1. Molecular Genetics First step: DNA is the genetic material – DNA, NOT protein of the chromosomes/chromatin –PROVEN WITH TRANSFORMATION AND TRANSDUCTION, THAT WE JUST MASTERED 1

2. First, conceptual basis of the connection between mutation (Gene, or DNA change), and phenotype Mutation 2

3. One gene one enzyme hypothesis. The first exciting insight into the function of genes

4. 4 MutantOrnithineCitrulineArginine arg-1 arg-2 arg-3 + + + - + + - - + Supplement Precursor --------ornithine----------citruline----------arginine ARG1 ARG2ARG3 One gene one enzyme hypothesis. The first exciting insight into the function of genes Precursor XYZ Gene1Gene2Gene3 xxx

5. NOT like above, where the DNA must recombine and replace the endogenous copy, BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.) In vitro In bacteria - Introduce an isolated (cloned) gene by transformation 5

6. Because many drug-resistant mutations are recessive, cloning by complementation is often feasible x Genomic DNA library Each mutant is transformed with the library x x x x x Mutation in this gene is responsible for the drug resistance phenotype in mutant #1 Mutant #1 Complementation of the drug resistance phenotype 6

7. Plate to select for phenotype of interest Complementation groups

8. 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

9. 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

10. 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

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13. One gene – one gene product (here a protein) gtg cat ctg act cct gag gag From gene to gene product function, from mutation to phenotype Peptide bond 13

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17. A single b.p. change in the hemoglobin gene leads to all the phenotypes of Sickle Cell Anemia 17

18. gtg cat ctg act cct gag gag gtg cat ctg act cct g t g gag MUTANT –Sickle cell anemia gtg cat ctg act cct g t g gag From gene to gene product function, from mutation to phenotype 18

19. wild type Hemoglobin-S sickle cell 19

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22. From gene to gene product function, from mutation to phenotype 22

23. gtg cat ctg act cct gag gag gtg cat ctg act cct gug gag MUTANT –Sickle cell anemia אנמיה חרמשית gtg cat ctg act cct gug gag gene ->gene product function, mutation to phenotype, In bacterial enzyme,... or in gene for complex Human syndrome 23

24. 24 Xeroderma pigmentosum

25. המופיליה B חוסר ב- Factor IX Simple (even spontaneous) changes, huge consequences – how to find them? 25

26. Factor IX (HemoB) Gene for Factor IX (HemoB) 26

27. Phenotypic Rescue: Introduce an isolated (cloned) gene by viral (engineered) Infection with Hemo.B gene (encodes clotting Factor IX). 27

28. Evolution from “1 gene, 1 protein” to… 1 gene, 1 protein –Haemoglobin 1 gene, 1 polypeptide 1 gene, 1 gene product –tRNAs, rRNAs, snRNAs, miRNAs, … Additionally – next: (e.g. for proteins)- many sites are susceptible to mutations outside of coding region 28

29. Genes contain much more information than the structural region (or "coding region for proteins") of the gene product. They include information directing the proper timing and placement of the expression of the gene product 29

30. Regulatory Coding 30

31. Coding Promoter 31

32. Example: Prokaryotes and Eukaryotes have highly conserved necessary promoter elements specific distances from transcription start, enhancers which act from varying distances. Specifically: many eukaryotic genes have a "TATA box" 30 bases from the start site and a CCAAT box 70-80 base pairs from the start site in their promoter. Transcriptional controls on initiation, elongation, termination of RNA 32

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34. Translational controls on initiation, elongation, termination, localization Example: Here too, a large degree of control is exercised at initiation, and the "non-translated leader" portion of RNA includes control information. Starts are always at Met (AUG), but specifically, in Prokaryotes: true start sites are preceded by a consensus sequence know as a ribosome binding site, or Shine - Dalgarno sequence - AGGAGGU (prok.) 34

35. Ribosome Binding site 35

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37. Animation ch-9 transl…nSteps TRANSLATION/Shine-Dalgarno 37

38. Shine-Delgarno 38

39. Example: Eukaryotic RNA's are spliced - one of several post transcriptional alterations Post transcriptional modifications of the RNAs 39

40. Splicing site 40

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42. Polyaden. site 42

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45. Post-translational modifications, processing, packaging and trafficking of proteins 45

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47. 47 Darwin’s Finches