elsasser at flash.net
Sat May 27 15:04:55 EST 2000
You might want to check out the papers from the Guarente lab on yeast aging. In a
1997 Cell paper (90% sure of the year, but it's absolutely a Cell paper and
Sinclair and Guarente), the authors demonstrated that episomal elements of rDNA
accumulate as yeast ages. This results from recombination of one or several 9.1
kB repeats looping out of the rDNA locus which contains 50 to 100 direct repeats
of the ribosomal DNA. As I remember it, the plasmid sizes that they were looking
at were in the 20 kB range (two repeats), and possibly higher. The supercoiling
was assessed by 2D gels (chloroquine in both dimensions, surprisingly enough, but
at different concentrations). The supercoiling was also assessed by treating DNA
preps with topoisomerase then running forever on a standard 0.7% agarose gel
(something like 30 or 40 hours at low voltage). There was a clearly visible shift
of the putatively circular version after topoisimerase treatment.
"Dr. R O'Kennedy" wrote:
> Thanks for the prompt reply.The host strains I'm am using need to be
> RecA- and rarely if ever do I get evidence dimer formation.
> Your suggestion was in the direction that I have been doing . Do you
> have info /ref on optmising chloroquine concentrations.
> rogier wrote:
> > Hi Ronan,
> > I'm using pBR as a supercoiling reporter. In my recA+ strain,
> > the plasmid sometimes forms dimers, which are about 8 kbp.
> > Although individual topoisomers are visible in the 8 k dimer,
> > differences in supercoiling between different samples are hard to
> > spot.
> > But you might give it a shot with low % gels and/or long run
> > times. With such a big plasmid the range of possible topoisomers
> > may be very wide, tho. So if you can use a smaller plasmid I
> > would try that first.
> > Good luck,
> > Rogier Stuger
> > Mol Cell Physiol, Free U of Amsterdam
> > E rogier AT bio.vu.nl
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