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Lecture 25.pdf

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Biology (Sci)
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BIOL 202
Daniel Schoen

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Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 BIOL 202 - Basic Genetics Lecture 25- Mutations I: Types & Consequences Weʼre going to cover mutations, gene regulation, !"#$%&"'()*'' bioinformatics, applying the info from those three +%$,-./'0'1'234"5'6'#./5"7%"/#"5' things. 8",9:/;5!"#$%"&'"("))"**+*,-"&'"./")'",,0+,/1 Weʼve heard a lot about alleles, phenotypes etc. But what we havenʼt really heard about is the o  Does mutation occur & what does it mean? origin of those mutations. So what are the o  Spontaneous mutations consequences for gene function. !  Types o  Types of point mutations !  Consequences on gene function Mutation & Hipfner Biol2202 Mutants We have a sort of apocalyptic and dark view of mutation. Mutation certainly has a dark side (cancer for example). But thereʼs also a benign side to mutation which is what we will focus on. For example, coat color in mice. Weʼve also Hipfner Biol3202 Mutation & Mutants talked about peppered moths in England. Darwinʼs finches are mutants to adapt to different environments and different food sources. Similarly, we have a bunch of mutations in a flower to make them more attractive or more accessible to pollination. So while mutations may be disastrous at the level of an organism, they might be disastrous at the level of a population to allow species to adapt. In fact, mutation is one of the two main forces Mutation & HMutantsBiol 402 that allows for adaptation and evolution. So mutation creates the variation and recombination o  Genetic variation between reshuffles the things into different combinations and thatʼs individuals arises from: how adaptation takes place. The point being if youʼre an 1.  mutation (new variants of organism, you want to avoid mutation but it has to be genes = alleles) tolerated at some low level in order for the species to survive. Weʼre going to talk about how you keep mutations to a 2.  recombination (different minimum but they still have to happen. So what are we combinations of alleles) talking about more specifically when we talk about mutation o  Allows for adaptation/ and mutants? Pretty obvious that weʼre talking about changes in DNA sequence which CAN lead to altered coding evolution or altered phenotype. But they donʼt have to. Vast majority of Hipfner Biol 205 mutations have not effect. Basically what happens is you have a normal gene product with a ▯ 1 Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 normal coding sequence and that gets translated into whatʼs considered a normal Mutation & mutants protein product (there can of course be many versions of normal) and ultimately to a normal phenotype. If you have a mutational event, itʼs going to have some effect on the product of the gene: can make a less active or non-active form of the protein or it can prevent production of the protein altogether. The end consequence of this is that you will get an altered phenotype. o  Mutation = change in DNA sequence that can lead to an altered coding or gene regulatory sequence & altered phenotype Mutation & mutants Hipfner Biol 2026 In general we can distinguish between two types of mutations (two locations where mutations occur): Somatic mutations (not heritable - not passed on to the offspring but o  Somatic mutations fall within ‘body tissue’ of course once a cell has a mutation itʼs going !  Non-heritable to pass that mutation on to all of the cells that o  Germline mutations fall within cells that make derive from it so in that sense, mutations at gametes the level of cells are heritable so if you get a !  Passed on to some progeny (one copy) mutation in a single somatic cell itʼs going to Hipfner Biol 272 result in a patch of mutant cells). You can imagine that if that mutation was in a tumor suppressor gene that that would now be a population of cells that is now susceptible to a second hit, to an inactivation of a second copy of the gene which could eventually lead to tumor formation. Other type of mutation that we talk about is germ line mutations which are passed on to the next generation. Usually, the mutation will only occur in one copy of the gene so from one mutated stem cell, all of the gametes that it gives rise to half those will pass on the mutation to the next generation whereas the other half will not. So the question is since cells need some level Is mutation a normal ongoing process? of mutation, is it happening all of the time? That would kind of suggest that yes it must be. So o  In the past, thought that genetic change may occur in what is the source of this mutation. There have response to selective pressure been two schools of thought about this: the first one says that mutations just happen that just a o  Luria and Delbrück (1943) – “Fluctuation test” for fact of life and theyʼre kind of spontaneous and spontaneous mutation vs genetic adaptation theyʼre happening all of the time. The other !  Compare bacterial resistance to viruses (bacteriophage) between many independently grown samples of bacteria school of thought is that mutations donʼt just happen all of the time, they happen in response to selective pressure. So things are pretty stable but then you challenge, you put stress on that organism and that stress is going to lead to mutations that produce the variability Hipfner Biol 282 that allow that organism to survive the stress. ▯ 2 Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 About seven years ago, Luria and Delbruck came up with the Fluctuation test to tell whether mutations occurred spontaneously all of the time or whether mutations are only occurring in responses to stresses. The basis for their assay is that they took advantage of bacteriophage. These are just viruses that infect bacterial cells and kill them. So normally if you took a growing culture of E.Coli cells and you would add in a bacteriophage, the bacteriophage would infect and kill them all and you would then plate them on an agar plate and you would expect to get no colonies - all of the cells would die. But inevitably when you do this kind of a test, you get a few colonies! So some of the bacteria manage to become resistant to infection by the bacteriophage and thatʼs because of genetic mutations. So Luria and Delbruck took advantage of this fact to use an assay to try and figure out when those mutations are occurring. They took a starter culture of E.Coli, grow out up and then split it evenly into many cultures (like 30 or 40 individual cultures) and then you grow those up and just before theyʼre finished, you add the phage, let them go for a couple of more divisions and then Is mutation a normal ongoing process? plate them and see what the effect of o  Luria and Delbrück (1943) – “Fluctuation test” for the phage on all of the different spontaneous mutation vs genetic adaptation cultures is. They basically had two o  Hypothesis 1: hypothesizes: Hypothesis one was !  If virus treatment leads to genetic adaptation – resistance that if it was the viruses that were actually inducing the mutation in develops only upon exposure to environmental challenge - expect similar # of resistant colonies in all samples some way then because all of the cultures were grown under identical conditions, youʼre putting in the exact to phaget same amount of bacteriophage into Phage each culture, you expect all of the added cultures to look more or less the same in terms of the number of colonies of bacteria that will become Hipfner Biol 202 9 resistant. So graphically it would look like the figure (the green indicates cells that would form colonies on a place and the white ones die). Because everything is essentially identical in those colonies, youʼll get similar numbers in each case. The alternative hypothesis is that if itʼs spontaneous mutation that is responsible for the Is mutation a normal ongoing process? resistance to the bacteriophage mutation, then in principle those mutations can happen at any o  Luria and Delbrück (1943) – “Fluctuation test” for time. So out of your 20 or 30 or 40 cultures, in spontaneous mutation vs genetic adaptation o  Hypothesis 2: some of them youʼre going to get a mutation very early on during the growth of those cells and all !  If resistance results from random spontaneous mutation, the cells that derive from that cell that acquired addition of phage & expect to see widely varyingring/after [fluctuating] resistance between multiple samples that mutation will all acquire the resistance to bacteriophage. In other cases, itʼs going happen in later generations. The principle is that if you to phaget challenge these 20 cultures with phage, if the Phage mutations were occurring spontaneously added throughout the growth, youʼll get huge variability Hipfner Biol 102 in the number of resistant colonies that can grow. Thatʼs why we call them fluctuation tests. You ▯ 3 Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 have fluctuating resistance. And thatʼs what the saw in the experiment. So in some colonies, you would have 250 resistant colonies and in some you would get none, some 10, some 40 - so it was all over the map. So that was the first formal proof that we are undergoing continuos spontaneous mutation. Thatʼs in bacteria though. Can we see that in humans too? Yes. Spontaneous mutations There are lots of different ways of measuring o  How often? Pretty rare, but detectible frequency mutations in humans. The classical one would be to look for the frequency or the rate at which you get spontaneous mutations occurring in people who have no family history of genetic diseases and you look at that over a huge population (like all over scandinavia). That gives you some idea of how often these mutations are happening without an obvious cause and if you look at this for a number of genetic disease, itʼs fairly consistent in that the range is somewhere close to 1 per 10 million gametes results in a mutation in a disease Hipfner Biol 20211 gene going up to 100 mutations per 10 million gametes. So the mutation rate is Spontaneous mutations somewhere around 1 in a million to about 1 in o  How often? Pretty rare, but detectible frequency 10 thousand. Now we donʼt have to estimate mutation rates you can actually go in and look at it much more directly. So the figure on the right is just a little report about a paper that was published not so long ago. The researchers found two men at a village in China whoʼs great great great great grandparents were siblings (back 13 generations). And since the early 1800s those families have always lived in the same village. So they took these two men and they We each carry ~150 new mutations in our genome sequenced 10 million bases on the Y from Biol 212123210.htm chromosome and then compared the sequences. And if you take two people that are that closely related and you do this you end up finding four mutations out of 10 million bases General types of mutations over 13 generations. So yes, spontaneous o  Spontaneous vs induced mutations do occur but they occur at a pretty !  Spontaneous – “background” level of mutations, no rare rate. In fact if we would sequence each of purposeful or accidental exposure to mutagen our genomes and compare us to our parents, !  Induced – treated with mutation-inducing agent odds are we have about 150 new mutations that didnʼt occur in our parents. When you consider o  Scale !  Point mutations – changes or addition/deletion of how little of genomic DNA is functional and one or few nucleotides coding, this is extremely low. !  Insertional mutations – insertion of large chunks of So we just talked about spontaneous mutations. DNA – e.g. transposable elements (Lecture 27) The other type of mutation that you get is called !  Chromosomal mutations – losing, gaining or swapping large bits of or whole chromosomes Hipfner Biol 132 ▯ 4 Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 induced mutation. These are mutations that occur in response to exposure to mutagens (will talk about this next lecture). We can distinguish between different kinds of mutations based on the scale (the size) of the damage thatʼs done. It ranges from just really tiny changes (point mutations) which is changes or additions/deletions of one or more nucleotides. We would consider these single gene mutations if they occur in a gene because itʼs going to be localized to one specific gene. Types of point mutations Thereʼs also insertional mutations. Will talk about this o  Base substitutions in two lectures. On the other !  Transition – Purine"purine; extreme of the scale thereʼs pyrimidine " pyrimidine chromosomal mutations. What weʼre going to be focusing on ! e.g. G#C"A#T in the next few lectures is point !  Transversion – Purine"pyrimidine; mutations. So just mutations pyrimidine"purine that affect a few bases. ! e.g. G#C"T#A Different Types of Point Mutations Pierce Fig. 18.3 o Base additions & deletions There are two different types. !  Insertions/deletions (indels) One is base substitutions which is changing one base for another (or one base pair for a See Fig 16-2 different base pair). These can Hipfner Biol 202 14 be classified in two ways - theyʼre either transition mutations which means that a purine gets substituted for a purine or a pyrimidine gets substituted for a pyrimidine. So G.C -> A.T would be an example (the little dot means “base paired with” and the arrow means “transitioned”). The opposite of that is a transversion which is when a purine gets mutated to a pyrimidine and vice versa. The other type of point mutation is called an indel mutation so an insertion or deletion. Thatʼs when you get one nucleotide inserted into a strip of DNA or you get one nucleotide deleted from a stretch of DNA. You can see that the difference between base substitutions is that the length of DNA is the same youʼve only swapped one for another. But in the case of insertions and deletions, youʼve changed the length of the DNA so that everything downstream of the inserted mutation is now shifted. This will be important later. How do we get these different types of mutations? We have base substitutions we have indwells - how at the level of chemical reactions in DNA, do these things take place? And when weʼre talking about spontaneous mutations, there are two main sources of spontaneous mutations. When would be the most sensitive time in terms of generating mutations in DNA? It would be during replication because youʼre now trying to make a copy of a chromosome and thatʼs an error prone point in a cellʼs lifetime. One of the main sources of spontaneous mutations are errors that are made during replication of the DNA. ▯ 5 Sarah Margareta Ibrahim▯ Wednesday, March 13th 2013 Origins of Spontaneous Mutations Origins of spontaneous mutations 1. Replication Errors - Base Mispairing 1.  Replication errors – Base mispairing !  mispairing of newly incorporated bases during DNA These are errors that are made during replication DNA replication. An obvious source of replication errors is mispairing of the !  Occurs frequently (1/10 synthesized nt) !  due to misalignment (“wobble”) and base ionization, bases. So if DNA polymerase comes along and incorporates the wrong base leading to altered base-pairing properties by mistake then youʼve got a problem. It ou does this pretty frequently. When DNA p is being replicated then about 1 out of …e every 10,000 bases or 1 out of every 100,000 bases something in that range, DNA polymerase makes a mistake. There a couple of different ways. We Pierce Fig. 18.10 and 18.11 think of base pairing as this perfect Hipfner Biol 20215 process where Aʼs get matched with Tʼs and Gʼs get matched with Cʼs but in reality itʼs not that nice and clean. But in fact the DNA double helix is kind of flexible and what sometimes happens is that the bases come together misaligned and the consequence of that is that you can get different forms of base pairing so T can base pair with G through two H bonds if you get a bulge in the helix and these things come in at the wrong angle. Thatʼs called wobble. Thymine and guanine wobble occurs in DNA just because the things donʼt line up. Thatʼs one source. Another source of that is different chemical forms of the bases. So this is the way that theyʼre always drawn and the way we always think of these four nucleotides (these four bases) but in fact, (these are the predominant forms that you will find in cells) but there are other forms of the bases. For instance, adenine can be protenated, gets a positive charge and thatʼs going to change its Origins of spontaneous mutations base pairing properties and because it 1.  Replication errors – Base mispairing has a positive charge where it does (the little red dot in the figure thatʼs !  Can lead to transitions and transversions circled) , it can no longer base pair strand new strand with Thymine and is instead going to
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