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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. - +.

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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


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