erwin schrodinger what is life? 1944 nobel prize in physics 1933 atomic theory
TRANSCRIPT
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Erwin Schrodinger
What is Life?
1944
Nobel Prize in Physics 1933Atomic Theory
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Max Delbruck
Nobel Prize in Medicine 1969
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rII……rapid lysis…. makes large plaques
Seymour Benzer
rII
WT
K B
- +
+ -
E.coliT4
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103 106 109
Plate 0.1 ml and count # plaques
Example-plate has 50 plaques, therefore 500 phage per ml at 109
or total = 5x 1011 per ml
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Back to Benzer
rII
WT
K B
- +
+ -
E.coliT4
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 onStrain K
Get very rare plaques that result from recombination
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rII
rII-1
rII-2
Internal deletion
Does not grow on K
Does not grow on K
Does not grow on K
Grows on K
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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
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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
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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?
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GatewayTM TechnologyRecombineering 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
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Lysogeny-Lysis
Lysogen
attP
attB
attL attR
Int
Xis
Lysis
Lysis
Stress
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Step 1…generate an “entry clone” with YFG
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Step 2 recombine YFG into a destination vector
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Destination vectors for every use
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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
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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?
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Modal number of chromosomes= 82
Range = 70 to 164. 100% aneuploidy in 1385 cells examined.
HeLa cells karyotype from ATCC
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Recall yeast one-step gene replacements
YFG1
URA3
URA3
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in vitro approachUse TAP purifications tomake protein chips
GST tagged protein kinases
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Integrating KinaseExpression Arrayand TAP data