Molecular Genetics

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

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

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

5. One gene one enzyme hypothesis. The first exciting insight into the function of genes Supplement ARG1 ARG2 ARG3 Mutant Ornithine Citruline Arginine Precursor --------ornithine----------citruline----------arginine + + + arg-1 arg-2 - + + arg-3 - - +

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

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

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

12. Hemoglobin-S sickle cell wild type

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

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

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

18. In bacteria - Introduce an isolated (cloned) gene by transformation In vitro 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.)

19. Because many drug-resistant mutations are recessive, cloning by complementation is often feasible Mutant #1 x Genomic DNA library GENE1 Each mutant is transformed with the library x GENE1 x GENE1 x GENE1 x Mutation in this geneis responsible for the drug resistance phenotype in mutant #1 GENE1 Complementation of the drug resistance phenotype x GENE1

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

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

22. 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

23. Genes contain much more information than the structural region (or "coding region forproteins") of the gene product. They include information directing the proper timingand placement of the expression of the gene product

24. Coding Regulatory

25. Coding Promoter

26. Transcriptional controls on initiation, elongation, termination of RNA 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.

28. 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 ribosomebinding site, or Shine - Dalgarno sequence - AGGAGGU (prok.)

29. Ribosome Binding site

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

32. Shine-Delgarno

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

34. Splicing site

36. Polyaden. site

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