Mutation and Genetic Variation For every gene, there are many different alleles - alleles are versions of the same gene that differ in their DNA base sequence - some alleles differ in the protein product of the gene; others do not (more on this to follow) - your combination of alleles is unique, and makes you you Alleles are generated by mutations, which are changes in the DNA base sequence

Evolution and Alleles Define evolution: genetic change in a population over time But what does “genetic change” mean, exactly? 1. Mutation can create new alleles that were not previously present in that population 2. The frequency of alleles may change Today we will consider mutation in some detail; we will then then discuss in detail the forces that can change allele frequencies in natural populations

Mutation and Genetic Variation Changes in the DNA sequence create new alleles in every generation

The first mutation that was characterized at the molecular level (that is, at the DNA sequence level) was the sickle-cell anemia allele of the hemoglobin gene In people afflicted with the disease, a mutation was found at amino acid #6 of the 146-amino acid hemoglobin protein

The is an example of a point mutation = 1 of 2 sources: (a) random error during DNA replication (b) error in the repair of damaged DNA -- chemical mutagens, free radicals, radiation

Errors during DNA replication DNA polymerase occasionally (but rarely) adds the wrong nucleotide during DNA replication The mutation is a transition if.. - Purines Pyrimidines

Purines Pyrimidines Errors during DNA replication DNA polymerase occasionally (but rarely) adds the wrong nucleotide during DNA replication If one type gets swapped for the other type, it’s a transversion Transversions only show up in DNA sequences half as often as transitions

Why are transition mutations more common? inserted guanine Transversions stick two non-complimentary bases against each other - steric clash disrupts the shape of the DNA helix, making a bulge - the bulge in the DNA is more often noticed and fixed by repair enzymes

2nd source of mutations: error in the repair of damaged DNA (1) chemical mutagens - can intercalate (stick into) the DNA helix, distorting the shape and causing excision (cutting out) of bases (2) free radicals - uncharged oxygen atoms or OH molecules that have single electrons - extremely oxidizing (electron-hungry) - damage DNA bases (3) radiation - UV from sunlight can cause thymine dimers, which need to be excised (= cut out)

Mutations that change the corresponding mRNA codon to another codon for the same amino acid are called synonymous substitutions (“equivalent”) -C-A-A- -C-A-G- glutamine -- also called silent substitutions -- common when the change is at the 3rd position Note: Mutations may, or may not, change the amino acid sequence

- synonymous substitutions usually do not affect the phenotype of the organism - assumed to be neutral, meaning they have no effect on fitness fitness = how much an individual contributes to the gene pool of the next generation -C-A-A- -C-A-G- glutamine Neutral changes have no adaptive value to the organism Mutations may, or may not, change the amino acid sequence

HOWEVER – “silent: changes can affect the phenotype if they introduce a rarely used codon Cells make fewer tRNAs for rare codons Introduction of a rare codon into a highly transcribed gene can slow translation of the mRNA by the ribosome (Shields et al. 1988) -C-A-A- -C-A-G- glutamine If ribosomes stall mid-translation, this can lead to protein mis-folding that changes the properties of an enzyme (Kimchi-Sarfaty et al. Science 2007) So-called “translational selection” can act against silent changes, which are therefore not truly “neutral” as we often assume Mutations may, or may not, change the amino acid sequence

Mutations that change the corresponding mRNA codon to a codon for a different amino acid are called non-synonymous substitutions (“non-equivalent”) -C-A-A- -C-A-G- -C-A-C- glutamine glutamine histidine -- these mutations alter the protein product of the gene -- Mutations may, or may not, change the amino acid sequence

non-synonymous substitutions alter the protein product, so often affect the phenotype of the organism -C-A-A- -C-A-G- -C-A-C- glutamine glutamine histidine -- most changes are neutral or detrimental to the organism -- if the change is adaptive under some circumstance, then the mutation may lead to a higher fitness (example: sickle-cell allele and malaria) Mutations may, or may not, change the amino acid sequence

Loss-of-function mutations normalACA-ATG-GTA-CGACys-Tyr-His-Ala 1-BaseACA-GAT-GGT-ACGCys-Leu-Pro-Val insertion Protein sequenceDNA sequence Frameshift mutations change all subsequent amino acids, usually making a dysfunctional protein

Loss-of-function mutations normalACA-ATG-GTA-CGACys-Tyr-His-Ala 1-BaseACA-GAT-GGT-ACGCys-Leu-Pro-Val insertion 1-BaseACA-TGG-TAC-GACys-Tyr-Met-Leu deletion Protein sequenceDNA sequence Frameshift mutations change all subsequent amino acids, usually making a dysfunctional protein - some viruses rely on frameshifts to make a 2 nd functional protein for “genomic efficiency” (minimize amount of DNA needed)

Loss-of-function mutations normalACA-ATG-GTA-CGACys-Tyr-His-Ala 1-BaseACA-GAT-GGT-ACGCys-Leu-Pro-Val insertion 1-BaseACA-TGG-TAC-GACys-Tyr-Met-Leu deletion MutationACA-ATT-GTA-CGACys to a stop codon Protein sequenceDNA sequence

Loss-of-function mutations normalACA-ATG-GTA-CGACys-Tyr-His-Ala AGG-GGG-CTA InsertionACA-AGG-GGG-CTA-ATG Cys-Ser-Pro-Asp-Tyr - caused by a transposable element, or “jumping gene” - transposons inactivate the gene they disrupt, sometimes only temporarily; they may hop back out at a later date, restoring the correct coding sequence - many genomes are littered with transposons or “defunct” former transposable sequences Protein sequenceDNA sequence

Mutation rates vary… (1) among different species (2) between the sexes (3) among individuals (4) among genes - generally to mutations per cell division - can vary between individuals, depending on their alleles for: (a) DNA polymerase (some less accurate than others) (b) DNA repair enzymes (some less efficient than others) 

Linkage A B C a b c a B C A b c Crossing over between A and B is very likely to happen, so these loci are not linked Alleles will tend to get separated by recombination during meiosis When alleles are close together on a chromosome we call them linked, because crossing over is unlikely to separate them

Linkage When alleles are close together on a chromosome we call them linked, because crossing over is unlikely to separate them A B C a b c A B C a b c Crossing over between A and B is very likely to happen, so these loci are not linked Alleles will tend to get separated by recombination during meiosis

Chromosome rearrangements: Inversions When 2 double-stranded breaks occur in a chromosome, the part in between the breaks may flip around and get re-inserted This results in an inversion, where the gene order is reversed between the break points relative to the normal chromosome reversal of gene order DNA breaks

Inversions 2 Inverted regions cannot line up properly with the homologous chromosome during meiosis A B C D E F G a b c f e d g (1) regions will not align during synapsis, when homologs pair (2) crossing over would cause loss of part of the chromosome -so- (3) alleles f, e and d are now linked, and will be transmitted to offspring as one big “supergene”

Inversions 3 In natural populations, inversions are very common Studies on the fruit fly Drosophila have shown that natural selection favors certain inversions: - inversions that link alleles for small body size together are favored in hot, dry areas - inversions that link alleles for large body size together are favored in cold, wet climates 

Gene Duplication 1 Mutation can create new alleles, but how do you get new genes? Mistakes during meiosis can result in unequal crossing over, when a daughter chromosome inherits duplicated regions of a chromosome A B C for instance, crossing over between two transposons

Gene Duplication 1 Mutation can create new alleles, but how do you get new genes? Mistakes during meiosis can result in unequal crossing over, when a daughter chromosome inherits duplicated regions of a chromosome A B C A B B C

Gene Duplication 2 The viral enzyme reverse transcriptase can create gene copies from mRNA transcripts, which then get inserted back onto a chromosome A B C mRNA transcript for the A gene reverse transcriptase DNA copy of the A gene

Gene Duplication 2 The viral enzyme reverse transcriptase can create gene copies from mRNA transcripts, which then get inserted back onto a chromosome A B C mRNA transcript for the A gene reverse transcriptase DNA copy of the A gene inserts anywhere on any chromosome

Gene Duplication 2 The viral enzyme reverse transcriptase can create gene copies from mRNA transcripts, which then get inserted back onto a chromosome A B C mRNA transcript for the A gene reverse transcriptase DNA copy of the A gene = A B C A - random accidents can thus create functional copies of genes

Gene Duplication 3: polyploidy In plants, the production of diploid gametes during meiosis can result in tetraploid offspring - that is, offspring with 4 copies of each chromosome Tetraploid plants cannot produce viable offspring with normal 2N plants, but can breed successfully with other tetraploid individuals - new species can thus come into existence, as a population of plants that only interbreeds with itself

“Directed mutation” controversy First confirmation that mutation precedes adaptation came from Luria & Delbruck’s 1943 experiment, called the Fluctuation Test - tested two alternative hypotheses about how E. coli became resistant to a virus: (1) “acquired hereditary immunity” hypothesis: each E. coli cell has a small chance of surviving a virus, but survivors get “acquired immunity” that can be passed to their offspring - exposure to virus induces resistance (= adaptation) -

“Directed mutation” controversy First confirmation that mutation precedes adaptation came from Luria & Delbruck’s 1943 experiment, called the Fluctuation Test - tested two alternative hypotheses about how E. coli became resistant to a virus: (1) “acquired hereditary immunity” hypothesis: each E. coli cell has a small chance of surviving a virus, but survivors get “acquired immunity” that can be passed to their offspring - prediction: in each tube, there will be a few cells that get resistance, but only after they are plated out with the virus

“Directed mutation” controversy (2) “mutation” hypothesis: in some tubes, a random mutation will happen early on & get passed to most offspring, prior to virus exposure - will give rise to occasional “jackpot cultures” that luckily got the resistance mutation early in their family tree - prediction: there will be wildly different #’s of resistant colonies from different starting cultures, depending on how early the mutation occurred

“Directed mutation” controversy Lederberg & Lederberg (1952) – replica plating experiment Put a cloth over a “master plate” covered in bacteria; blotted it onto many replica plates coated with virus master plate Virus-resistant colonies appeared in the same place on each replica plate - thus, those colonies grew from resistant cells that were already present on the master plate - resistance was not induced by the virus...

“Directed mutation” controversy Cairns et al. (1988) argued in the journal Nature these experiments didn’t give cells a fair chance to “try to mutate” to survive, because the virus killed cells that were not already resistant Took cells that had a frameshift mutation in their lac gene, needed for growth on medium where lactose is the only energy source - normally, cells mutate from lac - to lac + at a frequency of 1× ×10 8 lac - cells growing in tube of normal media lactose plates – 3 colonies grow (they’ve mutated to lac + ) no lactose – no colonies grow

“Directed mutation” controversy Cairns et al. (1988) found that after mutants sat on lactose-containing plates for 2-4 days, many more colonies suddenly started to appear - only showed up if cells were sitting on lactose plates - were times more frequent than normal - no increase in rate of mutation from Val S Val R, so there was no across-the-board increase in mutation rate Day 1Day 3Day 5 lactose plates no lactose

“Directed mutation” controversy Cairns et al. (1988) proposed that cells were somehow sensing that they needed to mutate their lactose-metabolizing gene to survive..... were able to “choose which mutations will occur” by directing mutation towards the broken lac gene - from , at least 5 papers argued for “directed mutation” when cells were grown on a nutrient they couldn’t use This directly contradicts the fundamental premise of Darwinian evolution by natural selection, variation precedes adaptation - make sure you understand why this was so controversial!!

“Directed mutation” controversy Anderssen et al. (1998) published the following model in Science to explain the observations of Cairns: (1) lac- frameshift mutation still produces 1% of  -gal enzyme encoded by wildtype lac+ allele (2) Cairns’ expt was done with the lac- mutation on a plasmid, which could increase the odds of gene duplication events (3) if a cell acquires a duplication of lac- allele, it can grow a little more because of the small amount of enzyme produced; the more duplications that occur, the more growth is possible lac- lac- lac-

“Directed mutation” controversy Anderssen et al. (1998) published the following model in Science to explain the observations of Cairns: (4) this leads to invisible “micro-colonies” of slow-growing cells with up to 100 copies of the broken gene.... meaning, 100 targets for random mutation - you’re 100x more likely to get a reverse-mutation to lac+ when you have 100 copies of the broken gene lying around ! (5) once the reverse-mutation to lac+ occurs, the extra broken copies of lac- are lost and the lac+ cell rapidly forms a colony Under this model, all mutations happen by chance; duplications help the cell by producing more  -gal enzyme, which leads to growth & increases the odds of getting a lucky lac- lac+ mutation

“Directed mutation” controversy Anderssen et al. (1998) confirmed experimentally the predictions of their model, which the “directed mutation” hypothesis did not predict - residual  -gal enzyme activity was necessary in supposedly “lac-” mutants, or no late-appearing lac+ colonies ever appeared - early lac+ cells had up to 50 copies of lac- allele - the phenomenon required recombination, which is not needed to fix a frameshift mutation, but is needed for gene duplication This story is important because it shows that you can challenge the basic theory of evolution through scientific experimentation – but careful experiments done for the last 150 years have, so far, all supported the premise of Darwinian evolution by selection

Natural selection is the sole evolutionary force responsible for the adaptation of organisms to their environment Mutation is random with respect to the adaptive “needs” of individual organisms; you cannot “try” to mutate to survive Variation precedes adaptation Bottom line