KAR1 transfer of YACs from AB1380

Anthony Tomlinson tomlinson at pplros.demon.co.uk
Thu Jan 18 05:18:29 EST 1996


Hi,

I've been trying to use the protocol of Spencer et al (Genomics 22:118,
1994)
to transfer a 400Kbp YAC from AB1380 to YPH925 (Kar1).Basically the
technique takes
advantage of the phenomenon of cytoduction and the rare "chromoductant"
effect.
I have two questions/observations that, hopefully, someone can help me
with:

1. The YPH925 stab that arrived from ATCC was clearly non-clonal as it
had white
and red (ade-) areas. On streaking it was clear that I got white, red and
sectored
colonies. I have assumed that this is some effect of the Kar1 mutation, is
it? Is it possible to get a stable clonal culture of such a mutation? If I
colony purify, is there a simple way of testing for Kar1?

2. Upon mating my YAC strain (called HP8 for ease of writing/typing) and
plating
on ura drop out plates plus cycloheximide, I get a very high spontaneous
rate of Cy resistance. I do not see this with another strain containing
the
same YAC and made by traditional mating (to YPH499 or 500, I can never
remember their
mating types!). Is this something that others have seen? I don't think
its mentioned
in the paper. If AB1380 gives a high CyR background it makes the
technique a
hell of a lot more hassle.

Thanks in advance
Mike Dalrymple
PPL therapeutics Ltd
Scotland



More information about the Yeast mailing list