pombe tetrads

S L Forsburg nospamforsburg at salk.edu
Wed Mar 3 09:37:00 EST 1999



> Jennifer Buslepp (buslepp at med.unc.edu)
writes

> I have a diploid S. pombe strain which is heterozygous for my gene of
> interest (also homozygous for leu 1-32) , where loss of this gene is lethal. I have
> been attempting to isolate mutants of my protein which fail to complement this knockout in
> the haploid, using a LEU plasmid containing my gene with random
> mutations. I have isolated several potential mutants using random spore
> analysis and am now attempting to verify these by tetrad analysis. I
> have been plating the tetrad spores on pombe minimal media,
> and I still do not see any growth after almost two weeks. I am certain
> that the yeast have the plasmid. My question is whether or not pombe tetrad
> analysis can be done on minimal media, and if not,
> can the analysis be done on rich media without loss of the plasmid? Any
> help with this will be greatly appreciated.

Two points:
1) Most plasmids are lost at high frequency during meiosis in pombe. 
The likelihood of recovering tetrads with your plasmid in the spores is
therefore very slight.  Thus the advantage of random spore analysis
(RSA) which allows you to sample sufficient numbers to isolate the few
that keep the plasmid.  In general, you can do linkage of this sort just
fine by relying on RSA and it is perfectly acceptable.  Since RSA is a
much more common technique in pombe than cerevisiae, pombe folk are
quite comfortable with results obtained from it.  (cerevisiae folk tend
to view it with more suspicion, but that's more a matter of sticking
with what you're used to, than anything else). 

In this case, where a plasmid covers the disruption in yfg1, you could
cross the yfg1::ura4 p(LEU2) strain to wild type and plate the spores
selectively to demonstrate that the the only ura4+ isolates must have
the LEU2 marker;  however, half the Leu+ guys should be ura4-.  

2) pombe will germinate fine on minimal, although the efficiency of
germination may be somewhat reduced relative to YE.


-- 
-susan
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S L Forsburg, PhD  forsburg at salk.edu
Molecular Biology and Virology Lab          
The Salk Institute, La Jolla CA 
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