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Disruption an essencial gene in diploid yeast

R.Stevens rstevens at u.washington.edu
Tue Apr 11 16:37:50 EST 2000


Hey Y.Chen,
	Sorry for the delay in this reply.

	As you have suggested, one possibility is that your gene exhibits
haploinsufficiency.  One copy of the gene is not enough in the diploid
strain.  The yeast genes that have this property include ACT1
(osmosensitive haplosensitivity, PNAS 81:4889-4893) and MLC1 (lethal
haploinsufficiency, JCB 142:711-722).
	The solution to making knockouts is to attempt the one-step gene
replacement in a strain that has a third, plasmid-borne copy of the gene.

Take care,

Rick

On 3 Apr 2000, Y.Chen wrote:

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

--------------------------------------------------------------------------
         @..@             Rick Stevens
        (\--/)            Department of Pathology
       (.>__<.)           Box 357705
       ^^^  ^^^           University of Washington
   ''''''''''''''''       Seattle, WA  98195-7705
--------------------------------------------------------------------------


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