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Disruption an essencial gene in diploid yeast

Y.Chen yiling at u.washington.edu
Sun Apr 2 20:04:30 EST 2000

Dear Yeast People,

I use a PCR URA3 linear cassette to disrupt an essential gene in diploid
yeast. This yeast strain has a point mutation in URA3 gene and turns out
a URA- background. The disruption of this gene is lethal in haploid yeast 

Theoretically, I should at least get some transformants which remain only
one wild type allele. I have been working on both Lithium Acetate and 
electroporation transformation methods for a while, but these methods never 
bring me the right transformants. I always get few transformants with the
homologous wild type allele but never get the heterozygous one with one
wild type and one marker exchanged allele.

So a question comes out of my mind. Is it possible that the phenotype is
lethal even with a wild type allele in diploid yeast cells? Could any one
suggest me some relevant references in this case?

So many thanks!

Yiling Chen
Department of Botany, Box 355325
University of Washington
Seattle, WA 98195-1800


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