In article <80rpjs$6fq at net.bio.net>,
Yeast Laboratory 1 <Yeast1 at demogr.mpg.de> wrote:
> Hi everyone,
>> I am having a problem with sporulating my cells. They simply do not
make
> spores. They two strains that I have tried to sporulate look like
diploids
> under the microscope (they are larger than haploids and they bud from
each
> pole). The genotypes of the strains are:
>> Strain one: Mat a/Mat alpha; ura3-52/ura3-52; trp1delta63/TRP1;
> leu2delta1/LEU2; his3delta300;HIS3; GAL2/GAL2 (S288C background)
>> Strain two: Mat a/Mat alpha; ura3-52/ura3-52 (S288C background)
>> Unless there is some problems with sporulation in a ura3-52, I suspect
it
> could be the type of sporulation media or even the enzyme (10%
> Beta-glucuronidase). Has anyone else encounted this problem?
>> Thank you in advance for your help!
> Christi
>gendron at demogr.mpg.de
Hi Christi!
Nothing in the strains' genotypes should inhibit sporulation. Your
message doesn't have enough detail about the sporulation procedure
itself. Some questions:
1. How did you "make" the diploid? Do you even know for sure that they
are diploid (i.e., should be sterile)? I don't think the fact that the
cells are larger than the haploids is enough. If you know they came
from zygotes, and they are sterile, then they ought to be diploid.
Also, sometimes the diploid has complementing markers, so you can select
for it.
2. Have you checked the sporulation medium-treated cells under the
microscope BEFORE Beta-glucuronidase treatment? If so, you should see
tetrahedral structures, the asci containing spores. Depending on the
strain, sporulation might take 1-5 days. How long did you let yours go?
3. What is the sporulation medium composed of?
4. I realize that this is a long shot, but it sounds from your message
that you might have put 10% Beta-glucuronidase INTO the sporulation
medium from the start. If so, then your strains probably will not
sporulate at all, since they have become speroplasts.
Ed Davis, Ph.D.
National Cancer Institute
Frederick, MD USA
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