protocols for determining strain ploidy and plasmid copy number

Michael Andres McMurray mmcmurra at u.washington.edu
Tue Jan 18 16:01:59 EST 2000


On 18 Jan 2000 Alpha1lab at aol.com wrote:

> I am looking for protocols for the following.  
> 1. Test for yeast strain ploidy, ie how do I verify a strain as being diploid 
> vs. haploid?

If it is diploid:
- it should not mate (test with PT1 and PT2 mater strains)
- it should show bipolar budding (as opposed to axial in the haploid)
- the cells should be larger than the haploid
- it sould be able to undergo meiosis (sporulation)

The first two could be artifically affected by mutations that derepress
the mating type loci, leading to "pseudo-diploidy".  Hopefully all four
will allow you to make a definite decision.

> 2. Plasmid copy number  determination of 2 micron based vectors in S. 
> cerevisiae

This was done in a relative way in Cell 29:585 (1982), by isolating DNA
from an equal number of cells and estimating by Southern the amount of 2
micron DNA present in each strain.  I'm not sure if there are more
quantitative ways; you should check J. Bact. 157:283 (1984), in which the
copy number of 2 micron-based vector plasmids was examined.
 
> Also, can haploid strains be transformed by standard tecniques, ie LiOAc?  
> Can I maintain a full length 2 micron plasmid vector in a circle + strain by 
> maintaining selective pressure?

Yes, haploids transform fine by LiAc.  Also, being circle + doesn't
significantly interfere with maintenance of vector plasmids with 2 micron
replication systems, to my knowledge; there could be an effect on the copy
number of the vector (since the endogenous 2 micron might be "counted"
during the copy-number control), but I don't think it's significant unless
you're doing very careful analysis.  I think someone has done the
experiments to test this, so a literature search might give more info;
again, J. Bact. 157:283 (1984) could help.

> 3. What's a good protocol for curing endogenous 2 micron plasmid to make 
> circle 0 strains?

http://ycmi.med.yale.edu/YGAC/curing2micron_info.html
Basically, overexpression of the FLP recombinase screws up 2 micron
copy-number control, and inhibits growth of cells containing the 2 micron
plasmid -- hence, a screen for cells that have been cured of it!

Good luck,
Michael

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