espinoza at cgl.ucsf.edu (Hernan Espinoza) writes:
>Albert Spielmann <Albert.spielmann at bota.unine.ch> writes:
>>I'm working with the two-hybrid system from Clontech.
>>The yeast strains are CG1945 and Y190. Both have a ura3-52 deletion.
>>Normally they can't grow without uracil. But as said in the supplier's
>>protocol, it must grow!! Why?
> Your question is a bit ambiguous, under what conditions must the
>strains grow? I'm not familiar with the Clonetech system, but I would
>wager it's to select for a URA3 marker on a plasmid...
A followup to an email reply from David:
The genotypes of the strains in question are ura3-52 Ura3::GAL1uas
-GAL1tata-lacZ (for Y190) and ura3-52 Ura3::GAL417-mers3x-CyC1tata-lacZ
(for CG1945).
My theory:
The strains started off as ura3-52 (unable to grow on SD-ura) which,
if I remember correctly (and I'm sure I'll be reminded if I don't),
is a Ty insertion mutant. Integration of the lacZ constructs (which
carry a wild-type copy of URA3) at the URA3 locus rescued the ability
of the strains to grow on SD/-ura.
A diagram...(nowhere near to scale)
....LacZ.......
Plasmid (linearized): : :
: :
:== =URA3==>..:
X
Genome: ----------==++=URA3==>------ (++ = Ty insertion)
Result: ------===URA3==>......LacZ....==++=URA3==>-------
^^^^^^^^^^
Good copy
of URA3, thus
growth on SD-ura
Does that make sense? Hope this helps. -Hernan
