1. Mutation The process that produces a gene or a chromosome set that is differing than that of the wild type. The gene or a chromosome set that results from such a process.

8. Pairing between the normal (keto) forms of the bases Mismatched bases. Rare tautomeric forms of bases result in mismatches

9. Transition

11. MUTAGENS Radiation Chemical Agents Mobile Genetic Elements

13. Mutagens Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

14. 2-AP: 2- aminopurine Analog of adenine that can pair with cytosine in its protonated state Normal pairing 14

15. 2-AP: 2- aminopurine Analog of adenine that can pair with cytosine in its protonated state Normal pairing

16. 5-BU :5 bromouracil An analogue of thymine 5-BU can be mistakenly incorporated into DNA as a base. The ionized form base pairs with guanine Normal pairing

17. Transition

18. Rare form of BrU in Template

19. Mutagens מוטגנים Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

20. Alkylation-induced specific mispairing. Treatment with EMS alters the structure of guanine and thymine and leads to mispairings Transition

21. Alkelating agents

22. A powerful carcinogen originally isolated from peanuts infected with fungus. Alfatoxin attaches to guanine at the N-7 position. This leads to the breakage of the bond between the base and the sugar, thereby liberating the base and resulting in an apurinic site. Agents that cause depurination at guanine residues should tend to induce GC to TA transversions

23. Usually an A is inserted instead of the depurinated site

24. 2-AP EMS NG

25. Mutation rate, a question of balance

26. MutationsDNA repair

27. Mutagens Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

28. Flat planar molecules that mimic base pairs and are able to slip themselves in (intercalate) between the stacked nitrogen bases at the core of the DNA double helix. In this intercalated position, an agent can cause single-nucleotide-pair insertions or deletions Intercalators

29. Mutagens Radiation Chemical Agents Mobile Genetic Elements Ultraviolet (UV) Ionizing

31. UV light generates photoproducts Occurs between two adjacent pyrimidines on the same DNA strand The UV photoproducts significantly perturb the local structure of the double helix. These lesions interfere with normal base pairing. The C to T transition is the most frequent mutation, but UV light also induces other base substitutions (transversions) and frameshifts, as well as larger duplications and deletions. Pyrimidine: T, C

32. Transition Transversion

33. Mutagenes induce mutations by a variety of mechanisms. Some mutagenes mimic normal bases and are incorporated into DNA, where they can mispair. Others damage bases, which then are not correctly recognized by DNA polymerase during replication, resulting in mispairing MESSAGE

34. The Ames test A method that uses bacteria to test whether a given chemical can cause cancer. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound Bruce Ames 1928-today University of California, Berkely

35. TA100- sensitive for reversion by base pair substitution TA 1535/8 frameshift Ames test Strains inactive for BER and prone for Entry of molecules

38. Plate to select for phenotype of interest Complementation groups

39. First, we need to catalogue our mutants to complementation groups (Total of 138 mutants were isolated in the original CTF screen). xx Mate x x Diploid still shows CTF phenotype Mutant#1Mutant#2 Mutant#1 and Mutant#2 are mutated in the same gene Same complementation group Diploid

40. xx Mate Mutant#3Mutant#4 Diploid x x Diploid dont show CTF phenotype Mutant#3 and Mutant#4 are mutated in different genes Different complementation groups Complementation groups

41. Chromosome Transmission Fidelity (ctf) Mutants Total # of mutant isolates:138 19 Complementation Groups101 37 Undesignated (single member)37 Estimated total # of genes represented ~ 50 ctf genes Complementation groups

43. WOBBLE A situation in which the third nucleotide of an anticodon (at the 5’ end) can form two alignments. This third nucleotide can form hydrogen bonds not only with its normal complementary nucleotide in the third position but also with different nucleotide in the position.

45. I stands for inosine, one of the rare bases found in tRNA, often in anticodon

47. MESSAGE The genetic code is said to be degenerate because in many cases more then one codon is assigned to a single amino acid, and, in addition, several codons can pair with more then on anticodon (wobble)

48. Synthetic lethal- A screening method used to uncover mutations in a second gene that will require the cell to maintain a wild-type copy of the gene being studied in order to survive. 1st mutation + 2nd mutation = lethality This screen is commonly used in yeast genetics, but can be used in other model organisms as well.

49. Synthetic Lethality yfg1 yfg2 Dead Viable yfg1 yfg2

50. Synthetic Lethality A B aa B X A bb ViableLethal aa bb Wild-type Viable X X X Functional Relationships Essential biological function SL interaction Pathway B A2 A3 B1 B2 B3 Pathway A A1 A2 A3 B1B2B3 Complex AComplex B

51. B Mating Heterozygous diploid Tetrad BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Dissection Meiosis a b A B a b A B a b A B a b A Is this combination lethal?

52. Yeast tetrad analysis (classic method) tetrad Step1: separate spores by micromanipulation with a glass needle Step2: place the four spores from each tetrad in a row on an agar plate Step3: let the spores grow into colonies

53. Classical approach (tetrad dissection) BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Tetrad Dissection bni1∆ bnr1∆

54. a yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX Query gene Mating 4700 heterozygous diploids We ask which mutations are synthetically lethal with our query mutant The SGA approach allows to do it in a systematic way We ask which mutations are synthetically lethal with our query mutant The SGA approach allows to do it in a systematic way YFG1 AAAAA yfg1::KmX A YFG1 a

56. Meiosis Tetrads yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX a Selection for double mutants a A a A a A a A a YFG A A a yfg YFG yfg YFG aaaa

57. Select for haploid double mutants and score viability sts1 a yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX a aaaa

58. SGA Screening Lab (University of Toronto)

59. Pairwise genetic interactions can be represented by a graph 8 SGA Screens: 291 Interactions 204 Genes 8 SGA Screens: 291 Interactions 204 Genes

60. Genetic Interaction Network 132 Screens 4000 Interactions 1000 Genes ~200,000 Interactions/genome Amy Tong, Fritz Roth et al., Science 303:808-813 (2004)

61. ~2000 Quantitative SGA Screens

62. Yeast Genetic Interaction Network Global Level DNA replication

63. Yeast Genetic Interaction Network Global Level Vesicle- mediated transport DNA replication

64. Ribosome, Translation Mitochondria Vesicle- mediated transport Glycosylation & cell wall Polarity & cell morphogenesis DNA replication and repair Chromosome segregation and mitosis Nuclear-cytoplasmic transport Chromatin & transcription Peroxisomes Amino acid biosynthesis RNA processing Nuclear migration Protein Degradation Yeast Genetic Interaction Network Global Level

65. Can we recapitulate synthetic lethality in mammalian cells?

66. yfg1 yfg2 Dead yfg2 yfg1 yfg2 Normal Tumor yfg1 yfg2 SL interactions identified in yeast could be investigated as a candidate for novel therapeutic target

67. Potential therapeutic targets