Pyrovus
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Inducing mutations
I'm planning to do some accelerated evolution experiments (probably with yeast), and to do so I'm going to need a way to increase the number of random
mutations.
Unfortunately I don't have access to a source of ionising radiation (which would probably be the simplest means), so chemical means would probably be
the most practical.
One possibility which springs to mind is Fenton's reagent, which produces hydroxyl radicals and hydrogen superoxide:
Fe(2+) + H2O2 -> Fe(3+) + OH- + OH.
Fe(3+) + H2O2 -> Fe(2+) + HO2 + H(+)
However, I imagine H2O2 would probably be pretty toxic to yeast, plus with all that ethanol for the H2O2 to oxidise it might not be all that
efficient. Also this method would generate OH/HO2 in solution, which might have difficulty lasting long enough to get into the yeast cell and interact
with the DNA.
Any thoughts/ideas?
Never accept that which can be changed.
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not_important
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`Twould kill the yeast too readily, even though it will make mutations. UV light is better, as are alkylating agents such as methyl iodide or
sulfate, nitrosoguanidine, mustard gas, caffeine, colchicine, bromouracil, aminopurine, nitrous acid, acridine orange, proflavin, ethidium bromide.
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unionised
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Actually, yeast is quite well adapted to the presence of H2O2 because it catalyses the decomposition very effectively.
I don't generally recomend the use of mutagens at home- because thery are mutagenic.
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nitro-genes
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Hmmm, never seen musterd gas in any supplier catalogue, although a real mad scientist makes that himself of course...
IIRC, the ability of yeast to breakdown H2O2 depends mainly upon the amount of catalase which is present in the peroxisome which in turn depends on
the subtrate the yeast is growing on. Another problem may be that hydrogen peroxide is a know inducer of PCD in yeast, although this might just be due
to the damage it causes to the DNA. It is known that most mutagen treatments have a high lethality percentage...
A mutagen that might be usefull, and is very common is EMS (ethylmethane sulphonate). It causes transversional point mutions of mainly G->C and
A->T throughout the DNA, so it is not completely random. Especially if you are looking for an evolutionary model this migh be a problem, since
transitions are much more common to occur naturally, because these are purine->purine and pyrimidine->pyrimidine changes. In real life mutations
aren't limited to point mutations alone, so I'm not sure if there are any chemicals that can simulate this diversity, since most favor a particular
type of mutation...
So it all depends a bit on the goal of your experiment? 
[Edited on 25-9-2006 by nitro-genes]
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Twospoons
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Saw an article in New Scientist, where a researcher found that the mutation rate rises in response to cellular stress, with the mutations mostly
occuring certain parts of the DNA which were prone to change. So it may not be necessary to add a mutagen - just apply the stress to drive your
evolution in the direction you want, and the mutation rate will rise naturally.
Helicopter: "helico" -> spiral, "pter" -> with wings
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not_important
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Indeed, stress the beasties and when they get tension headaches, give them headache meds with aspirin and caffeine which also causes mutations. You'll
be turning out comic book super-heros in no time.
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nitro-genes
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| Quote: | Originally posted by Twospoons
Saw an article in New Scientist, where a researcher found that the mutation rate rises in response to cellular stress. |
That's interesting, I mean stress simply is the result of an organism to be unable to adapt to it's environment. From an evolutionary perspective the
increasing mutation rate under stressfull conditions could be a major driving force to help the species adapt to the stressfull conditions...
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Twospoons
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Precisely. Thats what made the article so interesting - it explained how the genetic material of the offspring could be different to parent(s), and
how new genes could spontaneously come into existance in an effort to deal with some new environmental factor. Another anti-evolution argument shot
down ...
Helicopter: "helico" -> spiral, "pter" -> with wings
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crazyboy
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What would be a good OTC source of UV light capable of inducing mutations in microorganisms? What type of stress exactly? High alcohol environments,
high/low temps?
I plan on making a yeast strain with high alcohol tolerance (similar to WLP 099) I plan to add yeast to a mixture of alcohol and water than propagate
the yeast that survived, add them to another alcohol water mixture with more alcohol and repeat. I believe a mutagen would be useful in this case but
it must be fairly mild or the yeast might die.
Any ideas? Ideally the mutagen would be UV radiation or ionizing radiation or possibly high temperatures (or anything that is safe to consume).
[Edited on 31-5-2009 by crazyboy]
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chemoleo
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I remember such research being conducted with E coli: Here, the stress was nutrient deprivation and high temperature - the bacteria were continuously
grown in the same medium while the temperature was gradually increased over the course of several days from 37 to 42 deg C. These bacteria had between
~15 mutated genes in order to adapt, as was shown by sequencing afterwards.
As to doing this with yeast, I'd do it as you propose - increase the substance you are after, and see what propagates. Add antibiotics to prevent
bacteria from growing. I would not just use EtOH and H2O, you got to have essential nutrients (vitamins) in there, as well as buffer, and at least
some glucose.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Rich_Insane
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If I remember correctly NaN3 is a common mutagen used to induce mutations in a laboratory.
There are some companies that sell sterilizing UV light, but that will kill most single-cellular organisms.
Benzene is a mutagen, as are many other aromatics.
If you want some form of ionized radiation, you could extract the radioactive compound in smoke detectors and build something, but I wouldn't
recommend it. I would go with NaN3.
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merrlin
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You might try cannabilizing a CRT or other electron beam source and pointing it at a thin target wall to produce x-rays. You would have to evacuate
the chamber, but the path length could be kept short to minimize beam scatter. It should provide a continuous duty source with a reasonable service
life. Acceleration voltage and beam current could be monitored.
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