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How Random is Random Mutation?

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Below is the abstract of an article in the latest edition of PLOS Biology. The scientists developed a method by which they could compare ‘evolved’ strains from the pure strains with which they’re been crossed. Under duress–that is, deprived of a glucose environment, and forced to live on galactose–they found that when four different strains of yeast were distressed in this way, all four strains developed the SAME type of adaptation in the SAME gene (GAL80), a gene which, in normal environments, suppresses the ‘galactose utilization pathway’.

Think about it: ALL four ‘evolved’ strains basically hit on the same mechanism. We certainly have change (mutation), but is it ‘random’ if each of the four strains reacts in the same way? How probable is it for a mutation to occur in the same place in all four strains while causing the same changed metabolic pathway to be set in motion? Random mutation? I think not.

High-Resolution Mutation Mapping Reveals Parallel Experimental Evolution in Yeast
Ayellet V. Segrè1, Andrew W. Murray1, Jun-Yi Leu1*¤

1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America

Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2–24 kb (0.1–10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene.


Here’s a PDF link:

Here’s the html link:

I haven’t read the entire article as yet, but I will shortly. I look forward to your reactions and thoughts.

Just one final note: isn’t it wondeful how evolution is so supple that it can explain all things. All you have to do is INVENT WORDS!!!

Here we have “parallel” adaptation (why not call it non-random, or directed mutation? That’s what is happening after all.), and then there’s “exaptation” (which really means that some anatomical structure that has a normal usage in some class of animals is now being employed for a completely different function in a related species with no known way of explaining how it came about because there is a lack of intermediate forms, and thus, ‘selective pressures’ to invoke. It’s really no more than another way of saying, “We don’t know how this change came about”. It just sounds better) and then, my favorite, “co-adaptation” (which is something that normally functions in one part, or organ, of an organism, and which is now found functioning in another part/organ of the animal that is performing a completely different function within the organism. This, again, is a word that is invented to get around having to explain how it is that one kind of gene is producing two kinds of effects. But simply inventing a word really doesn’t add to our understanding, does it?). Isn’t evolution great?

Comments
moderator: looks like another comment I sent is stuck somewhere in the queue, or perhaps in the spam filer? Indeed. No longer. -moderator ofro
September 6, 2006
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DaveScot: “Where I think ofro is wrong is that there was any random mutatoin based evolution involved. Fungi adapt to different food sources and they don’t do it by accident. If a gene actually changed sequence to go from glucose to galactose I’d be looking for a mechanism whereby that mutation was directed. It was not likely serendipity that did it.” I think it boils down to what you mean with “directed” and “serendipitous”. But before I get to that, it is important to distinguish between adaptive mechanism and genetic changes in response to an altered environment. The cells certainly have built-in adaptive mechanisms. The regulatory mechanisms that turn/off the proteins metabolizing glucose and those that turn on/off galactose metabolism are such mechanism ( and these two regulatory mechanisms share some components, I believe). That is why the cells could be switched from one substrate to the other. The explanation for why these mechanisms exist at all is related to the cell’s overall energy cost: in the long run, it is energetically more economical to shut off a certain metabolic pathway if there is no substrate around as opposed to synthesizing the proteins and maintaining that pathway. That is different from genetically adapting to a new environmental situation, as imposed here experimentally. The result was a change of the properties of one of the regulatory proteins such that it didn’t shut off the galactose-utilizing pathway, or at least not as rapidly, so that it could be re-activated more readily. As I explained in my response to PaV, this mutation was, from what we have known about the regulatory pathways involving glucose and galactose utilization, an experimental expectation. (I think the authors were actually not out to demonstrate that but were looking for a different phenomenon that I haven’t tried to understand yet). Back to directed and serendipitous. It seems to me that you are looking for some sort of telic mechanism that predisposes the yeast cell to change its DNA sequence in response to an altered environment, beyond the RM&NS paradigm. The reason why I don’t think such a mechanism exists, or at least is not necessary, is the following. At the end of the cycling protocol, when a mutated cell was isolated from each of the four batches, there were three different strains with different galactose phenotypes (of the four, 2 were identical). What would happen if we put them back to a steady diet of glucose when they don’t need to handle galactose? My interpretation of your notion is that you would expect them eventually to switch back genetically to the original DNA sequence, or perhaps just stay the same genetically. The latter case will, in the long run give them an energetic disadvantage because now they are keeping the galactose-utilizing pathway on without getting anything out of it. The former case is unlikely. In microbiology, there is a long history of looking for mutants in a certain phenotype and subsequently for revertants, i.e. new mutants in which the original phenotype was restored. In the great majority of the cases, the mutation in the revertants was not restored, but instead a different mutation was found that changed the protein’s function to nearly the same as the original wild-type. The reason is that there are many ways to restore a protein’s function compared to the one way to return to the original condition. It is simple statistics. In the same way, there is a chance that, if you wait long enough, there will be a revertant with a galactose phenotype similar to the original one, but it will most likely not be identical to the original strain. According to your directed mutation scheme, I would expect a return to the original genetic state. If I misunderstood it, and you would not make such a prediction, then there is no experimental way to distinguish between the two models, and I would prefer the simpler mechanistic description I outlined above.ofro
September 6, 2006
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PaV: (sorry if this post is going to be lengthy) It really depends on what you consider as random. The results of the study show that the best way for the yeast to switch faster to a galactose-utilizing metabolic pathway was to mutate the Gal80 protein, and to mutate it only in a certain ways. In that sense the mutation wasn’t random. However, that does not mean that the mutations were non-random in the sense of being targeted. If I let the cells grow for 700 generations, there are bound to be a small number of mutations in the genome of zillions of yeast cells, and these mutations will be randomly distributed throughout the genome (or at least close to random; there could be – due to some special DNA-structural properties – some regions with a higher and some regions with a lower mutational probability). But we would never detect these random mutations with the protocol used, since at the same time the cells were subjected to a nutrient-switching protocol. The beauty of natural selection is that only mutations that permit the cells to grow and multiply faster will have an advantage over the other cells. Damaging mutations will either kill or impair cells, and neutral mutations won’t have a consequence on the ability to switch to galactose. So among all the random mutations that occurred, this artificially imposed environmental stress selected, by necessity, those that gave the cells a selective advantage. That is why the results give the appearance of non-randomness. There is an additional level of random vs. non-random. We know now that the major advantage for a cell prospering in this protocol is achieved if a certain protein (called Gal80) is mutated. Are the mutations randomly distributed within this protein? This is preliminarily answered by asking: could a mutation anywhere in this protein give a selective advantage? The answer is no; if a mutation were neutral, there would be no selection. The only mutations that could be detected are those that downregulate the protein activity (and therefore de-repress the galactose pathway). And indeed, the experimental observation is that among the four independently raised strains there are three different sets of mutations in “conserved sites.” The implication of “conserved” is that among all the analogous Gal80 proteins found in other yeast strains that can differ in their sequence by as much as 60%, these amino acids are always the same because changing them would destroy function. So you would expect mutations in such critical sites. Two mutants have the same mutation in a single amino acid, and the other two are double-mutants where two amino acids were changed. Four of these amino acid sites were known from other experiments to weaken the repressor function of Gal80, one directly and three by not being able to go the nucleus where the protein is supposed to bind to DNA. So even though the results appear non-random to the casual observer, they were random. The five critical amino acid locations detected here were random selections from among the number of possible critical sites (I don’t know the exact number). Five of them were found, perhaps more would be found with an extended search.ofro
September 6, 2006
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The (highly scientific) language used in the article and in some of your posts exceeds my knowledge, so it may be I didn’t understood all that genetic stuff, but… I read all the posts, and I didn’t find any clue about WHAT triggered the mutations… I mean HOW the yeast’s DNA “knew” that there is a need for mutations… My logic (maybe be wrong, nevertheless) says to me that the yeast somehow DETECTED the environmental change and somehow triggered some VERY SPECIFIC type of mutation, and this had an immediate effect on adapting the bug for this new environment… Please correct me if I’m wrong…Sladjo
September 6, 2006
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HodorH "I think it’s misleading to call it directed. Accelerated would be more clear." I think it's misleading to call it random mutation. Induced mutation would be more clear. In fact the researcher at Scripps who discovered the LexA pathway in e.coli calls it induced mutation. http://biology.plosjournals.org/perlserv/?request=get-document&doi=10%2E1371%2Fjournal%2Epbio%2E0030176DaveScot
September 6, 2006
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PaV wrote: "And, for a missense mutation, a minimum of two nucleotide errors is needed. And, these mutations have to occur next to each other....... So, tell me, how did these mutations–all in the same GAL80 gene–come about ‘randomly’?" I'm not sure why you claim that a missense mutation requires two "nucleotide errors". It doesn't. And, in fact, the mutations listed in the four strains only required one mutation each to make missense mutations. The rest of your calculation assume that the specific mutation would occur in ONE yeast cell. There are preciously few experiments done using a single microorganism. A typical experiment would contain millions if not billions of cells. I don't think your numbers in post #8 bear out the fact that we’re not dealing with anything random here.Hawks
September 6, 2006
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PaV - where do you get the idea that two mutational events are needed to get a mis-sense mutation? I would have expected that they would most commonly arise when the wrong nucleotide is added at replication, which would be a single event. BobBob OH
September 5, 2006
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PaV writes:
Michael Denton would likely agree with you. But he takes a Platonic view of all of this and takes the position that all of creation is “optimal”, and therefore directed by the “natural laws “ (with the presumption that God is responsible for those laws) to their rightful “end”. But I have a feeling you wouldn’t go along with him here. Am I right?
No, I'd say I agree with Denton more or less on this point. Almost. All of creation is optimal for fulfilling the Creator's purposes except those with free will (probably only humans).
HodorH further wrote: That said, if you’re telling me that these positive mutations occurred at a higher rate where they became beneficial to the yeasts than elsewhere in the genome, then I’m telling you to go back to the lab and do that experiment. Sorry, but I’m not getting the point you’re trying to make. Perhaps you can rephrase it.
Without good evidence, we should assume that mutations occur in this gene at the same frequency as in other genes. This paper offers no such evidence.
That’s not quite accurate. The mutations occurred in the same gene (GAL80), but the number of missense mutations differed between the four strains.
Two of the four strains had the same mutation. The other two had 2 mutations each, all different from the shared mutation from the other two.
An environmental stress is imposed on each of these four strains of yeast. Each of these strains developed a missense mutation(s) in the GAL80 gene. The article says that were dealing with an area of about 1kb, i.e., 1000 nucleotide bases. Now the length of the yeast genome is probably 10^8 nucleotides long. The 1kb therefore represents 10^3 nucleotide bases. The mutation rate of eukaryotes is around 10^-8 errors/nucleotide base. So, roughly, every time it replicates ( and since its genome size is 10^8), 1 error (mutation) occurs. And, for a missense mutation, a minimum of two nucleotide errors is needed. And, these mutations have to occur next to each other.
I'm afraid this isn't right at all. Missense mutations only require 1 base pair to change. Where did you get this idea? It would be a good idea to track down the source of this misconception; basic errors are the last thing we need.
So, the odds of getting ‘one’ mutation in the 1kb region, that is, the ‘first’ mutation (error), will take, on average, at least 10^5 generations of the yeast (and thus surpass the number of possible nucleotide sites that lay ‘outside’ the 1kb region). Now, to get the ’second’ mutation, it isn’t sufficient for it to lie in the 1kb region; it needs to be ‘next’ to the ‘first’ mutation. So, if the genome size is 10^8 bases, and the error rate is 10^-8 errors/base, then, again, we get 1 error/replication (generation). So now we need 10^8 generations for the ’second’ mutation to occur since there is only one place in 10^8 locations that lie ‘next’ to the ‘first’ mutation). If we assume that the generation time of yeast is 20 minutes, 10^5 generations represents (10^5 generations x .333 hrs/generations)/24 hrs/day/365 days/yr=3.8 years. 10^8 generations is a thousand times more; so it represent 3800 years. So, tell me, how did these mutations–all in the same GAL80 gene–come about ‘randomly’? Has the lab been in existence for 3800 years?
Hmm, you seem to be doing calculations for a constant population size of 1. My above objection obviously applies to this section as well.HodorH
September 5, 2006
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DaScot says
Regardless, directing mutations at a few genes vs. a large number is the difference between hunting with a rifle and hunting with a shotgun. Both are still directed methods.
More like an atomic bomb. There's no evidence from either paper that ANY specific genes or chromosomal regions were targetted. I think it's misleading to call it directed. Accelerated would be more clear. PaV, my response is forthcoming, but may take a bit longer.HodorH
September 5, 2006
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hodor "Increasing the rate of mutation is not the same as directing the location of mutations." I don't entirely agree. I doubt the mechanism in question accelerates mutations in critical sequences like those that define the genetic code. That's certain death. So there is probably at least some very rough targeting. Regardless, directing mutations at a few genes vs. a large number is the difference between hunting with a rifle and hunting with a shotgun. Both are still directed methods. Turning up the mutation rate in response to the environment is, in and of itself, directed mutation.DaveScot
September 5, 2006
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To Hawks, and ofro, and HodorH: please read my last post. I think it's obvious that, as I stated in the post, MUTATION has happened, but the question is whether it was 'random' or not. I think my numbers in the last post bear out the fact that we're not dealing with anything random here. But if I'm wrong, then please point out where I have erred.PaV
September 5, 2006
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HodorH wrote: All mutations are directed. They are directed by the laws of the created universe, in which their organisms reside. Michael Denton would likely agree with you. But he takes a Platonic view of all of this and takes the position that all of creation is "optimal", and therefore directed by the "natural laws “ (with the presumption that God is responsible for those laws) to their rightful "end". But I have a feeling you wouldn't go along with him here. Am I right? And, of course, you know full well what I meant by 'directed', so why the need for equivocation? HodorH further wrote: That said, if you’re telling me that these positive mutations occurred at a higher rate where they became beneficial to the yeasts than elsewhere in the genome, then I’m telling you to go back to the lab and do that experiment. Sorry, but I'm not getting the point you're trying to make. Perhaps you can rephrase it. And lastly HodorH wrote: You should also note that not all 4 strains received the same mutation. That's not quite accurate. The mutations occurred in the same gene (GAL80), but the number of missense mutations differed between the four strains. Let me, however, just verbally clarify things here: An environmental stress is imposed on each of these four strains of yeast. Each of these strains developed a missense mutation(s) in the GAL80 gene. The article says that were dealing with an area of about 1kb, i.e., 1000 nucleotide bases. Now the length of the yeast genome is probably 10^8 nucleotides long. The 1kb therefore represents 10^3 nucleotide bases. The mutation rate of eukaryotes is around 10^-8 errors/nucleotide base. So, roughly, every time it replicates ( and since its genome size is 10^8), 1 error (mutation) occurs. And, for a missense mutation, a minimum of two nucleotide errors is needed. And, these mutations have to occur next to each other. So, the odds of getting 'one' mutation in the 1kb region, that is, the 'first' mutation (error), will take, on average, at least 10^5 generations of the yeast (and thus surpass the number of possible nucleotide sites that lay ‘outside’ the 1kb region). Now, to get the 'second' mutation, it isn't sufficient for it to lie in the 1kb region; it needs to be 'next' to the 'first' mutation. So, if the genome size is 10^8 bases, and the error rate is 10^-8 errors/base, then, again, we get 1 error/replication (generation). So now we need 10^8 generations for the 'second' mutation to occur since there is only one place in 10^8 locations that lie ‘next’ to the ‘first’ mutation). If we assume that the generation time of yeast is 20 minutes, 10^5 generations represents (10^5 generations x .333 hrs/generations)/24 hrs/day/365 days/yr=3.8 years. 10^8 generations is a thousand times more; so it represent 3800 years. So, tell me, how did these mutations--all in the same GAL80 gene--come about 'randomly'? Has the lab been in existence for 3800 years? I think you see my point here. I welcome your response.PaV
September 5, 2006
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Ofro is sort of right. Fungi are masters at optimizing digestive enzymes for the substrate they find themselves on or in and they have a large repository from which to choose the kind and amount of enzymes. Labs where commercial strains of mushrooms are kept in culture know this and it's common practice to both periodically change the agar recipe so they don't become adapted to potato dextrose and stop producing enzymes to digest malt extract (for instance) and to put a small amount of their fruiting substrate into the agar (sawdust or straw for example) so they continue producing the proper enzymes to digest those lignin rich nutrient sources. Once they've adapted to any particular nutrient source it can take quite a while for them to adapt to something else. Where I think ofro is wrong is that there was any random mutatoin based evolution involved. Fungi adapt to different food sources and they don't do it by accident. If a gene actually changed sequence to go from glucose to galactose I'd be looking for a mechanism whereby that mutation was directed. It was not likely serendipity that did it.DaveScot
September 5, 2006
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PaV: I think you fell prey to a thorough misunderstanding of the experiments. You are interpreting the fact that all four “evolved” yeast strains had a mutation in the same gene was due to some form of directed, non-random event. The reason why the mutation occurred in one gene is one of experimental necessity. Just look at the conditions to which these yeast strains were conditioned. They were subjected to a protocol involving 36 cyclical changes of the growth media between containing either glucose or galactose as energy source. To go from metabolizing one sugar to metabolizing the other, the yeast has to turn on one metabolic pathway and turn off the other. Looking for a phenotype that exhibited a faster metabolic switch to galactose, the researchers found that all four strains had a mutation in a gene that regulates the expression of the galactose utilization pathway. (It normally suppresses the galactose pathway, and the mutations reduced the repressor activity). Now let’s ask where you would expect a beneficial mutation to occur. Take a random pick among the thousands of yeast genes. Would you expect that a mutation in a DNA repair or a drug resistance or an amino acid metabolic mechanism have a selectable effect on the galactose response? Or would it be more reasonable to expect a mutation in one of the regulatory proteins involved in turning on the galactose pathway? Nothing mystical about it.ofro
September 5, 2006
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If e.coli, a lowly bacteria, can control mutation rate in certain genes in response to something in the environment you should be reluctant to deny the same capability to other organisms which are equally or more complex without evidence.
Unless I misread, those mutations are not directed. Increasing the rate of mutation is not the same as directing the location of mutations. I also would expect bacteria to have more adaptable genomes, as (some of them) they persist in varied enviroments, and thus must be able to adapt quickly.HodorH
September 5, 2006
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Hodor - read this before you further dismiss directed mutations. If e.coli, a lowly bacteria, can control mutation rate in certain genes in response to something in the environment you should be reluctant to deny the same capability to other organisms which are equally or more complex without evidence.DaveScot
September 5, 2006
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PaV wrote: "Think about it: ALL four ‘evolved’ strains basically hit on the same mechanism. We certainly have change (mutation), but is it ‘random’ if each of the four strains reacts in the same way? How probable is it for a mutation to occur in the same place in all four strains while causing the same changed metabolic pathway to be set in motion? Random mutation? I think not." The article in question says also: "We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment." Since the adapted yeasts strains had already gone through SELECTION for the trait desired, it would hardly be surprising if any mutations discovered were not random. Your question "We certainly have change (mutation), but is it ‘random’ if each of the four strains reacts in the same way?" should really be restated as "Given that the four strains react in the same way, should we expect any mutations found to be random?" The answer is no.Hawks
September 5, 2006
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This could be (a definite could be) evidence for Dr Spetner's "built-in responses to environmental cues". Perhaps "Not By Chance" needs to be revisited...Joseph
September 5, 2006
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All mutations are directed. They are directed by the laws of the created universe, in which their organisms reside. That said, if you're telling me that these positive mutations occurred at a higher rate where they became beneficial to the yeasts than elsewhere in the genome, then I'm telling you to go back to the lab and do that experiment. You should also note that not all 4 strains received the same mutation.HodorH
September 5, 2006
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