intro3 mutation 2012 - dalhousie...

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“You’re lucky nobody was injured. Your base pairs are out of alignment and that has your reading frames all messed up.

⎯ Doc, have you been trying to do your own repair work?”

Some objectives for the student: 1.  Understand the relationship between mutation and recombination.

2.  Understand relationship between mutation, polymorphism and substitution.

3.  Understand relationship between mutation and phenotype

4.  Understand that mutations are relevant to different scales of genetics [nucleotides to genomes].

5.  Begin to develop an understanding of the relationship between the process of mutation and the process of evolution.

Also see the website for review questions about mutation and recombination!

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Mutation (mostly) and recombination

“Whatever the cause may be for each slight difference in the offspring from their parents ⎯and a cause for each must exist⎯ it is the steady accumulation, through natural selection, of such differences, when beneficial to the individual that give rise to all the more important modification of structure …”

We make a strict separation between the process that generates mutation and the processes that influence evolutionary fate of such variation

Mutations are random: 1.  The probability of a mutation does NOT depend on whether or not it is

advantageous.

2.  The probability of a mutation is NOT directed by the environment in a favorable way.

3.  Specific mutations cannot be predicted.

4.  The process of mutation is stochastic (predictions can be made over the long run).

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Mutations arise by: 1.  Error in repair (missincorporation, spontaneous decay, etc. can be repaired) 2.  Error in replication (including steps in recombination)

Two broad categories of mutation: 1.  Point (nucleotides) mutations 2.  Insertions, deletions, or other rearrangements (scale is hugely variable)

The fate of a mutation is either: 1.  Fixation in a population 2.  Loss from a population

Polymorphism: genetic variation due to the transitory period where a mutation has not yet been fixed or lost from a population. Mutation: A genetic variant resulting from a mutation event. Holds no meaning about the evolutionary fate of a genetic variant. Substitution: A mutation that has completed the process of being fixed in a population.

mutation vs. substitution

[“population thinking”]

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A

C

G

T

Purines: Adenine and Guanine

Pyrimidines: Cytosine and thymine

Within category:

Transition (ts)

Between categories:

Transversion (tv)

Within category:

Transition (ts)

Two rates:

ts > tv

1. Synonymous (silent) 2. Nonsynonymous (replacement)

Two broad categories of change among codons

Copyright © 2009 Paul O. Lewis 71

The Genetic Code

http://www.langara.bc.ca/biology/mario/Assets/Geneticode.jpg

5'-ATG|TCA|CCA|CAA-3'

3'-TAC|AGT|GGT|GTT-5'

5'-AUG|UCA|CCA|CAA-3'

N-Met|Ser|Pro|Gln-C

First 12 nucleotides at the 5' end of the rbcL gene in corn:

coding strand

template strand

mRNA

translation

DNA double helix

polypeptide

transcription

Codon models treat codons as the independent

units, not individual nucleotide sites.

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Ser Ter Seq 1 ATG CTG GTC AAG TTG AGA AGT TAA

↓ (1) (A) Seq 2 ATG CTG GTC AAG TTG AGA AGC TAA

Ser Leu Ter

Seq 1 ATG CTG GTC AAG TTG AGA AGT TAA ↓ (2) (B)

Seq 2 ATG GTG GTC AAG TTG AGA ACT TAA Val Lys Ter

Seq 1 ATG CTG GTC AAG TTG AGA AGT TAA ↓ (3)

Seq 2 ATG CTG GTC TAG Ter

Two sequences showing the different types of mutations (1) synonymous mutation, (2) nonsynonymous mutation, (3) nonsense mutation, (A) transition, (B) transversion.

Synonymous transition

Nonsynonymous transversion

Changes among codons can be classified according to nucleotide change

Nonsense transversion

Because of the structure of the code, transitions are more likely to be synonymous than transversions. Most degenerate sites occur at third codon positions; here, all transitions are synonymous whereas only some transversions are synonymous

Structure of code determines some effects of mutation on protein

Copyright © 2009 Paul O. Lewis 71

The Genetic Code

http://www.langara.bc.ca/biology/mario/Assets/Geneticode.jpg

5'-ATG|TCA|CCA|CAA-3'

3'-TAC|AGT|GGT|GTT-5'

5'-AUG|UCA|CCA|CAA-3'

N-Met|Ser|Pro|Gln-C

First 12 nucleotides at the 5' end of the rbcL gene in corn:

coding strand

template strand

mRNA

translation

DNA double helix

polypeptide

transcription

Codon models treat codons as the independent

units, not individual nucleotide sites.

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TTT (Phe)

TTC (Phe)

CTT (Leu)

TGT (Cys)

TAT (Tyr)

TCT (Ser)

TTG (Leu)

GTT (Val)

ATT (Ile)

TTA (Leu)

Mutational pathways among codons:

Relative proportion of different types of mutations in hypothetical protein coding sequence. Expected number of changes (proportion) Type All 3 Positions 1st positions 2nd positions 3rd positions Total mutations 549 (100) 183 (100) 183 (100) 183 (100) Synonymous 134 (25) 8 (4) 0 (0) 126 (69) Nonsyonymous 392 (71) 166 (91) 176 (96) 50 (27) nonsense 23 (4) 9 (5) 7 (4) 7 (4)

Assumptions: (1) ts = tv; and (2) codon frequencies = 1/61; complelty neutral evolution

Mutational pathways among codons

3 x 3 x 61 = 549 mutational pathways

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1.  Nucleotide indels

2.  Genic (exon) indels

3.  Chromosomal segments

4.  Genomic compliment

Insertion-deletion events (Indels)

insert T 1↓23 Leu Arg Ser Ter

Seq 1 ATG CTG AAG TTG AGA AGT TAA … ↓

Seq 2 ATG CTG ATA GTT GAG AAG TTA AGA Val Glu Lys Leu Arg

The insertion of a T causes the amino acids encoded beyond the insertion to change and since no stop codon is found it would continue until one is reached resulting in a longer polypeptide.

Insertion-deletion events (Indels): nucleotides

Frame–shift mutations are highly deleterious (consider hemoglobin)

•  common in non-coding sequences and loop regions RNA encoding genes

•  rare in coding sequences and stem regions of RNA genes

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Insertion-deletion events (Indels): chromosome segments


Robertsonian or non-reciprocal translocation

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Reciprocal translocation

Euchromatin chromatin

vs.

Heterochromatin chromatin

Gene

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•  Occur in viruses, prokaryotes and eukaryotes

•  Very deleterious if breaks occur within genes

•  Position effect variegation (PEV)

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Insertion-deletion events (Indels): genomic scale events

Haploidy: the condition of having only ½ (n) the of the two haploid sets (2n) of chromosomes. •  normal in some species; e.g., drone honeybees

Polyploidy: the condition of having more than two haploid sets of chromsomes (>2n) in some multiple of n.

•  lethal in most animals; but, known in a few reptiles and fish •  as much as 70% of modern angiosperms have polyploid origins

Autopolyploidy: polyploids having all sets of chromosomes from the same species Allopolypoidy: polyploids having sets of chromosomes originated in different species Aneuploidy: a change in number of chromosomes.

•  e.g., trisomy 21

Genetic variation + differential success ⇒ Natural selection

Fitness: an individual’s ability to survive and reproduce

A reminder:

We will cover these topics in much more depth in PopGen Topic 6, Natural selection

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Phenotypic effect of mutation

1.  Deleterious (vast majority of mutations; R. A. Fisher, 1930’s)

2.  Slightly deleterious (Tamoko Ohta [1972])

3.  Neutral (M. Kimura [1969]; King and Jukes[1969])

4.  Slightly beneficial (Darwin [1859] and R. A. Fisher [1930’], rare but important; Ohta [1970’s] more common, less important in finite population sizes)

5.  Major beneficial effects (Episodic; Mendelians; LGT)

FIVE KEY QUESTIONS ABOUT MUTATION:

WWhhaatt iiss tthhee nnaattuurraall rraattee ooff mmuuttaattiioonn?? WWhhaatt aarree tthhee eeffffeeccttss ooff mmuuttaattiioonn oonn ffiittnneessss?? IIss mmuuttaattiioonn rraattee iittsseellff uunnddeerr ggeenneettiicc ccoonnttrrooll?? IIss tthhee mmuuttaattiioonn rraattee ssuubbjjeecctt ttoo nnaattuurraall sseelleeccttiioonn?? IIss eevvoolluuttiioonn eevveerr lliimmiitteedd bbyy tthhee aavvaaiillaabbiilliittyy ooff nneeww mmuuttaattiioonnss??

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Note that mutation rate is measured in different ways 1. The rate of nucleotide substitution at sites believed to be free from natural selection

pressure (neutral). 2. The rate at which new mutations occur at a gene locus (or per genome) per generation. 3. The rate of accumulation of lethal or deleterious mutations on a chromosome. 4. The rate at which new phenotypic variance is generated by mutation.

"   What is the natural rate of mutation?

….Be careful !

Two approaches to measuring mutation rates:

1.  Direct

2.  Indirect


Direct methods: 1.  Quantify the number of new mutation that occur within the timeframe of the

study. 1.  Visible effect on phenotype in lab population 2.  Phenotypic effect from dominant mutants in a pedigree 3.  Now cost effective for direct sequencing!

2. Requires very large numbers; mutation is slow

Microorganisms: 10-1 to 10-10

Mice: 8 x 10-4

Fruit flies: ~4 x 10-6

Human genealogies: 10-6

Typical rate: 10-6

per bp

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Indirect methods:

1.  Estimate the number of substitutions along lineages since they shared a common ancestor.

2.  Must be neutral (substitutions used as a proxy for mutations)

3.  Date of common ancestor must be known; must convert to generations

4.  If substitutions are not neutral, rates will be influences by: 1.  Strength and type of selection 2.  Effective population size

We will deal with these issues later in the course


Substitution rate is not the same as mutation rate when the mutations are subject to natural selection pressure

New mutations

Fixation in a population

natural selection and genetic drift acts as a “sieve”

The substitution rate can be used as an indirect measure of the mutation rate if the mutations are selectively neutral

Indirect methods:

Cause the sieve effect to differ in different lineages

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Indirect methods:

1.  Pairwise, or along a phylogeny. 1.  Pairwise approach has lower power than using a known phylogeny 2.  Unknown phylogeny has estimation errors

2.  Define (assume) which sites are neutral

3.  Choose a model and correct for multiple substitutions

Problems:

1.  Errors with assumptions, models, tree.

2.  Conversion of results to per generation units of time


• In case 1, simply counting the number of changes as inferred under parsimony might work, but the divergence (branch lengths) must be low.

• In case 2, the divergence is so large (branch lengths so long) that there are at least

two sources of error: (i) the uncertainty of the ancestral reconstruction; and (ii) how many substitutions actually occurred along a branch.

• In case 2, model based methods that provide a correction for multiple substitutions

at one site along a branch will provide better estimates of the rate.

ACG TAC TAA

ACG TAT TAA

ACG TAT TAA

C

T

ACG TAC TAA

ACG TAT TAA

ACG TAT TAA

C

T ? → A → G → T ?

C → A → G → C ?

Case 1 Case 2

Rooted phylogenies and ‘ancestral character-state reconstruction” can be used to indirectly infer the number and direction of substitutions.

Ancestral character states

Indirect methods:

(1MY) (100 MY)

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Discrepancies: •  Differences between methods can be large and in either direction. •  Power can be low in some lab-based experiments •  Environments in lab could facilitate or reduce the mutation process relative to

natural populations Human example: •  Neutral indirect method yields estimate of 10-8 (Kimura 1983) •  Human genome is 6.4 x 109 bp •  Estimated new mutations per zygote is 64

–  Too high if most new mutations are deleterious

So, what is the effect of new mutation on fitness?

"   What are the effects of new mutations on fitness?

•  We can answer this question with mutation accumulation (MA) experiments.

•  Terumi Mukai developed an approach where a large portion of a genome can be “shielded” from natural selection via heterozygosity

•  Run experiment long enough to “collect” mutations

•  Can check the fitness effects of the “collected” mutations at any time by making them homozygous

•  Approach is based on balancer chromosomes i.  Multiple overlapping inversions (suppress recombination) ii.  Carries an allele that is lethal in the homozygous state iii.  Carries a dominant marker that is easily identifiable

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Heterozygous balancer (homozygotes are lethal)

mutation

Select a heterozygous offspring with balancer marker

Modified from St. Johnson (2002) Nature reviews 3: 176-188

Breeding with a balancer chromosome to shelter a chromosome from recombination


MUKAI’S EXPERIMENT:

1. Establish a set of 101 genetically identical lines of flies (starting with wild type chrom 2). 2. Cross each generation to a reference stock bearing a balancer for chromosome 2.

a. A single male is used as the parent in each generation. b. Select the balancer heterozygotes based on the marker phenotype c. Deleterious recessives on chrom 2 are sheltered from selection because (i) they are only

allowed to exist as heterozygotes AND (ii) grown in optimal conditions

3. Lines are maintained independently for 60 generations, and mutations are allowed to accumulate. a. Independence allows divergence of the lines in numbers and effects of mutation b. Allows measurement of accumulation of variance among lines in a fitness trait over time

4. Measure fitness at regular intervals in the study

a. Breed sheltered chromosome to yield homozygous configuration (het x het) b. Viability index: deviation from 2:1 ratio [see figure below]


With proper breeding:

•  mutants are trapped on the “wild-type” chromosome

•  mutants sheltered from selection because are maintained as heterozygotes

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No fitness effects: 2:1 Fitness effects: < 2:1 Modified from St. Johnson (2002) Nature reviews 3: 176-188

Die as larvae Viable

heterozygote Homozygote

recessives could influence fitness

1 2 1 : :

Viability index measures depression in fitness as a deviation from 2:1 ratio


>

Mean and variance of the viability index over the course of the Mukai et al. (1972) experiment.

Adapted from Mukai et al. (1974) Genetics 72:335-355.

NOTE: Indices were standardized to a value of 1 in generation 0


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Results: •  Astonishing decline in viability (about 15% in just 40 generations) •  About 1% decline in fitness per haploid genome per generation •  Mutation is a destructive force •  Natural selection is important defence mechanism [purifying selection] •  Highlight the concept of mutational load Mutational load: the reduction in fitness (via death, failure to reproduce, or reduced

reproductive success) incurred by the presence of harmful mutations. Genetic load: the reduction in fitness that is the sum of the effects of:

i. Mutational load ii. Segregational load iii. Substitutional load

Re-start here in 2012!

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"   Is mutation rate itself under genetic control?

Mutator genes/alleles: •  Genes that function in replication and repair.

–  Mutation in such genes leads to a “Mutator phenotype”

•  Mutator genes/alleles elevate the genomic rate of mutation –  Well known in bacteria

Classic example: MutT gene of E.coli

“Normal” MutT:

•  Hydrolyzes 8-oxo-dGTP (8-oxo-dGMP)

•  8-oxo-dGTP will pair both C and A

•  Important in preventing specific mutations

•  Normal G:C pairs

Mutator phenotype of MutT:

•  no hydrolysis of 8-oxo-dGTP (8-oxo-dGMP)

•  8-oxo-dGTP inserted into DNA

•  pairs with both C and A

•  8oxoG:C and 8oxoG:A

•  rate of C⇒A and G⇒T transversions increases

Note: all mutator variants are defective copies; no variants have been discovered that decrease mutation rate

G

C

G8OXO

G8OXO

C

G8OXO

A

G8OXO Free 8-oxo-dGTP because MutT not functioning

Some get inserted

DNA Replication 2

C DNA Replication 1

T

A DNA Replication 3

G ⇒ T

C ⇒ A

Two possible outcomes:

8-oxo-dGTP pairs with C

or

8-oxo-dGTP pairs with A

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Transposable elements:

•  pieces of DNA that are capable of self replication

•  1000 to 10000 bases long

•  encode proteins, including a transposase

•  move about within a “host” genome

•  transposition activity is NOT under the control of the host genome

•  transposition activity is mutagenic

Diagram of “simple transposition” of a segment of DNA catalyzed by the Tn5 transposase.

Target DNA

Donor DNA

Transposase enzyme

Cleavage

Capture of target

Strand transferred to target DNA

Tn5 Transposon DNA

Note: in “replicative transposition” the sequence element replicates itself from place to place, thus leaving behind


Transposase has dual function: •  excisionase •  integrase

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YES. But, the involved genetic and protein machinery is sometimes under the control of the genome and sometimes it is not.

"   Is mutation rate subject to natural selection?

•  Mutation rate has a genetic component

•  There is standing genetic variation in those genetic systems

•  Mutation is the ultimate source of variation required for adaptation

Many have speculated that it should be subject to natural selection

Hypotheses fall into two broad categories:

H1: Mutation rates are adjusted to levels that promote adaptation

H2: Mutation rates are adjusted to minimize the cost of fidelity

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Some problems:

1.  The vast majority of new mutations are likely to be detrimental to fitness.

2.  The input of deleterious mutations might cause selection to favor lower genomic mutation rates; i.e, there is a conflict.

3.  Short term gains in fitness from new mutations would be offset by long term accumulation of deleterious mutations.

4.  Natural selection cannot maintain mutation rates in a population for the sake of future adaptive value; it can only act to maintain mutation rates as a consequence of their adaptive value in the present conditions.



There might yet be a way.

We need to invoke a process called “hitchhiking”

Mutator allele Strongly beneficial allele

Chromosome

Physically very close so likelihood of recombination breaking up this configuration is lowest

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“Hitchhiking” of a mutator gene with and without recombination

Adapted from Sniegowski et al. (2000) BioEssays 22:1057-1066.

No recombination

Recombination

Mutator allele that increase the mutation rate

Beneficial allele subject to strong positive selection

Indirect effect of selection




Models suggest a possibility for indirect selection, but only in clonal lineages

How does this hypothesis fit real data?

•  mutator phenotypes are exceptions rather than rule

•  mutators in experimental populations do not appear to increase the rate of adaptation

•  germ-line mutation rates (in mammals) are 3 fold lower than in somatic cell lines

Why aren’t mutator phenotypes selected?

•  It seems the negative effects of deleterious mutation over long periods of time outweigh any short periods where the higher rate was beneficial.

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A. H. Sturtevant (1937):

•  Why does the mutation rate not evolve to zero?

M. Kimura (1967):

•  Reductions beyond observed rates are impossible because of physiochemical constraints?

•  The physiological cost of further reductions are so high that they impose prohibitively high fitness costs to individuals ?

Remarkable conservation of genomic mutation rate among DNA-based microorganisms

Adapted from Sniegowski et al. (2000) BioEssays 22:1057-1066.

RNA viruses

Mut

atio

n ra

te

Genomic mutation rate

Base-pair mutation rate

physiological limit ?

Higher eukaryotes


or

cost of fidelity

or

beneficial rate ?

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Drosophila experiment:

600 generations:

X-ray irradiation

Results: 1.  X-ray induced mutation decreased over

course of experiment.

2.  Repair systems seemed to evolve to compensate for additional mutations

3.  Mutation rates returned to wild-type levels after irradiation was stopped!

•  The cost of maintaining the extra repair systems was probably too great after the extra mutation was removed from the system.

•  Suggests genomic rate is due to trade-off between cost of mutations and cost of repairing them.


H3: Natural selection for high mutation rates without incurring an excessive increase in mutational load in special cases.

1.  Elevate mutation rate at certain loci only: •  called contingency loci

•  generally related to evading host immune system

•  variation more valuable than conserved structure of protein

•  well supported examples; not very controversial

2.  Restrict elevation of genomic rates to times when they extra mutations are needed:

•  called “adaptive mutation”

•  Mutation rate only increases when needed; e.g., stress

•  Some bact. exhibit elevated mutation rates when starved

•  Difficult to distinguish direct effects from those that evolved under natural selection.

•  Very little modelling; very controversial idea

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"   Is evolution limited by the availability of new mutations?

Work by Trudy Mackay on fruit flies directly address this question

•  Fruit flies + P elements (transposable elements)

•  P elements have highly mutagenic effect

•  Designed one of the “classic” experiments

•  utilized hybrid dysgenesis

Hybrid dygenesis: Result from cross of P-strain (male) and M-strain (female) flies

P strain:

•  30-50 copies of P-element in genome

•  Repressor protein

•  No P element activity

•  P x P cross = normal progeny

M strain:

•  No P-element in genome

•  No Repressor protein

•  M x M cross = normal progeny

P egg cytoplasm: repressor proteins M egg cytoplasm: no repressor protein

Matings within M and P strains produce normal progeny

Normal progeny

M = no P elements P = P elements in genome

Normal progeny

Certain Mating between M and P strains produce hybrid dysgenisis

M = no P elements P = P elements in genome

Normal progeny

Reduced fertility

abnormalities

Dysgenic effects are asymmetric

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MACKAY’S EXPERIMENT:

1. Establish populations derived from dysgenic and non dysgenic crosses 2. Apply artificial selection for both high and low bristle numbers (divergent selection)

3. Carry out artificial selection for 16 generations by choosing the 10 most extreme phenotypes in a

generation to be the parents of the next generation.

4. Conduct assays of the effect of this variation on fitness (we will not cover this result in this lecture)

Rational for MacKay’s experiment:

•  movement of P elements causes mutations

•  some mutations will, by chance, affect quantitative characters

•  dysgenic lines will have higher P element activity

•  dysgenic lines will have higher standing genetic variation

•  if selection is limited, the response to artificial selection will differ between dysgenic lines and non dysgenic lines


Adapted from MacKay. (1985) Genetics 111:351-374.

Non-dysgenic fruitflies Dysgenic fruitflies

Generational means for abdominal bristle score in dysgenic and non-dysgenic lines of Drosophila subjected to artificial selection


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The response to artificial selection was twice as fast in the dysgenic lines as compared with the non-dysgenic lines.

The general inference has been that evolution can be constrained by the availability of new mutations

Remember, this result is for artificial selection. It is not clear how important this is for wild populations. However, consider the long term consequence of this result for an endangered species.

FIVE KEY QUESTIONS ABOUT MUTATION:

WWhhaatt iiss tthhee nnaattuurraall rraattee ooff mmuuttaattiioonn?? WWhhaatt aarree tthhee eeffffeeccttss ooff mmuuttaattiioonn oonn ffiittnneessss?? IIss mmuuttaattiioonn rraattee iittsseellff uunnddeerr ggeenneettiicc ccoonnttrrooll?? IIss tthhee mmuuttaattiioonn rraattee ssuubbjjeecctt ttoo nnaattuurraall sseelleeccttiioonn?? IIss eevvoolluuttiioonn eevveerr lliimmiitteedd bbyy tthhee aavvaaiillaabbiilliittyy ooff nneeww mmuuttaattiioonnss??

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A B

a b

a B

A b

Parental contribution Resultant offspring

A B C D

a b c d

A B C D

a B C d

Gene conversion between the same gene Gene conversion between different genes

Gene A Gene A Gene A

Gene B

Reciprocal recombination:

Non reciprocal recombination:

Recombination as a source of variation: 1.  It can give rise to astronomical numbers of combinations of alleles at

different loci.

2.  In many organisms [especially sexual organisms] recombination generates far more variation than does mutation.

3.  Recombination also breaks up favorable gene combinations!!!

4.  Recombination constrains variation displayed in polygenic characters [we do not go over this in this course].

5.  Gene conversion is a force for homogenization

6.  Unequal crossing over changes the amount of DNA, leading to tandem duplications or deletion of DNA.

Remember: mutation is the ultimate source of variation.

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Recombination as a source of variation: “Toy” example:

Drosophila: ~10,000 functional genes

Assume: 10% of loci are heterozygous

Number of unique allelic configurations: 10300

Note that Avagadro’s number = 6.023 x1023

Some objectives for the student: 1.  Understand the relationship between mutation and recombination.

2.  Understand relationship between mutation, polymorphism and substitution.

3.  Understand relationship between mutation and phenotype

4.  Understand that mutations are relevant to different scales of genetics [nucleotides to genomes].

5.  Begin to develop an understanding of the relationship between the process of mutation and the process of evolution.

Also see the website for review questions about mutation and recombination!

intro3 mutation 2012 - dalhousie...

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