What is Life? Erwin Schrodinger 1944 Nobel Prize in Physics 1933 Atomic Theory
Max Delbruck Nobel Prize in Medicine 1969
Seymour Benzer rII……rapid lysis…. makes large plaques E.coli T4 K B + - WT - + rII
Plate 0.1 ml and count # plaques 103 109 106 Example-plate has 50 plaques, therefore 500 phage per ml at 109 or total = 5x 1011 per ml
E.coli Back to Benzer T4 K B + - WT - + rII 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
rII Does not grow on K rII-1 rII-2 Does not grow on K Does not grow on K Grows on K Internal deletion
In the early days of phage genetics….. • Two types of mutants… • Altered plaque size and shape • 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
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
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?
GatewayTM 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
Lysogeny-Lysis Lysis attP attB Int attR attL Lysogen Stress Xis Lysis
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
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?
HeLa cells karyotype from ATCC Modal number of chromosomes= 82 Range = 70 to 164. 100% aneuploidy in 1385 cells examined.
Reducing expression by shRNA Libraries
URA3 URA3 Recall yeast one-step gene replacements YFG1
in vitro approach Use TAP purifications to make protein chips GST tagged protein kinases
Integrating Kinase Expression Array and TAP data