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Gene disruption in S. pombe ?

Steve K. Cho cho2 at simmons.swmed.edu
Fri Mar 26 17:28:10 EST 1999


Dear yeast lovers,

I've been working with yeast only for a few months now. I got a result I
can hardly understand.
So, I need your help!!!

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???


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.

Any possible explanation and help will be welcomed.

Thank you for your time.

Steve.





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