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Luria delbruck 1943 experiment smc, Essays (university) of Biochemistry

The Genetic Material

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S. E. Luria and M. Delbruck (1943). Mutations of bacteria from vrius sensitivity to virus resistance.
Genetics 28:491
[Presented by: Steve Carr (scarr@mun.ca), 14 January 2014]
Background
Max Delbruck (1906 -1981) & Salvador Luria (1912 -1991)
Shared 1969 Nobel Prize in Physiology or Medicine
Bacteriology in 1940s not heavily influenced by genetic thinking
Bacteria have no nuclei: do they have "genes"?
Bacterial "phenotypes" are the manifestations of 106s of bacteria simultaneously
Bacteria don't have sex: crosses not possible
[Discovery of bacterial sex led to 1958 Nobel Prize]
bacteriophages ("phages") - "subcellular parasites that infect, multiply within, & kill bacteria."
T1 phages are active on E. coli
[phage] >> [bacteria] no bacterial colonies grow: bacteria are Tons ("T-one sensitive")
[phage] ~ [bacteria] some bacterial colonies grow: bacteria are Tonr ("T-one resistant")
Tonr phenotype is stable
all descendant bacteria are Tonr
phenotype persists in the absence of T1
Two Hypotheses (d'Herelle 1926 vs Brunet 1929)
1. Tonr phenotype is induced by exposure of bacteria to phage
Each bacterium has a (small, finite) chance of survival (~ 1 / 107);
Survivors have altered metabolic phenotype, which is transmitted to offspring
[distinction between phenotype & genotype not clear]
Bacteria adapt to their environment :
a Lamarckian hypothesis: inheritance of acquired characteristic
2. Tonr phenotype occurs spontaneously, prior to exposure of bacteria to phage
Some rare bacteria (say ~ 1 / 107) are already Tonr
These bacteria have undergone genetic mutation to a stable genotype
[phenotype persists in absence of phage]
a Darwinian hypothesis: Tonr bacteria are selected
Materials & Methods
Hypotheses make different predictions as to
numerical distribution of Tonr phenotypes among bacterial cultures.
Induction (Adaptation) Hypothesis predicts: n / N = a
where n = number of Tonr bacteria observed out of
N = number of Tons bacteria plated, and
a = probability of conversion from Tons to Tonr
Then, n should be a constant fraction of N
Mutation Hypothesis predicts: n / N = ga2g / 2g = ga
where a = mutation rate (# mutations / cell / generation)
g = # generations to go from 1 N bacteria, so that
N = 2g doublings occur, of which
n = ga2g produce mutant Tonr bacteria
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S. E. Luria and M. Delbruck (1943). Mutations of bacteria from vrius sensitivity to virus resistance.

Genetics 28:

[Presented by: Steve Carr (scarr@mun.ca), 14 January 2014]

Background

Max Delbruck (1906 -1981) & Salvador Luria (1912 -1991) Shared 1969 Nobel Prize in Physiology or Medicine

Bacteriology in 1940s not heavily influenced by genetic thinking Bacteria have no nuclei: do they have " genes "? Bacterial "phenotypes" are the manifestations of 10^6 s of bacteria simultaneously Bacteria don't have sex: crosses not possible [Discovery of bacterial sex led to 1958 Nobel Prize]

bacteriophages ("phages") - "subcellular parasites that infect, multiply within, & kill bacteria." T1 phages are active on E. coli

[phage] >> [bacteria] no bacterial colonies grow: bacteria are Ton s^ ("T-one sensitive")

[phage] ~ [bacteria] some bacterial colonies grow: bacteria are Ton r^ ("T-one resistant")

Ton r^ phenotype is stable all descendant bacteria are Ton r phenotype persists in the absence of T

Two Hypotheses (d'Herelle 1926 vs Brunet 1929)

  1. Ton r^ phenotype is induced by exposure of bacteria to phage Each bacterium has a (small, finite) chance of survival (~ 1 / 10^7 ); Survivors have altered metabolic phenotype , which is transmitted to offspring [distinction between phenotype & genotype not clear] Bacteria adapt to their environment : a Lamarckian hypothesis: inheritance of acquired characteristic
  2. Ton r^ phenotype occurs spontaneously , prior to exposure of bacteria to phage Some rare bacteria (say ~ 1 / 10^7 ) are already Ton r These bacteria have undergone genetic mutation to a stable genotype [ phenotype persists in absence of phage] a Darwinian hypothesis: Ton r^ bacteria are selected

Materials & Methods

Hypotheses make different predictions as to numerical distribution of Ton r^ phenotypes among bacterial cultures.

Induction (Adaptation) Hypothesis predicts: n / N = a

where n = number of Ton r^ bacteria observed out of N = number of Ton s^ bacteria plated, and a = probability of conversion from Ton s^ to Ton r Then, n should be a constant fraction of N

Mutation Hypothesis predicts: n / N = ga2g^ / 2g^ = ga

where a = mutation rate (# mutations / cell / generation) g = # generations to go from 1 N bacteria, so that N = 2 g^ doublings occur, of which n = ga2g^ produce mutant Ton r^ bacteria

[because a mutation in the i th generation contributes a

i

g-i

= a

g mutants] Then, n should increase wrt N , as g increases

How can differences in n be evaluated?

Suppose c cultures are started from a single Ton s^ mutant each after g generations there are N = 2 g^ bacteria in each culture

Statistical foundations of Luria - Delbruck experiment

Thought experiment:

Consider four cultures each started from a single bacterium after g = 4 generations, expect 16 cells from 15 divisions @, total 64 cells from 60 divisions plate each culture separately w/ T1, count total # Ton r Suppose 10 Ton r^ colonies observed: what distribution (" fluctuation ") expected?

Induction Hypothesis:

Ton r^ induction occurred only in fourth generation upon exposure to T probability of induction (a) is uniform / bacterium a = 10 inductions / 64 cells = 15% observe = 3, 1, 5, & 1 Ton r^ colonies mean = 10 / 4 = 2.5 Ton r^ per culture variance = 2. Follows a Poisson Distribution: variance = mean

Mutation Hypothesis

Ton r^ mutation has occurred spontaneously, prior to exposure to T mutation rate (a) = 2 events / 60 cell divisions = 0.033 mutations / cell / generation mean = (2 + 0 + 8 + 0) / 4 = 2.5 Ton r^ as before After 4 generation, earlier Ton r^ mutations leave more offspring (as in Culture 3) variance = 10. after 5 generations, when the number of Ton r^ cells has doubled in each culture: variance = 48.

Mutation Hypothesis predicts variance >> mean, as g increases

Experimental procedure:

"The first experiment was done on the following Sunday morning. (In a letter dated January 21 [1943], Delbruck exhorted me to go to church"

Twenty x 200 ul "individual cultures" One x 10 ml "bulk culture" Inoculate with ~ 10^3 bacteria @ Grow for g = 17 generations ~10^8 bacteria / ml Plate entire "individual cultures" & 200 ul aliquots of "bulk culture" on petri dish w/ T