3. Erwin Schrodinger What is Life? 1944 Nobel Prize in Physics 1933 Atomic Theory

4. Max Delbruck Nobel Prize in Medicine 1969

9. rII……rapid lysis…. makes large plaques Seymour Benzer rII WT K B - + + - E.coli T4

10. 10 3 10 6 10 9 Plate 0.1 ml and count # plaques Example-plate has 50 plaques, therefore 500 phage per ml at 10 9 or total = 5x 10 11 per ml

11. Back to Benzer rII WT K B - + + - E.coli T4 Plate concentrated wild type on strain B to select rII mutants Grow rII mutants to high density (strain B) Plate onto strain K to select rare revertants Some rII alleles revert (low frequency) others never revert Two different non-revertable alleles and do mixed infection on Strain K Get very rare plaques that result from recombination

12. rII rII-1 rII-2 Internal deletion Does not grow on K Grows on K

13. In the early days of phage genetics….. Two types of mutants… 1.Altered plaque size and shape 2.Host range…..grow on certain strains of E. coli Ultimately the goal became to identify every gene in the genome Filling in the map with conditional mutants Temperature sensitive mutants Nonsense mutants

14. From the very beginning of Molecular Biology and Genetics The goal has been to have a complete understanding of the genome This means assigning a function to every gene in the genome genotype phenotype DNA Function

15. Assigning functions to a gene When is it expressed? Where is it expressed? Is the protein modified? Protein-Protein Interactions? Phenotype when protein is reduced? Phenotype when the protein is overexpressed?

16. Gateway TM Technology Recombineering in vitro A diversion to phage lambda Life style choice… Lysis vs lysogeny Lysis-plaques Lysogeny-phage infects the cell but is dormant …The cell survives until there is some stress (uv light) …Lysis Lysogens (bacteria with dormant phage) are phage resistant

17. Lysogeny-Lysis Lysogen attP attB attL attR Int Xis Lysis Stress

19. Step 1…generate an “entry clone” with YFG

20. Step 2 recombine YFG into a destination vector

21. Destination vectors for every use

22. Assigning functions to a gene When is it expressed? Microarray experiments Where is it expressed? Epitope tagged protein Is the protein modified? Gel shifts and mass spectrometry Protein-Protein Interactions? GST or other affinity purifications Phenotype when protein is reduced? siRNA Phenotype when the protein is overexpressed? Strong promoter But problems remain for tissue culture cells

23. Transfection (not transformation) Stable (hard) or transient (easy)? Transient: Fraction of transfected cells is variable Expression levels differs in individual cells What cell types do you choose? What is the isogenic wild type control?

24. Modal number of chromosomes= 82 Range = 70 to 164. 100% aneuploidy in 1385 cells examined. HeLa cells karyotype from ATCC

25. Reducing expression by shRNA Libraries

26. Recall yeast one-step gene replacements YFG1 URA3

36. in vitro approach Use TAP purifications to make protein chips GST tagged protein kinases

46. Integrating Kinase Expression Array and TAP data