Wolfram
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Help with isolating mutants
Do you have any idéa how it could be possible to isolate mutants (bacteria, yeast or HELA cells) that have mutated in the way so that they have
mutations that prevent further mutations? For example exelent DNA-repair systems or very accurate DNA-pols compaired to the wildtype.
As I know DNA pol makes about one misstake in each 100000 bp copied. Diffrent repair systems repair the wrong bps so that in the end there only is
about one misstake in each 1000000000 pbs. I have heard that its about the same mutationrate in almost all organism and if this is true one could
suspect that the DNA-copy and repair machinery has reached the maximum of its possibility in accuracy. So could it really be true that mutationrate is
the same in all organism?
If not do you have any statistics over which organisms get more mutations and which get less?
Thanx for any help.
[Edited on 14-2-2004 by Wolfram]
[Edited on 14-2-2004 by Wolfram]
[Edited on 14-2-2004 by Wolfram]
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chemoleo
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Hmmm,. that's a difficult question.
To answer your first question, I doubt it would be easily possible, if possible at all.
How would you select for mutants with a better repair system? You'd need to induce mutation with UV, chemicals etc and then screen billions of
cells many generations later for a marked decrease of DNA mutations (i.e. differences in DNA sequence compared to the original cells that were
mutated). That'd mean to sequence the whole genomes of billions of cells! Totally unfeasible for todays technology.
Regarding the accuracy of the DNA replication systems, yes, I also remember them being almost equally accurate in all organisms. However, I don't
know about the DNA Repair systems, a genome such as the human one is MUCH larger than the prokaryotic bacterial one. Greater success in DNA
repair should be required in organisms with bigger genomes, else you'd introduce many more mutations each time the cell divides than in organisms
with smaller genomes.
Also, a certain mutation rate isdesired, that is, if the DNA replication/repair mechanisms are TOO accurate, mutations will
not accumulate fast enough for the organism to evolve speedily enough! If an organism (unlike all the others) doesn't evolve as fast
(due to lesser mutations, resulting from a very efficient DNA repair/replication machinery), it is likely that faster evolving species would
eventually exterminate the slow-evolving species.
It's all in equilibrium, even in this case! Organisms that have a much greater mutation rate than the others may evolve faster, but more
will die due to lethal mutations (only very few of all mutations are beneficial, most are harmless (silent), and a few deleterious). If there are less
members of a species that can mutate (the previous round killed off many, due to a higher mutation rate), chances are that less members will survive
with beneficial mutations. One of those beneficial mutations would be, of course, a better repair/repication system - which would allow the
organism to replicate more and produce more viable offspring. That's how the species will return to equilibrium!
So - I wouldnt think that DNArepair/replication has reached it's maximum accuracy. There was just no evolutionary necessity for an even greater
accuracy, in fact a greater accuracy is undesired, for it essentially slows down evolution!
Are you wondering whether humans should get some kind of gene therapy to make their repair mechanisms/replication mechanisms more accurate? It would
be certainly beneficial to the individual, but most certainly it wouldnt be beneficial to the evolution of our own species!
But then, modern medicine essentially stopped a part of that progress anyhow...
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Wolfram
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How about this instead..
"Hmmm,. that's a difficult question.
To answer your first question, I doubt it would be easily possible, if possible at all.
How would you select for mutants with a better repair system? You'd need to induce mutation with UV, chemicals etc and then screen billions of
cells many generations later for a marked decrease of DNA mutations (i.e. differences in DNA sequence compared to the original cells that were
mutated). That'd mean to sequence the whole genomes of billions of cells! Totally unfeasible for todays technology. "
Something like this was also my first thougt but this is impossible to do.
There are more clever approaches i guess. Less mutations must mean a mutation in mutationrepair or DNA pol. gene, so no sequencing of total genome is
needed.
If you isolate diffrent populations of cells with mutations in this genes you can grow each mutant in colonies and scan short sequences of the genome
of the colonies for mutations. Less mutations means that the mutation was beneficial.
Or how about this: Shortlived organisms such as fruitflies, or C. elegans are habited in stessfull envirement with alot of radiation, UV-light and
mutagens those who thrive and survive the longest for many generations probably have better DNA repair then the rest. Since most DNA-polymerases are
quite similar the data could be usefull also for other organisms.
[Edited on 15-2-2004 by Wolfram]
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Geomancer
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Wolfram: you seem to be addressing to slightly different issues here: measuring mutation rate and selecting for it. Selection is clearly the easier of
the two--but still difficult. To do it naturally, you want to find an environment that the organism is perfectly adapted to already. Sex might screw
with this.
As far as measurment goes, I freely admit my ignorance of the teqniques in this field, but, if it's possible, you could try this:
Subculture each organism to create a separate population
Grow that population for a certain amount of time
Extract the DNA
Cut up into little bits and "unzip" to get single stranded DNA
Anneal back to double stranded
Some of the fragments will have a very hard time finding a partner. These will be the bits somehow affected by mutations. Presumably, the unpaired
segments could then be titrated. The result would have some bearing on the mutation rate.
Obviously, this is all theoretical. Expect to spend quite a while actually developing techniques to do this.
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Wolfram
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Answer
Organisms or cells which live under harsh mutagenic conditions will soon accumulate beneficial mutations that improve their dna repair system.
You could screen those cells for this mutations. Once you find a cell with a beneficial mutation you can culture this cell and compaire the
mutationrate in this population with the mutationrate in wildtype popultion. You dont need to scan the entire genome just a smaller part so that you
get statistic data enough to compaire mutant and nonmutant mutationrate.
[Edited on 16-2-2004 by Wolfram]
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Lugh
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I was wondering, how would you know what is a benificial mutation and how would you know where to look?
Also, if you suspected that there might be some cells with the mutation your looking for you'd have some job trying to isolate it.
If you treat the cells with harsh conditions you might also have trouble differentiating between mutations which affect DNA repair and mutations
which affect other proteins which might help it's survival e.g. heatshock proteins.
But then again the above might be just trash, cell culture is not really my area.
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Wolfram
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Say I got 1000 cells that have surived and thrived in the harsh conditions for a long time.
I would take each cell and first look for mutations in genes for proteins that are known to repair mutation or participate in the dna-replication. But
ofcourse I could be wrong, and there could be a mutation in some other place that affects the mutationrate. So once I have a possible DNA repair
beneficial mutation I take the gene and insert it in a wildtype cell. If the new mutant cell has lower mutationrate copaired with the wildtype the
mutation must be beneficial.
[Edited on 16-2-2004 by Wolfram]
[Edited on 16-2-2004 by Wolfram]
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chemoleo
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Yes that could work. I like your ideas Wolfram  - although I am sure this has
been done already.
Take Escherichia Coli, subject them to strong UV light so that 95% of all cells die.
Culture the remaining 5 % under normal conditions (so they can recover), and repeat process, so that 95% die again. By the end you should have
selected extra resistant cells.
I am not sure though whether you'd pick up many DNA polymerase/DNA repair mutants, though. This system is already highly developed/evolved, and
somehow I doubt that there are many more beneficial mutations. More likely for instance, is that the bacterium develops a specific resistance against
the radiaiton - such as producing more of a bacterial dye that would absorb most of the UV.
I am pretty sure if you took those UV-hardened cells and subjected them to a chemical mutagen such as 5-bromouracil, the vast majority of them would
die once again. Probably it would make sense to subject the cells to various mutagens, sequentially, and 'iterate' the system to
superresistant cells.
Still I have this nagging doubt that this won't work.
For multicellular eukaryotes, I should think chances are low you could ever get this to work. Firstly, you will deal with 10000s of fruitflies, not
billions of bacteria! Much less likely to find a beneficial mutation there. THen, the generation time is far too long. Also, repair mechanisms are
much more complex, and not all their respective proteins are known. If one fly survives, this could be due to hundreds of different mutations across
the genome, all acting together. Would be very hard to decide what the resistance is due to.
Anyway, why dont u try it? grow a few thousand fruitflies in bottles, and add after some time some mutagen ( I can't think of one right now -
that would work well, and is OTC). If nothing happens, add more.
until flies start to drop dead. leave them, and grow the surviving ones in a separate flask. Repeat, and then send a specimen to a DNA sequencing
facility
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Wolfram
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Answer
Yes may bee multicellular organisms are not a good choice I think HELA cells or yeast would be the best. Once you have found a benefical mutation you
could look for similar sequences in mouse or fly. If this sequences are mutated you could possibly end up with a mutagen and radiation resistent
mouse.
I dont think the replication machinery is yet optimized since like you said yourself it is in equilibrum and there must be a small mutationrate for
spieces to evolve.
[Edited on 17-2-2004 by Wolfram]
[Edited on 17-2-2004 by Wolfram]
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chemoleo
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Ok you caught me on that one - I thought for myself lateron that the fact that
the mutation rate is desired, things are probably not optimised for a zero mutation rate.
But... a zero mutation rate probably does not exist.
So yes, let's assume it can be optimised.
How would you go about it when it comes to selecting for extra-resistant human cells?
HeLa cells (for those who don't know, they are cells from a lady called Henrietta Lacks, who died in the 50s due to ovarian/uterine cancer and
whose cells have been cultivated ever since - i.e. these cells will never stop dividing, no Hayflick limit and such) - HeLa cells are not natural, and
if they keep on dividing it could mean anything, but not necessarily a mutation in the DNA pol gene (in case of deliberately introducing mutagenss).
I dont know... it would be interesting to see (though somewhat sad) how the Tschernobyl victims managed... were there survivors that had mutations in
the aforementioned genes, and thus survived? In fact I think most died. Actually that's a pointless argument. You'd need a germline
mutation... to produce any effect in the offspring.
Anyway... if managed to create superresitant cells/organisms/mice/humans etc, I doubt it would be down to a single gene, and I doubt it would be down
just a few rounds of selecting... it'd need many many generations, and constant selective pressure!
In fact, I am thinking of trying this experiment myself... I got plenty of ecoli lying about! UV - no prob either... anyone cares to comment?
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Wolfram
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Hmmm..
Hmmm.. maybee I wouldn´t use the HeLa cell after all as you said they are already unnatural and mutated, and have as large genome as humans. I would
stick to yeast I suppose. After a while when many mutation resistent mutations would be known I would try to insert them in the equvalent proteins of
a mouse or a fly.
I guess if one mutation make a organism 5x more resistant to mutations and another makes it 4xmore resistent, the orgaism you get when you insert them
both would be 20x more resistant.
Now comes the interessting thing: There are theories about aging beeing a accumulation of mutations harmfull mutations, so if mutation resistant mouse
have longer lifespan than wild type that would support that theorie. If not it would cause serious problems for the suporters of the theorie.
"...mutation resistent mutations" that really sounds stupid but makes sence.
Now another question..
Have you heard of Deinococcus Radiodurans? Could you explain what this bacteria uses its repair system for? It appearently has an ability to survive a
radiationdose that "shatters glass", accoring to my microbiology book. I see no use for this ability in normal life so why does this
organism use resources to maintain such a rapair system?
[Edited on 18-2-2004 by Wolfram]
[Edited on 20-2-2004 by Wolfram]
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guaguanco
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This board seem to be swarming with mutants, and they seem pretty isolated to me. 
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chemoleo
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Wolfram, that's very interesting what you mention. I will have a look into it. Sounds like a fascinating organism.
Guaguanco.... what are you on about? Sorry I dont get the joke.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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guaguanco
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It's just a little joke. Not much to get. 
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j_sum1
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