The Nature of Mutation

1. The Nature of Mutation E3: Lecture 3

2. Deniz-Somer-Clementine Hypothesis Closest prime greater than or equal to I, if I is odd Closest prime greater than I, otherwise 0, if I is odd 1, otherwise Y, if I=O1 N, otherwise O1= Neem’s Hypothesis O2= I+1, if I<5 I, otherwise I-1, if I<5 0, otherwise N, if I<5 Y, otherwise O1= O3= BLACK BOX 1 BLACK BOX 2 O2= O3= BLACK BOX 3 BLACK BOX 4 Deniz-Toni-Somer Hypothesis BLACK BOX 5 BLACK BOX 6 Random in {I-1, I, I+1} O1 modulo I Y, if  I>4 N, otherwise O1= O2= BLACK BOX 7 BLACK BOX 8 O3= The Returnof Black Box Should we input 3 or 7?

3. Richard Lewontin Natural Selection • Richard Lewontin (1970) laid out Darwin’s postulates: • 1. There is variation between individuals • 2. There is heritability across generations • 3. There is differential survival/reproduction • Thus conceived, natural selection destroys variation by weeding out unfit individuals. • Regarding the first condition, there must be a process that generates variation or else selection grinds to a halt. • This process is mutational change and is conceptually separable from selection.

4. Natural Selection and Mutation M • In The Origin of Species, Darwin noted the following: • “Some [writers] have even imagined that natural selection induces variability, whereas it implies only the preservation of such variations as arise and are beneficial to the being under its conditions of life.” • Darwin conceptually separated the process of variation generation from natural selection. • In the Vignieri et al. study on dorsal coat coloration in mice, in order for selection to favor different heritable colors under different substrates, there must have been mutational changes that affected coat color. mainland mouse colonizes beach several generations The “sieve” of natural selection light mutant arises selection favors light mutant The “generator” of mutation

5. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

6. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

7. The Central Dogma 5’ 3’ T T T T T T C C C C G G G G G G A A G G G G G A A A A A A A A C C C C C C C C T T DNA transcription U mRNA translation Glutamic Acid Proline Glutamine protein An example of a mutation 3’ 5’ parental strand new strand • The central dogma suggests that information flows from nucleic acid sequences (DNA to RNA) to protein, but not in the reverse direction. • Crick (1970) stated: • “Although the details of the classification proposed here are plausible, our knowledge of molecular biology, even in one cell, let alone for all the organisms in nature, is still far too incomplete to allow us to assert dogmatically that it is correct.” • - Reverse transcription (RNA to DNA) • - Prions (protein mediated change of proteins) • Mutations are changes to the genetic material (DNA or RNA) due to: • Errors during replication • Exposure to radiation • Exposure to mutagens • If mutations remain unrepaired, then the change can be inherited.

8. Types of Mutation 5’ 3’ T T T T T T T T T T T T T C C C C C C C G G G G G G G G G G G G A A A A G G G G G G G A A A A A A A A A A A A A C C C C C C C C C C C C T T T T wild-type sickle-cell wild- type insertion non- motile wild- type point mutation deletion • Classifying mutation by effect on structure • - Point mutation • Synonomous (codes for the same aa) • Non-synonomous • - Missense (codes for new aa) • - Nonsense (codes for stop) • - Insertion (of bases or longer stretches) • - Deletion (of bases or longer stretches) • Classifying mutation by effect on function • Loss-of-function mutation (due to missense, nonsense, or frameshift mutations) • Gain-of-function mutation • Lethal mutation • Other classifications (e.g., coding vs. regulatory) CCC still gives Proline CAU now gives Histidine white- eye UAG is stop codon

9. Fitness Effects of Mutation Experimental Results control treatment fitness generation • Most mutations are either neutral or deleterious. • This can be shown by performing a mutation accumulation experiment: • Experimental treatment: A random offspring individual is picked to establish the next generation • Control treatment: Several individuals continually establish the next generation. • As an analogy, think of two versions of the game “telephone” treatment control I know glass for 81 is fun! I know classes for 81 infants! I know class 481 is fun ! Aye, no gas for 81 infants! I know class 481 is fun ! I know glass for 81 is fun! I know class 481 is fun! *$#% &%#@% “not-for-parties” version standard version

10. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

11. The Mutational Trigger mutant pea plant wild type mutant fruit fly wild type • Zoologists and botanists are often able to see mutants arise that do not bring fitness benefits to their bearers. • Early microbiologists were less fortunate: mutants of the microorganisms they studied could often be revealed only under selective conditions in which the mutation allowed survival. • Consider antibiotic resistance (something we’re interested in from a medical point of view): • - Let’s say you have a growing culture of bacteria in a test tube. • - You then add an antibiotic to this tube. • - Your culture quickly clears (due to the death of a large number of cells) • - However, after further incubation, the culture recovers (due to the growth of drug-resistant cells). • But what was the trigger of these drug-resistant mutants? • - Did the presence of the antibiotic stimulate the resistance mutations? • - Did the mutations occur randomly (e.g., even before the application of the antibiotic)? ?

12. Two Hypotheses Max Delbrück Salvador Luria • Imagine a single cell placed in a test tube with growth media. This cell doubles and then its offspring double and so on… This can be represented as an inverted tree. • At a certain point in the population growth, the selective pressure (an antibiotic) is applied (represented by the red dashed line). • Extreme versions of our hypotheses: • Directed Mutation: Mutations occur only after application of the selective pressure • Random Mutation: Mutations occur spontaneously at any doubling event. • The problem is that resistant cells and sensitive cells don’t look different– we can only tell them apart by imparting the selective pressure (e.g., after a “red line”) • The stakes are big– are mutation and selection linked or independent? • This question was addressed in 1943 by Salvador Luria and Max Delbruck.

13. The Luria-Delbruck Experiment • The key insight that Luria and Delbruck had, was to look at several replicates to see how mutant numbers were distributed across replicates. • The Directed Mutation hypothesis predicts a fairly even distribution of mutants across replicates. • The Random Mutation hypothesis predicts an uneven distribution of mutants across replicates, with a few replicates having unusually high numbers of mutants (these are called jackpots).

14. A Signal to Distinguish Hypotheses • After growing up several replicate populations and then placing each population under selective conditions, Luria and Delbruck could calculate the average number of mutants and the variance in the number of mutants. • Relative to the mean, jackpots tend to inflate the variance across replicates in the number of mutants. • By looking at the ratio of the variance over the average, we can distinguish these hypotheses. This ratio is expected to be one for the directed mutation hypothesis and is expected to be greater than one for the spontaneous mutation hypothesis.

15. An Exercise Random Mutation Simulation 2 2 2 2 2 2 2 2 2 2 2 2 2 6 6 6 6 6 6 6 6 6 6 6 6 1 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 5 4 5 5 4 5 1 2 1 2 1 2 2 1 4 1 4 4 6 6 1 1 Directed Mutation Simulation 2 6 1 4 1 3 3 5 1 4 6 • We’re going to simulate the Luria-Delbruck experiment in class. You will receive a piece of paper with two inverted trees on it and a die. • In the “Random Mutation Simulation” start at the top of the tree and make your way down always doing the following: • Record the roll of your die in the box • If the roll was a 1, then fill in all boxes and circles to the bottom right of the box. • If the roll was not a 1, then do nothing. • As you move down the tree, skip all filled in boxes. • In the “Directed Mutation Simulation” simply roll your die 8 times and record the numbers in the 8 boxes. If the roll was a 1, fill in the circle to the bottom right of the relevant box. • Circles represent bacterial cells, boxes represent replication events, and filled circles represent mutants.

16. Their Data • In all of their replicated experiments, Luria & Delbruck found a significantly higher variance than average– consistent with spontaneous mutation.

17. An Independent Check Esther Lederberg Joshua Lederberg Random Mutation replica plating tool • In 1952, Joshua and Esther Lederberg provided independent support for the Random Mutation hypothesis. • They grew up bacteria on an agar-filled Petri dish until it formed a confluent lawn. • Then they replica plated this population onto several new agar plates with selective media (e.g., the presence of an antibiotic). Directed Mutation different locations of mutant colonies velvet some same locations of mutant colonies

18. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

19. Directed Mutation Strikes Back John Cairns Julie Overbaugh replicate 1 replicate 2 replicate 3 replicate 4 replicate 5 replicate 6 DNA DNA mRNA mRNA protein protein Cairnsian revision? central dogma • John Cairns, Julie Overbaugh and others suggested that for some executions of the Luria-Delbruck protocol (considering other mutations), the variance was too low to be accounted for solely by spontaneous mutation. • Indeed, they argued that the distribution of mutants in replicated populations looked like a combination of the distributions predicted from the spontaneous mutation and directed mutation processes. • Further, they claimed to provide independent evidence that directed mutation played at least some role: • - They saw E. coli unable to use the sugar lactose (Lac-) mutate into two types ofLac+ bacteria (with different colony shapes). • - The first type (fast colony former) had a high variance for its mean across replicates. • - The second type (slow colony former) had a variance similar to its mean across replicates. • The authors claimed the second mutant was the result of directed mutation (mutation to Lac+ in the presence of lactose) • The mechanism? “[Perhaps] the cell could produce a highly variable set of mRNA molecules and then reverse-transcribe the one that made the best protein.”

20. Data Consistent with Directed Mutation Spencer Benson • An E. coli strain was studied by Spencer Benson that lacked the ability to take up large maltodextrins (Dex-). • Two point mutations can restore this ability in E. coli (either in the OmpC locus or OmpF locus). • Benson (1988) found Dex+ mutants via OmpF are generated at a much higher frequency than Dex+ mutants via OmpC. • Benson suggested that this bias might be due to directed mutation that causes the OmpF change (under appropriate selective conditions), but not the OmpC change. • Take 5 minutes to talk to your group about the following: • 1. Is Benson’s suggestion the only plausible process to explain the pattern? • 2. If not, what other hypotheses would you suggest? • 3. How might you experimentally test your hypotheses?

21. The Return of Random Mutation Random Mutation where Mutant Grows Slowly Directed Mutation • The Luria-Delbruck random mutation hypothesis makes an important assumption: The mutant (whenever generated) duplicates at the same rate as its ancestor. • Benson et al. (1991) checked the growth rates of OmpF and OmpC Dex+ mutants and found OmpF mutants to have a higher growth rate. • Could slow growth explain Cairnes’ “two-variance” observation? • If the mutant grows slower, then the number of mutants decreases and the variance drops (looks more similar to the pattern predicted under directed mutation). • Interestingly, the colonies producing an even distribution of mutants in the Cairns experiment were slow growing colonies…

22. Some Lessons from the Mutation Controversy Experimentally: • It is extremely important to remember (and test for) any underlying assumptions of your hypotheses. • Running appropriate controls is important: • - The directed mutation hypothesis suggests that mutation occurs specifically at loci where changes will improve the fitness of the organism under current selective conditions. • - In order to test whether the mutation under consideration is directed, it becomes important to consider mutations at other loci that are not under selection (think how you would do that…) Biologically: • Given no molecular mechanism for “trying out” mutations within the lifetime of an organism and some experimental problems from the data supporting the directed mutation hypothesis, the random mutation hypothesis is the current working model. • Are there many different mutations that could produce a phenotype that would prosper under selective conditions? Do these different classes of mutants differ in their growth rates (fitness) under non-selective conditions? Under selective conditions? • Do stressful conditions raise the mutation rate of organisms? Is this an adaptive response or simply a by-product of poor organismal condition?

23. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

24. Variation in Mutation Rate mitomycin C caffeine E. coli adenine glycosylase • Mutation rate varies between and within species. For instance, mutation rate can be sensitive to environmental conditions: • - Some organisms experience a higher mutation rate under stressful/starvation conditions • - Chemical mutagens can increase the rate of mutation • - Genetic changes can affect mutation rate • Surveys of natural populations of bacteria suggest that more than 1% of isolates are mutator strains. These strains are often deficient in some DNA repair component. • Given heritable variation in mutation rate, it is reasonable to consider the possibility that mutation rate could evolve through natural selection… • Take 5 minutes to talk to your group about the following: • 1. Under what circumstances would you expect a higher mutation rate to be adaptive? • 2. What are the costs to being a mutator? • 3. How might you experimentally test your hypotheses?

25. Experimental Test for Adaptive Mutation Arjan de Visser Richard Lenski wild-type mutS mutY 1.3 1.2 fitness 1.1 1.0 0 1000 200 400 600 800 generations • Arjan de Visser, Richard Lenski and colleagues (1999) explored the success of mutator strains under novel selective conditions. • Controlling for genetic background, they constructed three strains, a wild-type, a mutY mutator, and a mutS mutator. • They grew these three strains up separately under conditions to which the strains were not adapted. • They found that both mutator strains evolved a higher fitness after 1000 generations than the wild-type. • The same result was not found under conditions to which all strains were adapted. • This experiment gives some evidence that there may be short-term advantages to higher mutation under novel selective conditions. • Potential trade-offs include long-term deleterious effects of a higher mutational load.

26. The Results of Your Simulation…

27. The Nature of Mutation • Lecture Outline • Introduction to genetic mutation • The Luria-Delbrück experiment • A mutation controversy • Selection for mutation rate • Summary

28. Summary • The Neo-Darwinian perspective separates the process of mutation from the process of selection—that is, mutations occur without respect to their selective consequences. • Most mutations have either neutral or slightly deleterious effects, but rarely mutants are selectively favored. • Luria and Delbruck designed a classic experiment using the variance in mutants between replicate populations to distinguish between the random mutation hypothesis (neo-Darwinian perspective) and the directed mutation hypothesis (Cairnsian perspective). • Luria and Delbruck (& others) found support for spontaneous mutation. • The evidence presented by some scientists in support of directed mutation illustrates the importance of proper controls and checking the assumptions of your hypotheses. • However, the directed mutation controversy has led to greater exploration of the (potentially adaptive) changes in mutation rate.