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CEN sequences on plasmids (yeast)


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#1 Ikar

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Posted 22 November 2010 - 09:25 AM

Hi, my question is:

If I transform a plasmid (with an ORI) into a yeast cell the plasmid will replicate once per cell cycle.
How can I be sure that the daughter will get one of this two plasmids?
Therefore I need to integrate a CEN-DNA-Sequence in the plasmid, so the mitotic spindle
can be attached.
-> That is only a consederation of me. I would be happy if you could tell me whether I'm right or wrong!

br,
Sebastian

#2 HomeBrew

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Posted 22 November 2010 - 10:35 AM

In thinking about this, I'm not sure you need to worry about it, depending on your actual experiment. On your vector, you're likely going to have a gene that complements an auxotrophic mutation in your yeast host strain, in addition to a yeast ori, right? So essentially, this is the "everything else dies (or doesn't grow)" method of clone selection -- if a particular daughter cell fails to receive a plasmid, it won't grow on your plate, because it will still have the mutant auxotrophic phenotype. So, if your host strain, vector construct, and media are all correctly coordinated, any yeast cell that doesn't have a plasmid won't grow and thus can be ignored...

#3 Ikar

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Posted 22 November 2010 - 01:37 PM

I think that a daughter cell without a plasmid won't be a problem, because it would vanish because of the negative
selection pressure(auxotrophy) as you just said.

I think the problem would be a mother cell with a lot of these plasmids with my target gene.
This could lead to overexpression and falsify the experiment.

#4 HomeBrew

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Posted 22 November 2010 - 04:21 PM

In bacterial genetics, this is called a gene dosage effect and depends on the copy number of the plasmid in the host cell. I don't know what the copy number is of a plasmid in a eukaryotic host, but if it's not too high, it shouldn't be too much of a problem. What's out there in the literature? I'm sure there's a lot of examples of gene cloning in yeast, how was this effect controlled for?

#5 Ikar

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Posted 23 November 2010 - 01:48 PM

I think that without such a CEN sequence the spindle apparatus cannot attach.
Then plasmids should be given to the daughter at random.

I think it also depends on the structural condition how much the mother cell is able
to accumulate before sharing plasmids with daughter cells.

I have also read that special Plasmids (called ERC = extrachromosomal rDNA cycles) are
factors that can accelerate the aging process in s.cerevisiae. Besides that, experiments
are supposed to have proven that it is just the number of plasmids which is responsible for
the faster aging. It doesn't matter wether it's rDNA or another plasmid: high amount of plasmid = old cell.

PS: I am not doing any experiments yet. I am just a inquisitive student (and also annoying I think ;) )




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