= 2 = 8 = 0 = 14 Rose and Petals game = =. Assignment 1 Peer-reviews due today I will email everyone reviews of their draft proposals When you revise

= 2= 8= 0= 14

Rose and Petals game

==

Assignment 1• Peer-reviews due today• I will email everyone reviews of their draft proposals• When you revise your proposal, please include a separate document that

includes a point-by-point response to substantial comments raised in the reviews

Assignment 2• Meetings to go over experimental design and analysis this week – if your

group hasn’t organized a meeting with me, do that today• Each group needs to prepare a ~12 minute presentation to the class for

February 24. This presentation should:– Identify the specific question you’ll focus on– Convince the audience that the question hasn’t yet been comprehensively answered and

that it is worth answering (what is its significance?)– Present your proposed experimental design. Include: a hypothesis; a prediction, the

independent variables that you will manipulate (e.g., population size and mutation rate;) the dependent variable(s) that you will observe (e.g., rate of fitness increase); replication; etc.. Note that you will have to ‘operationalize’ (convert from abstract to something practical) your measure of rate of fitness increase – e.g., you might measure fitness change over the first 25,000 time units or time taken to evolve X number of logic functions.

– Identify and justify any aspects of your design that are not straightforward – you might want to elicit feedback

– Propose a strategy for analysis of experimental outcomes to answer your original question.

• You should aim to give critical feedback to the presenting group

• To know what we need to know to conduct a study with the same goal as: de Visser et al. (1999) Diminishing returns from mutation supply in asexual populations. Science 283: 404-406

o The experiment manipulates supply of beneficial mutations (population size, mutation rate and degree of initial adaptation) and measures the effect of these changes on the rate of adaptation

o We need to know: (i) the basics of mutation and how it can be manipulated, (ii) how mutation supply is expected to influence adaptation, (iii) the basics of an evolution experiment, (iv) how the rate of adaptation can be measured, and (v) how the data that is generated can be analyzed to evaluate the hypothesis.

• How did the authors approach the design and analysis of the experiment?

• Are there any aspects of the study you think should be improved? If so, can you suggest how those improvement could be made?

This week…

Read by Thursday

Questions that motivate this paper

• We see high mutation rates in lots of microbes – especially in disease causing bacteria. Why?

Can we understand the circumstances under which high mutation rates provide an advantage (E.g., increases the rate of adaptation)?

What are the implications of pathogens having high mutation rates – does it make it easier for them to evolve drug resistance or the ability to cause disease?

de Visser et al. (1999) Science 284: 404-406

Observation: A high frequency of ‘mutators’ in many natural populations

• What is known…

Disease causing bacteria are often ‘mutators’ – why? Does it matter?

Hall and Henderson-Begg (2006)


Observation: A high frequency of ‘mutators’ in

natural populations

Inductive Conclusion: A

possible explanation for high frequency

of mutators

Complications: Unknowns whose influence on the

inductive conclusion should be examined

Complication I: Genetic linkage

complicates assignment of

cause and effect

• Note that the Inductive Conclusion (probably true) and follow up presentation of complications (why it might not be true) effectively serves to: (i) identify the research question and (ii) identify gaps in our knowledge and why it is

important they are filled




Complication 2:

Interference between

competing mutations

Predictions:1. Increasing mutation supply

will increase the rate of adaptation if a population has a limited supply of beneficial

mutations

2. Increasing mutation

supply will not increase the rate of

adaptation of a population that is not

limited by its supply of beneficial mutations

3. A fitter starting populations will have fewer beneficial available to it

so will be mutation limited at a higher mutation supply than will a

less fit population

• Evolution by natural selection is the process that drives the origin and success of the great variety of species we see around us

• The basic requirements of evolution by natural selection are very simple:

1. Variation in fitness

2. Competition between variants

3. Differential production of offspring by variants

4. Inheritance of beneficial traits between parents and their offspring

• Without mutation we’d still be self-replicating molecules in a primordial soup!

Why does mutation affect evolution?

The ultimate source of variation is mutation!

http://www.phidelity.com/photos/v/Artwork/Fractal+Flames/primordial_soup.jpg.html

Mutations are changes in an organisms genetic material (usually DNA/RNA)

atgtatcatcatcatcat

met tyr his

atgtaacatcatcatcatnonsense

met stop

his …atgtaccatcatcatcat

synonymous/silent

met tyr his his …

atgtgtcatcatcatcatnon-

synonymous/missense

met cys his his …

mutation

tatgatcatcatcatcat

indel – frameshift

(here a deletion)

met thr thr thr …

• We can classify mutations in several different ways (e.g., the effect they have on a protein, the type of change to the DNA sequence)

• Here we are mostly concerned about the effect of a mutation on the fitness of an organism – does a mutation increase (beneficial), decrease

(deleterious) or have no effect (neutral) on fitness

The basis of mutational change: mechanisms of mutations - spontaneous

• Spontaneous mutations: changes in DNA sequence that happen without any external physical or chemical cause

• DNA polymerase incorporates the wrong nucleotide into a growing DNA strand about every 104 – 105 base pairs (more later)

• Transitions can occur when rare tautomeric forms (= alternative structures) of bases are produced – these forms pair with a base different

from the original

• E.g., the usual keto (RC(=O)R’) form of guanine can sometimes convert to an enol (RC(OH)R’) form that pairs with thymine (instead of cytosine)

Sanders Fig. 12.11 (edited)

Original matched base pair

The basis of mutational change: mechanisms of mutations - spontaneous

• Mutations can also be induced by external agents (“mutagenic” chemicals, UV, radiation, etc.)

Mutation – induced

The basis of mutational change: repairing mutations

• If mutations are constantly introduced into DNA, how do organisms remain reasonably ‘error-free’ from generation to generation?

• Most biological molecules are replaced if they become damaged, new molecules will take their place – DNA is not replaced, it needs to be

repaired

• There are many biological mechanisms that act to repair DNA


• How important is error repair?

• By itself, DNA polymerase incorporates a wrong

nucleotide every 104-105 base pairs

•Many DNA polymerases have an intrinsic ‘proofreading’ ability (3’-5’ exonuclease)

that allows them to remove mispaired bases

• Proofreading increases DNA polymerase fidelity to one

wrong nucleotide per 106-107 base pairs (i.e. a 100-fold

improvement)

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Polymerase_Figure2.jpg


• DNA polymerase makes ~1 mutation per 106-107 base pairs but actual mutation rates are much lower than this – usually ~1 mutation per 109-1010 base pairs – error correction systems account for this ~1000-fold

improvement

mut

ation

rate

(cha

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from

par

ent t

o off

sprin

g pe

r bas

e pa

ir)

Bioessays 2000

• each point shows an experimental

estimate of the mutation rate for a

given species

•actual mutation rates are much lower than expected from DNA polymerase alone

• in E. coli we expect about 1 mutation per 500 cell divisions; in humans we expect about 1 mutation every generation

(although the mutation rate per

base pair is similar, humans have larger

genomes)

RNA viruses have no proofreading or error

correction

This difference is due to error correction

This difference is due to polymerase

proofreading

Repairing mutations – methyl mismatch repair (MMR)

• Several repair pathways recognize damaged bases and direct removal and repair

• What if the mutation is a mismatch of two normal bases (e.g. G-T, A-C)? How can the cell determine which base is the error and which base is supposed to be

present?

• The mismatch repair (MMR) system allows this to be accomplished…it can distinguish the original from the new base – and remove the new base

In E. coli and many other bacteria:

• Genomes contain ‘GATC’ motifs ~256 bases apart (44)

• The ‘A’ in these motifs is methylated by the enzyme

Dam (DNA adenine methyltransferase)

• There is a lag of a few minutes after a strand is synthesized before methylation occurs – during this time only the old

strand is methylated

Repairing mutations – methyl mismatch repair (MMR)

• The crucial condition for distinguishing new and old DNA strands is that the Dam enzyme

takes some time to find and methylate GATC sites – when only one strand is methylated this can

be recognized as the newly synthesized strand

• The mismatch repair systems starts with MutS recognizing a

mismatch and binding to the DNA helix

• MutS recruits other enzymes including MutH, which binds to a nearby ‘GATC’ site to distinguish

the new and old DNA strands

• The new strand is removed between MutS and MutH – this

gap is then repaired

• If a gene involved in MMR is non-functional, fewer errors

will be fixed and the mutation rate will be increased (a

“mutator”)

for a few minutes after a

GATC site is replicated only the old strand

will be methylated

(hemimethylated) – this fact is

used by the MMR system to

distinguish the new and old

strand

• Adaptation describes how well an organism does in its environment – quantified as the relative number of its offspring that have offspring themselves

• How does mutation influence adaptation? Genetic mutations sometimes change the phenotype of an organism and sometimes these changes will increase fitness.

Mutations and adaptation

A mutation that causes an increase in the fitness of an individual is called ‘beneficial’ and will tend to increase in frequency

NOTE: Especially in asexual organisms,

simply observing that a mutation increases in

frequency doesn’t mean that it is beneficial – WHY

NOT?

WQ = 1, WP = 1 + s (s = 0.01); generation 0 p:q = 1:1000

If you know the benefit of a mutation and its initial frequency, it is simple

to predict how it will change (t = time; s = selective benefit; p and q

different alleles)


Observation: A high frequency of ‘mutators’ in

natural populations

Inductive conclusion: A

possible explanation for high frequency

of mutators



Complication I: Genetic linkage

complicates assignment of

cause and effect

• Complete genetic linkage in asexual organisms makes it hard to distinguish between a high rate of adaptation:

1. …being caused by mutators: Mutator alleles allow more beneficial mutations to be produced as the beneficial mutations increase in frequency (causing a high rate of adaptation), so do the mutator mutations

2. …providing a background that benefits mutators: If beneficial mutations are actually quite common (as they would be if the rate of adaptation is high) as beneficial mutations increase in frequency they ‘drag’ along any other mutations they are linked to on a genome, including mutator mutations

Complication 1: Genetic linkage (cause or effect…)

Sniegowski et al. Bioessays (2000)Mutator mutation Beneficial mutation

tim

e

Mutator mutation Beneficial mutationor is it…

1.

2.

Can’t an experimenter just

look at the mutation

dynamics to see whether a

mutator or a beneficial mutation

occurred first? Actually, this is hard (see last

figure for intuition).

• To test if mutator alleles can be selected, Chao and Cox mixed otherwise identical mutator (mutT) and non-mutator (mutT-) bacteria and followed the change in ratio of the two types as the population adapted to a new environment

• They found that mutators tended to outcompete non-mutators as long as they were initially present at a frequency above ~10-4

• Why does this finding support the hypothesis that mutators can cause an increase in the rate of adaptation?

Is there any evidence that mutators can cause an increase in adaptation rate?

Chao and Cox (1983) Evolution

muta

tor

win

s if init

ially

pre

sent

above ~

10

-4

muta

tor:

non-m

uta

tor




Complication 2:


competing mutations



mutations







less fit population

Crow and Kimura, 1965 (after Muller, 1932)

Complication 2 & Prediction 1,2: competition between beneficial mutations

*’A’, ‘B’ and ‘C’ indicate newly arising beneficial mutations• Theory: o When the supply of new beneficial mutations is low, the population spends a lot

of time waiting for new beneficial mutations to

occur – the *waiting time* for mutations limits the

rate of adaptationo When the supply of beneficial mutations is

high, mutations interfere with one another and many go extinct – called clonal

interference – the *fixation* of mutations

limits adaptation (this is the “speed-limit” that de

Visser talks about)

• Prediction: a decelerating (non-linear) relationship

between mutation supply and rate of adaptation

?

mutation supplyrate

of

adapta

tion




Complication 2:


competing mutations



mutations







less fit population

• If the ratio of beneficial:deleterious mutations can change, the mutation supply that the “speed-limit” starts at might also change

• This ratio is not known – still, some models of adaptation allows to predict how it might change

Prediction 3: the ‘speed limit’ depends on initial fitness

trait 1

trait 2

starting phenotype

optimum phenotype

Fisher’s Geometric model of

adaptation – one of very few models that

try to predict the general underlying basis of adaptation

optimum phenotype

trait 1

trait 2

starting phenotype

Theory: The proportion of all mutations that are beneficial will decline as a population moves toward the phenotypic optimum

Prediction: Increasing the mutation rate might have a bigger benefit for populations close to the optimum because these populations spend most of their time waiting for new beneficial mutations to occur

• If the ratio of beneficial:deleterious mutations can change, the mutation supply that the “speed-limit” starts at might also change

• This ratio is not known – still, some models of adaptation allows to predict how it might change

Prediction 3: the ‘speed limit’ depends on initial fitness

How to test the three predictions



mutations







less fit population

Test:

1. Measure the rate of adaptation of a genotype

evolved at increasing levels of mutation supply.

Note: the prediction is qualitative – the authors don’t know exactly where

mutation supply stops limiting adaptation, so

start at very low mutation supply and increase to

very high levels!2. Repeat evolution

starting with a second genotype that is better adapted (closer to the

optimum phenotype) to the evolution

environment. Note: the authors don’t know how

much closer to the optimum phenotype this second genotype is – but

they do know that it is closer!

Methods – how do you measure the rate of adaptation?

AncestorAncestor split into

populations evolved at different mutation supply

(1000 generations) Compare fitness of evolved populations

Methods – how do you measure the rate of adaptation?

Ancestor

Why might it be

important to evolve replicate

populations at each mutation supply?

In practice, mutation supply was manipulated by a combination of:

1. deleting the mutY or mutS genes, which prevents mismatch repair and increases mutation rate by ~3- and ~30-fold, respectively

2. Evolving populations at different population sizes

Together, these manipulations allowed de Visser et al. to cover a range of mutation supply of ~1000-fold difference – hopefully enough to include the

onset of the speed limit of adaptation

Ancestor split into populations evolved at

different mutation supply (1000 generations) Compare fitness of

evolved populations

Results

Ancestor poorly adapted – higher

proportion of total mutations are beneficial

Note: arbitrary time chosen to assess the rate of adaptation (like asking:

“how fast does your car

accelerate?” – which would

depend on the distance you

are observing it over)

Ancestor well adapted – lower

proportion of total mutations are beneficial –

speed limit happens at a

higher mutation supply

Here’s the raw data – how do they fit the predictions? (What questions should you be asking yourself?)

Results








Speed limit





The ‘best fit’ lines seem to support expectation of a speed limit…

Results






Speed limit

The ‘best fit’ lines look good, but do they describe the data better than the null hypothesis?

Just what is the null hypothesis, anyway?







Conclusions

Null model (no speed limit =

linear relationship

between mutation

supply and fitness

increase)

Alternative model:

Speed limit modeled as a plateau in

fitness

Comparison of models – which one better explains the

data

Conclusions

Documents

= 2 = 8 = 0 = 14 Rose and Petals game = =. Assignment 1 Peer-reviews due today I will email everyone reviews of their draft proposals When you revise