Lenore Neigeborn wrote:
> this new procedure for making gene
> replacements that involves PCR-ing a selectable marker using oligos
> homology flanking your locus on the 5' end and homology to the marker on
> end (thus when PCR is complete you have a fragment with homology to your
> on the ends and a selectable marker in the middle). Then you transform in
> fragment for a one-step replacement.
>RESPONSE FROM SANDY Silverman::
>>We've also had trouble with this. I think the key is to use a strain with
>a complete deletion of sequences of the marker (i.e. his3-delta-200, as in
>the Lacroute paper). The problem here is that the YPH499, YPH500 strains
>that have that allele are known to act funny.
We (Linda Riles, actually) have been using this method with great
success (i.e., most of the transformants go the the right locus). You are
right, Sandy, the key is to use a strain carrying a complete deletion of
the selectable marker, and we use his3-200. The bad thing about his3-200
is that the deletion removes some of the promoter of the adjacent gene,
PET56, and presumably reduces its expression. This causes the cells to
throw off petites at a higher than normal frequency (according to Piotr
Slonimski, who knows about these things, and verified ancedotally by us,
(and by many others, I'm sure)). This might be the cause of your strain
problems, Sandy. (Or do these strains "act funny" in other ways?) In the
long run we plan to solve this problem by making a different big deletion,
but for now the petite problem hasn't bothered us too much, so we continue
to use the his3-200 strains.
Department of Genetics Box 8232
Washington University Medical School
4566 Scott Ave.
St. Louis, MO 63110
E-mail: mj at sequencer.wustl.edu