How can I disrupt ascospores of yeast?

Beth A.Montelone bethmont at ksu.edu
Mon Feb 7 11:24:38 EST 2000


>I am looking for a protocol to handle spores after I have mated two
>strains of yeast and induced sporulation.
>How can I get rid of remaining zygotes? How can I disrupt the 
>spores to grow them on selective medium?
>
>Any idea?

I am assuming that you cannot or do not want to dissect asci.  
This leaves you with random spore analysis.

I also assume that you initially selected the diploid by some sort of 
complementation selection by streaking on medium upon which 
only the diploid could grow.

According to Fred Sherman's chapter "Getting Started with Yeast" 
in Methods in Enzymology vol. 194, 1991, there are a number of 
ways to select spores and eliminate unsporulated diploids:

1) Sporulate the diploid on 2% (w/v) potassium acetate--this kills 
vegetative cells of many strains

2) Treatment with an equal volume of diethyl ether will preferentially 
kill vegetative cells

In either case, you would treat with glusulase to disrupt the ascal 
wall, sonicate lightly, and dilute and plate for single colonies.  
Those capable of mating would be haploids.

If your strain is heterozygous for a marker such as can1R, the 
diploids will be canavanine-sensitive, as will half the haploid spores, 
but the half that get the can1R allele will grow on canavanine.

Hope this helps.


Beth A. Montelone, Ph. D, Associate Professor
Division of Biology, Ackert Hall
Kansas State University
Manhattan, KS  66506-4901
phone (785) 532-6092; fax (785) 532-6653
web site http://www-personal.ksu.edu/~bethmont
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