pombe disruption

S L Forsburg nospamforsburg at salk.edu
Mon Mar 29 10:34:12 EST 1999



> Gene disruption in S. pombe ?
> 
> Steve K. Cho (cho2 at simmons.swmed.edu)

> I recently disrupted my gene of interest (yfg1) which I thought it is
> not an essential gene. I knock out yfg1 in both haploid and diploid
> heterothallic strains.
> In haploids, the transformants grew slowly in the beginning but formed
> colonies. I selected colonies from h+ and h- plates and perform Southern
> analysis. At the same time, I checked mating type and ura4 gene
> insertion for those colonies by PCR. All of them are positive as I
> expected (showed either h+ or h- product and ura4 product).
> However, on the Southern blot all of them are diploids showing 2.2 kb wt
> allele and 4 kb knock-out allele.
> I re-checked mating type again with gDNA by PCR. It showed only one band
> indicating either h+ or h-. The morphology of cells ... I would say ...
> they are like diploids.
> So, my question is....
> 
> Can they become diploids with same mating type? either h+/h+ or h-/h-.
> I heard pombe likes being as a haploid and they don't mate with same
> mating type.
> So, what am I looking???

Yes, they can become homozygous diploids in a process called
endoreduplication--basically they miss out an M phase along the way.  It
occurs even without selection at a low but detectable frequency.  You
can see it if you simply plate cells to single colonies on
YE-phloxin--the occassional dark pink, diploid colony.  However, because
they are homozygous at the mating type locus, they are unable to sporulate.

The concern is how you managed to get so many of them.  Usually for an
essential gene, the result is no colonies with the correct insertion on
the haploid transformation plate.


> It looks like my gene is essential in S. pombe. I got later diploid
> knock out transformants (h+/h- diploid checked by PCR and Southern
> analysis) and failed to isolate a haploid by random spore analysis.
> 
> I am performing tetrad analysis now.

Good!  It is always the safest, and easiest in the long run, to go
through a diploid for gene disruptions.


-- 
-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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