1. The origin of mutations Lamarck Darwin www.princessleia.com/Lamarck.php freethinkersasylum.com/2010/03/freethinkers-book-club-darwins-sacred-cause/ Beneficial mutations are somehow induced by the environment in which they are useful Mutations are ‘directed’ Mutations occur spontaneously independent of their effects Mutations are random Until 1943 there were two hypotheses concerning the origin of mutations:

2. The basis of mutational change: How do mutations arise? Luria and Delbruck came up with an ingenious experiment to test whether mutations arose: i) randomly – mutants pre-existed in a population before exposure to a selective environment (e.g., an antibiotic) ii) by direction/adaption – induced in the population by exposure to a selective environment Genetics 1943 Distinguishing between these two possibilities is difficult – requires knowing if an individual has a mutation before it is exposed to a selection pressure – once an individual is exposed to selection it is unclear whether or not the selection pressure caused the mutation (directed) or the mutation was already present in the individual (random) E.g., think about how you would determine if a bacteria was antibiotic resistant without exposing it to the antibiotic

3. The basis of mutational change: How do mutations arise? Luria and Delbruck started populations of bacteria from a small number of cells… … and allowed them to grow up to reach a large number of cells (~10 billion) then determined the number of bacteriophage resistant cells: resistant cells live and form colonies, sensitive cells die but – cells can only be identified as being resistant (having a mutation) after exposure to the selective environment – how does this experiment solve our problem? Each level represents a round of cell division bacteriophage on the plate kill sensitive cells

4. The basis of mutational change: How do mutations arise? If mutations are induced in response to selection they will occur only in the final generation of cells – the ones that are exposed to phage If mutations occur randomly they will occur throughout population growth – i.e., prior to selection grow up population without selection (e.g. no bacteriophage) expose the population to selection and count the number of mutant individuals (colonies) Directed/Adaption hypothesis

5. The basis of mutational change: How do mutations arise: the fluctuation test If mutations are directed in response to selection, each cell has an independent probability of becoming resistant – predicts a Poisson distribution of mutants across populations (low variance; variance = mean) If mutations are random cells have different probabilities of being resistant (it depends on their parent) – if mutations happen early, a large number of mutants are present (high variance; variance >> mean) A ‘jackpot’ – a population happened to get a mutation early in its growth. Because descendants of this early mutant inherit the mutation a large number of mutants will be present Directed mutation/ Fig. 12.1

6. The basis of mutational change: How do mutations arise? Luria & Delbruck, Genetics 1943 A ‘jackpot’ – a population happened to get a mutation early in its growth. Because descendants of this early mutant inherit the mutation, a large number of mutants are present The random mutation hypothesis predicts the variance should be much higher than the mean – the directed mutation hypothesis predicts the variance and mean should be similar

7. The basis of mutational change: How do mutations arise? Luria & Delbruck, Genetics 1943 Do you buy it? Do (all?) mutations occur randomly and irrespective of their usefulness? Recap of the experiment: Hypothesis: If mutations are directed, then we expect them to be Poisson distributed across replicates; If mutations arise randomly, then we expect them to be distributed with high variance across replicates Prediction: As for hypothesis but specific to phage resistance tested in this experiment (any reason that what is true for phage may not be true for mutations generally?) Test: Determine distribution of phage resistance across replicate populations Interpretation: Use a statistical model to determine if observed distribution is Poisson or has higher variance (more specifically, they falsified the completely directed mutation model, but does this necessarily mean that the random mutation model is true?)

8. For the next 45 years it was largely accepted that the probability that a mutation occurs is not influenced by whether or not it will be useful, then…

9. Summary of Luria- Delbruck (L-D) finding Pointing out that concluding from L- D and co. that ALL mutations are spontaneous is an example of the logical fallacy affirming the consequent Even worse… knowing what we now know, the L-D experiment couldn’t have detected a signal of directed mutation Cairns et al. 1988 [Luria-Delbruck (L-D) and Lederberg and Lederberg]

10. Can you put these plots into words? How does the distribution of mutations over independent populations differ under the directed and random mutation models? Random mutation Directed mutation probability of having X or more mutants number of mutants X 1 1 Mean number of mutation events per culture Mean number of mutation events on the plate

11. Just what distribution of mutants should you expect if both directed and random mutations are present? Random mutation Directed mutation probability of having X or more mutants number of mutants X 0.5 1 2 4 8 16 1 2 5 10 20 30 Mean number of mutation events per culture Mean number of mutation events on the plate

12. Just what distribution of mutants should you expect if both directed and random mutations are present? Random mutation Directed mutation Directed and random mutation probability of having X or more mutants number of mutants X Mean number of mutation events per culture 0.5 1 2 4 8 16 Mean number of mutation events on the plate 1 2 5 10 20 30 1 2 5 10 20 30 0 1 random mutation + X directed mutations What do you take away from this plot?

13. An experiment that aims to identify random and directed mutations should use a “marker” that is expressed immediately after a mutational change  e.g., resumption of the ability to grow on the sugar lactose (lac- to lac+) Experimental design lac- lac+ (independent 1 bp deletion mutations restoring the proper reading frame are shown) Addition of a ‘C’ to lacZ creates a frameshift mutation that reveals an ochre stop codon (taa) and prevents functional LacZ being formed – without LacZ, a cell can’t grow on lactose start codon selection for growth on lactose

14. Preliminary observation lac+ mutant distribution influenced by random mutations a mutator (uvrB-) strain appears to have an increased contribution of induced mutations (a higher μ)

15. Observation: (i) distribution of lac+ mutants on plates suggests influence of random and directed mutations (ii) lac- cells plated on minimal lactose medium continue to produce lac+ colonies at a constant rate – suggests that mutations occur during selection on plates (directed?) Experimental design add lac- cells and lactose immediately Mutant colonies rise at an approx. constant rate over time Is this consistent with the new mutations being directed (as suggested by Cairns et al.)? Is there any other explanation? If so, what? How could you test for the influence of directed mutations (in addition to random mutations) in this experiment? time number of lac+ mutants ~20 colonies on day 2 and ~8 cells per day thereafter count lac+ mutant colonies arising over time (only lac+ can form colonies on minimal lac medium)

16. Experimental design add lactose immediately wait 3 days before adding lactose Count colonies arising after addition of lactose (nothing to grow on before it is added) What would you predict if mutants arose before lactose addition (random)? What would you predict if mutants only arose after lactose addition (directed)? add lac- cells immediately and: Observation: (i) distribution of lac+ mutants on plates suggests influence of random and directed mutations (ii) lac- cells plated on minimal lactose medium continue to produce lac+ colonies at a constant rate – suggests that mutations occur during selection on plates (directed?)

17. Cairns et al. 1988 Predictions: If lac+ mutants are being induced by lactose – so only happen in its presence, then mutants will not be formed prior to lactose being added If lac+ mutants occur randomly on the plate – independent of the presence of lactose, then mutants will be formed prior to lactose being added time number of lac+ mutants 0246810 lac- cells added at t=0 lactose added experiment 1 lactose added experiment 2

18. Cairns et al. 1988

19. Bjedov et al. Read by next Tuesday (March 3) and come to class with a list of points you did not understand. You’ll discuss and attempt to resolve each others concerns in a small group. The aim is for us to discuss the paper on Thursday (March 5) and evaluate experiments and interpretations. To do this, you need to have a working knowledge of the experimental design and analysis. E.g.

20. Bjedov et al. Read by next Tuesday (March 3) and come to class with a list of points you did not understand. You’ll discuss and attempt to resolve each others concerns in a small group. The aim is for us to discuss the paper on Thursday (March 5) and evaluate experiments and interpretations. To do this, you need to have a working knowledge of the experimental design and analysis. E.g. In the third column, what’s the meaning of values above vs. below 1? Why choose the genes listed in the first column? What was done with them?

21. An independent research project Each group (3-4 people, unless something else makes more sense in a particular instance) should meet to come up with some research topics of interest. Research topics can be: experimental, bioinformatic/computational, theoretical, conceptual… The week of March 3 I will meet with each group to refine and develop a project. Before this meeting each group should do some research on their ideas to evaluate what is feasible. Assignment 3