CsCl centrifugation of DNA over time developed by Meselson and Stahl

4. CsCl centrifugation of DNA over time developed by Meselson and Stahl

6. In class question (extra credit) for Quiz #4 Question 1: (3pts) Why does one add EtBr to CsCl gradients for the isolation of plasmid DNA? Question 2: (4pts) Is an 8kb supercoiled plasmid more dense than a 3kb supercoiled plasmid. Yes/No (circle one) Will an 8kb supercoiled plasmid have more EtBr bound to it? Yes/No (circle one)

7. We will talk about this again in a later lecture: But CsCl gradients are not the same thing as Sucrose Gradients or Agarose Gel Electrophoresis.

9. CsCl centrifugation of DNA over time

10. N15is heavier than N14-Can be resolved in CsCl

11. pulse-chase Experiment: Incubator with N15containing medium for time, then chase with N14medium Expt 1 grows Slowly Expt 2 Bacteria Grow Faster Why?

12. N14 N15 N14 N15 only only Why would they do 2 different growth rates? Experiment 1 Experiment 2

13. N14 N15 only Fuse Results from Expt 1 and 2 Cell Divisions

14. N14 N15 only Experiment 1 observations Watson-Crick Model Does Expt 1 prove hybrid formation?

15. What about N14/N15hybrid? N15 dsDNA N15 ssDNA Critical Experiment: Hybrid Strand Separation And CsCl centrifugation Looks like control below N15 ssDNA N14 ssDNA

17. Evolution?

19. The Molecular Basis of Mutation-Evolution Mutations alter the nucleotide sequences of genes in several ways, for example the substitution of one base pair for another or the deletion or addition or one or a few base pairs.

20. Tautomeric Shifts

21. Tautomeric Shifts AffectBase-Pairing

22. Mutation Caused by Tautomeric Shifts

23. Base Substitutions • A transition replaces a pyrimidine with another pyrimidine or a purine for another purine. • A transversionreplaces a pyrimidine with a purine or a purine with a pyrimidine.

24. Previously discussed Frameshift Mutations

25. Factors Influencing the Rate of Spontaneous Mutations • Accuracy of the DNA replication machinery • Efficiency of the mechanisms for the repair of damaged DNA • Degree of exposure to mutagenic agents in the environment

26. Induced Mutations • Induced mutations occur upon exposure to physical or chemical mutagens. • Hermann J. Muller and Edgar Alternburg measured the frequency of X-linked recessive lethal mutations in Drosophila. • Muller demonstrated that exposing Drosophila sperm to X-rays increased the mutation frequency.

27. Chemical Mutagens

28. Types of Chemical Mutagens • Chemicals that are mutagenic to both replicating and nonreplicating DNA (e.g., alkylating agents and nitrous acid) • Chemicals that are mutagenic only to replicating DNA (e.g., base analogs and acridine dyes)

29. A Base Analog:5-Bromouracil…it is more like Thymine!

30. Mutagenic Effects of 5-Bromouracil

31. Nitrous Acid Causes Oxidative Deamination of Bases

32. Intercalation of an Acridine Dye Causes Frameshift Mutations

33. Alkylating Agents • Alkylating agents are chemicals that donate alkyl groups to other molecules. • Alkylating agents induce transitions, transversions, frameshifts, and chromosome aberrations. • Alkylating of bases can change base-pairing properties. • Alkylating agents can also activate error-prone DNA repair processes.

34. Hydroxylamine • Hydroxylamine is a hydroxylating agent. • Hydroxylamine hydroxylates the amino group of cytosine and leads to G:C A:T transitions.

35. The Electromagnetic Spectrum • X-rays induce mutations through ionization. • Ultraviolet light induces mutations through excitation- Energy addition.

36. Irradiation Dosage and Mutation Frequency

37. Ionizing Radiation Causes Changes in Chromosome Structure • Ionizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations. • These types of mutations display two-hit kinetics.

38. Thymine Dimers Mutagenesis by Ultraviolet Irradiation • Hydrolysis of cytosine to a hydrate may cause mispairing during replication • Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate error-prone DNA repair mechanisms.

39. Mutations Induced by Transposons Wrinkled Pea

40. Previously discussed Expansion of Trinucleotide Repeats • Simple tandem repeats are repeated sequence of one to six nucleotide pairs. • Trinucleotide repeats can increase in copy number and cause inherited diseases. • Examples: Fragile X Syndrome, Huntington disease, spinocerebellar ataxia • These diseases are characterized by anticipation, the increased severity of disease or earlier age of onset in successive generations as the trinucleotide copy number increases.

41. Mutations are induced by chemicals, ionizing irradiation, ultraviolet light, and endogenous transposable genetic elements. • Point mutations are of three types: • Transitions—purine for purine and pyrimidine for pyrimidine substitutions, • Transversions—purine for pyrimidine and pyrimidine for purine substitutions, and • Frameshift mutations—additions or deletions of one or two nucleotide pairs, which alter the reading frame of the gene distal to the site of the mutation. You must know for your future

42. Evolution?

46. Alpha and Beta chain mutants…some of them

47. Phylogenetic relationships

50. How could we use GFP fluorescence to figure out-codon optimize GFP?