Pombe question

SLF notmyaddress at hotmail.com
Mon Dec 18 13:24:38 EST 2000

> Hello, people!
> I have a classical yeast genetics question that I am not sure.
> The problem is I have a heterozygote knock-out diploid cells in h90 background (homothallic strain). {It's a long story why I didn't start with a heterothallic strain.}
> Anyway, this mating type switching causes some potential problem. So, I thought about crossing with heterothalic strain to move the knocked-out allele to the heterothallic background. Then, I got confused.
> h90 cells are h90 leu1-32,ura4D18, ade6-M210/h90 leu1-32,ura4D18,ade6-M210, KanMX6(G418 selection)::my gene.

I'm a little unclear how you are maintaining these cells as diploids.  pombe in general
prefers being haploid, and h90 cells are usually haploid.  They are simply switching, so t
hat any population contains a mixture of functionally h+ and functionally h- haploids that
can mate.  But they will only mate if starved, and immediately sporulate and form haploids again.

In the absence of complementing markers (e.g., ade6-M210 /ade6-M216) you won't have
diploids.  The strain you have given us the genotype for would not exist as a diploid since it
doesn't have any complementing markers.  Its real genotype is
h90 leu1 ura4 ade6 yfg1::kanMX
I suppose if your gene disruption is in an essential gene, you might be able to maintain this as a
diploid, but I'd wonder how you made it to begin with....

> I want to have cells in the genotype of h90(+) leu1-32, ura4D18, ade6M210/h- leu1-32,ura4D18, ade6M216 KanMX6::my gene. So, I can select the diploids using Ade marker. Then, I would like to isolate haploid cells of h- leu1-32, ura4D18,ade6M216, KanMX6::my gene. My gene is located on 2nd chromosome which mat1,2,3 genes are located. I got more confused at this point whether this can be done or not.

Simply being on the chromosome will not  affect segregation unless they are closely linked
on that chromosome.  Yeast are highly recombinogenic, and markers on the same
chromosome are usually genetically unlinked.  Even if two markers are tightly linked,
you can isolate recombinants (using random spore analysis, which allows you to assay
a large population).

I think you are misunderstanding  the nature of the h90. An h90 is just
another allele at the mating type locus, as is h- or h+.  The h90
strain can be treated as an h+ or as an h- in crosses, although it is so efficient
at mating with itself that you need to first make a diploid using complementing
markers.  That is, if you want to move your gene of interest from an h90 to an h+
background, you cross it  to an h+ strain with the opposite ade marker, select
diploids, induce sporulation, and screen for h+ kanMX offspring (this can
be done using random spores).  Half the offspring will be h+, and half
h90.  SImilarly, you can cross the h90 to an h-, make
a diploid, then sporulate.

More information can be found on our web site.
DON'T REPLY to the email address in header.
Use the one below, replacing AT with @
(this inconvenience is for spam control)
S L Forsburg, PhD  Associate Professor
Molecular Biology and Virology Lab
The Salk Institute, La Jolla CA

Women in Biology Internet Launch Page
"These are my opinions.  I don't have
time to speak for anyone else."


YEAST bionet newsgroup see: http://www.bio.net/hypermail/YEAST/
YEAST e-mail: messages sent to yeast at net.bio.net
subscribe: e-mail biosci-server at net.bio.net with: subscribe yeast
unsubscribe: e-mail biosci-server at net.bio.net with: unsubscribe yeast
YEAST on the WWW: http://genome-www.stanford.edu/Saccharomyces/VL-yeast.html
problems with the YEAST newsgroup? E-mail the moderator: francis at cmmt.ubc.ca

More information about the Yeast mailing list