Mutations - can change how codons are read missense nonsense 4

5. Types of Mutations Translation of a nonsense mutation 5

6. Types of Mutations Point Mutations - may not be obvious due to code redundancy neutral silent 6

7. Types of Mutations Point Mutations - can have polar effects Frameshift: insertion or deletion 7

8. Types of Mutations Deletion - Null Mutant - ‘knock out’ Large segment or entire gene lost No functional product possible Reverse mutation impossible unless gene replaced 8

9. Suppressor Mutations Second mutation cancels out effects of first restores wild-type phenotype to mutants Intragenic suppression Both mutations in same gene UGU (cys) - UGA (stop) - UGC (cys) Intergenic suppression Two different genes involved 9

10. Intergenic Suppressor Mutations Second mutation often in tRNA gene 10

11. Intergenic Suppressor Mutations Nonsense suppressor 11

12. Intergenic Suppressor Mutations Missense suppressor 12

13. Classifying Mutations Conditional - mutant phenotype expressed in certain conditions Temperature sensitivity - tyrosinase (melanin production) Useful for studying genes required for essential functions 13

14. Classifying Mutations Somatic - mutation occurs in body cells affects only the individual Germ line - mutation in gamete producing tissues passed on to next generation 14

15. Classifying Mutations Spontaneous - random mistake rate 1 in 104 to 109 mutations/cell/generation Induced - caused by exposure to mutagen mutagenesis 15

16. Causes of Spontaneous Mutations Tautomeric Shifts enol form of G with T imino form of A with C imino form of C with A enol form of T with G 16

17. Causes of Spontaneous Mutations Consequences of Tautomeric Shifts - transitions 17

18. Causes of Spontaneous Mutations DNA looping-out during replication (replication slippage) Deletion Insertion

19. Causes of Spontaneous Mutations Replication slippage in trinucleotide repeat regions Repeat expansion Anticipation Huntington disease Fragile X syndrome 19

20. Causes of Spontaneous Mutations Deamination C:G > U:A > T:A A > Hypoxanthine:C methylcytosine > T C:G > T:A Transitions 20

21. Induced Mutations Base analogs - 5-bromouracil incorporated into DNA during synthesis higher incidence of tautomeric shifts 21

22. Induced Mutations Base analogs - 5-bromouracil Transitions 22

23. Induced Mutations Intercalating agents - misalignment mutagens proflavin, acridine orange, ethidium bromide 23

24. Induced Mutations Intercalating agents - addition of nucleotide (base) insertion frameshift 24

25. Induced Mutations Intercalating agents - deletion of nucleotide (base) deletion frameshift 25

26. Base Modifying Agents Nitrous acid - oxidative deamination Transitions 26

27. Base Modifying Agents Hydroxylamine (NH2OH) Transitions 27

28. Base Modifying Agents Alkylating agents CH3 Nitrogen mustard Cl-CH2-CH2-N-CH2-CH2-Cl Ethylmethanesulfonate CH3-Ch2-O-SO2-CH3 Nitrosoguanidine HN=C-NH-NO2 O=N-N-CH3 Transfer methyl or ethyl group to bases 28

29. Base Modifying Agents Alkylating agents Methylmethane sulfonate Transitions, Mispairing, Crosslinking and Breakage 29

30. Mutagenic Effects of Radiation Nonionizing radiation - Ultraviolet light (UV) - 260 nm Absorbed by bases - pyrimidine hydrates, pyrimidine dimers Mispairing, Lethal if not repaired 30

31. Mutagenic Effects of Radiation Ionizing radiation - Xrays, Radioactive Isotopes, Neutrons (Radon gas, Radium) High energy - penetrates tissues, displaces electrons creates positively charged free radicals Base changes, breaks in backbone, crosslinking Results of exposure: Base substitutions, Deletions, Duplications, Inversions, Translocations, Chromosome breakage 31

32. Self-Induced Mutagenesis Radium - glows in the dark - watches, clothing mouth cancer, build-up in bones, anemia Nuclear energy - Chernobyl - 200x increase mutations in voles X-rays - physicians - bone cancer shoe stores - Tanning salons - UVA/UVB both dangerous Cigarette smoking - lung, pancreas, bladder, esophageal, etc. Radon gas - lung disease, cancer 32

33. Identifying Mutagens The Ames Test liver extract mimics metabolism reverse mutations induced 33

34. Detecting Mutations Visible - direct observation Nutritional - auxotrophs replica plating Resistance - selective media 34

35. Repair of DNA Damage Spontaneous damage to DNA ~ 1 change/ 109 bp/min 10,000 mutations per cell every 24 hr If not repaired, cells and individuals would die rapidly 35

36. Light Repair - Photoreactivation Direct repair of UV-induced pyrimidine dimers Photolyase (phr) - activated by visible light Error free repair - prokaryotes, simple eukaryotes 36

37. Repair of Alkylation Damage O6-methylguanine methyltransferase (ada) - E. coli removes methyl group restoring guanine Similar mechanism for repair of alkylated thymine 37

38. Base Excision Repair Glycosylase recognizes and removes damaged base by cleaving bond between base and sugar Other enzymes remove the sugar leaving gap in DNA DNA polymerase and DNA ligase repair gap 38

39. Nucleotide Excision Repair (NER) NER - Dark Repair - Repairs any damage that distorts DNA helix E. coli UvrA (uvrA), UvrB (uvrB), UvrC (uvrC), UvrD (uvrD) UvrA and B recognize damage UvrC and B cuts backbones on both sides of lesion UvrD unwinds and releases region between cuts DNA pol I and DNA ligase fill gap 39

40. Nucleotide Excision Repair (NER) E. coli 40

41. Nucleotide Excision Repair (NER) Mammalian systems - products of ~ 12 genes involved Deficiency in repair - Xeroderma pigmentosum light sensitivity 41

42. Methyl-Directed Mismatch Repair Recognizes mismatches in newly synthesized DNA E. Coli - mutS,mutL,mutH Exonuclease creates gap DNA pol III and ligase repair gap 42

43. Methyl-Directed Mismatch Repair Humans - hMSH2, hMLH1, hPMS1, hPMS2 involved Mutations in any of these genes - HNPCC Hereditary Nonpolyposis Colon Cancer Autosomal Dominant - Predisposition to cancer Heterozygous cell suffers mutation in good allele No repair capability remains Mutations begin to accumulate rapidly 43

44. Double-Strand Break Repair Homologous and Non-homologous recombination repair Defects - familial breast and ovarian cancer 44

45. Recombination Repair Postreplication - Recombination repair - recA DNA pol and ligase Nucleotide excision repair 45

46. Translesion DNA Repair - SOS Response E. coli - lexA, recA Too much damage for repair, RecA is activated induces LexA self-destruction no more repression of 17 genes for SOS repair DNA polymerase for translesion replication introduces errors into DNA 46

47. Transposable Elements Mechanism for Movement - insert into nonhomologous regions of chromosomes Transposase: move DNA elements Eukaryotes and Prokaryotes Reverse Transcriptase: RNA > DNA > RNA Eukaryotyes 47

48. Transposable Elements in Prokaryotes Phage mu - integration disrupts genes R plasmids - antibiotic resistance genes move accumulate on plasmids - Multiple Resistance ge *mu* ne ampRES tetRES 48

49. Transposable Elements in Prokaryotes Insertion Sequences (IS) - 768 bp (IS1) , 4-19 copies terminal inverted repeats (IRs), transposase transposition into genes inactivates them, alters expression 49

50. Insertion Sequences in Prokaryotes IS movement into a nonhomologous target site staggered cut direct repeats 50