1. Mutation

2. Means change Definition: An event that gives rise to an alteration in the genotype It also can be the change itself Mutation does not mean “bad” Mutations are IMPORTANT for a variety of reasons

3. Mutation ‘Levels’ DNA Level Chromosomal Level Genome Level

4. DNA Level At or below the gene level 1 ) Addition or Deletion of one or two nucleotides (+ and – frameshifts) 2) Substitution of one nucleotide for another 3) Inversion within a gene 4) Crossingover within a gene – Equal – Unequal 1 and 2 are often referred to as POINT MUTATIONS

5. Chromosomal Level 1) Duplications 2) Deletions 3) Translocations – Reciprocal – Non-reciprocal 4) Inversions ? ‘Standard’ crossing over ? 3 and 4 may result in POSITION EFFECTS

6. Genome Level These result in ploidy changes Aneuploidy Plus or minus one or a few chromosomes Euploidy (true polyploidy) Involves whole SETS of chromosomes – Autopolyploidy – Allopolyploidy

7. Additional Points ALL genes can mutate Observed levels are usually low Some genes have very high rates of mutation (Mutable Genes) Some genes seem to increase the rate of mutation in adjacent genes (Mutator Genes)

8. What Causes Mutations ? 1) Spontaneous (?????) 2) Chemically induced 3) Radiation induced Most (if not all) really come down to a chemical level No such thing as a “spontaneous” mutation “Spontaneous” means…

9. Chemicals Many chemicals are mutagens; many others are promutagens Chemicals (just like radiation) can cause point mutations. Chemicals (just like radiation) can cause chromosome breakage (e.g. - LSD, Mercury, Caffeine --- at least in cell culture)

10. DNA Level Mutations + and – Frameshifts and their effects + or – one nucleotide + or – a whole codon Multiple frameshifts The effects depend on WHERE it occurs The effects depend on what the change is ?????? One change equals one change, or does it ??????

11. DNA Level Mutations Substitutions Transitions (more common) Purine (A, G) for a purine OR pyrimidine (T, C) for a pyrimidine Transversions (less common) Purine for a pyrimidine (or pyrimidine for a purine GENERALLY have less chance of an effect than frameshifts. WHY?

12. Other DNA Level Mutations Inversions within a gene Crossingover within a gene

13. Substitution Mutations Results from a temporary type of isomerization (tautomerization) during replication Base enters a “rare” state (unstable) Can be in a base already in the DNA strand Can be in one of the incoming bases We will look at both for a TRANSITION Requires two divisions to get the “stable” mutation

14. Substitution Mutations One nucleotide is replaced by another Transition --- insertion of a one purine base for the other or one pyrimidine base for the other (results in an “odd” purine-pyrimidine pair) Transversion --- replacement of a purine base with a pyrimidine or vice versa (results in a temporary purine-purine pair or pyrimidine- pyrimidine pair)

17. Substitution Mutations Base analogs increase the rate of formation of substitution mutations 5-bromouracil (5BU) Use of 5-fluorouracil

18. Radiation Induced Mutations By non-ionizing radiation (UV) Dimer formation (especially T  T dimers) Light Repair System (enzyme breaks dimers)

19. Radiation Induced Mutations By non-ionizing radiation (UV) Dimer formation (especially T  T dimers) Dark Repair System (excision repair) – Endonuclease, exonuclease, DNA polymerase, ligase

20. Radiation Induced Mutations By ionizing radiation Mechanisms - “Target Hypothesis” - “Physical-Chemical” Hypothesis Temperature, hydration, and oxygen content all have an effect

21. Mechanism

23. Ionizing Radiation and Mutation Late 1920s Stadler – working on barley and corn Muller – working on Drosophila Muller later received a Nobel Prize (1948) Why H. Muller and not Stadler?

24. SOME GENERALIZATIONS FOR IONIZING RADIATION Mutation frequency is DIRECTLY proportional to the dose Doesn’t matter if given in one large dose or several small ones No lower limit (no threshold) Safe amount?????

27. Additions and Deletions Chemical basis Caused by intercalation (insertion) of some nucleotide like chemical into the chain Before replication --- increases length Results in a Plus (+) frameshift mutation During replication --- decreases length Results in a Minus (-) frameshift mutation Acridines are capable of doing this

28. Additions and Deletions Requires 2 divisions to get the “stable” mutation Proflavin Acridine Orange (very potent)

29. Additions and Deletions

30. - frameshift Intercalation of Proflavin DURING DNA replication

31. + frameshift Intercalation of Proflavin PRIOR TO DNA replication

32. Chemicals Besides Acridines Too many to mention…but… Mustard gas (adds a methyl group) Formaldehyde Phenol Nitrous Oxide (replaces NH 2 with O)

33. Mutation Concluded Temperature has an effect Carcinogens and Mutagens Somatic vs. germinal mutations Somatic are “dead-end” in animals In plants somatic can be “passed on” asexually by vegetative propagation (runners, bulbs, corms, cuttings, grafting) In plants somatic mutations can get into the germ line!

34. Testing for Mutagens & Promutagens Ames test Uses a histidine deficient (dependent) strain of Salmonella Looks for reverse mutation to histidine independence above the background rate For suspected promutagens: first treat substance with a liver (human or rat) extract and then do the test

35. Teratogenesis Teratogen – an agent that induces a non- inheritable defect during embryonic development (Proteratogen) In humans --- about 4% of births How do they work? – Interfere with mitosis or cell migration – Interfere with differentiation (disturb gene regulation, translation or activity of ultimate gene products)

36. Teratogens Radiation Viruses (Rubella and pregnant women) Thalidomide (really a proteratogen) Diethylstilbestrol (DES) (taken by pregnant women - results in genital tract abnormalities in their children) Dioxins (found as a contaminant in herbicides such as Agent Orange) Hexachlorophene (a bacteriocide)

37. Teratogens Smoking (CO reduces O 2 available to the fetal circulation; nicotine constricts blood vessels and reduces blood flow) Progestin (artificial progesterone-like compound; causes masculinization of female fetuses) Alcohol – microcephaly, low birth weight, some mental retardation) Caffeine, Aspirin and numerous legal and not so legal drugs

38. Some Other Birth Defects Cleft lip and/or cleft palate Spina bifida Club foot Heart disease and malformations Cataracts Deafness Intestinal tract disorders Low birth weight