inconsistence in B-galactosidase activity
EA_Golemis at fccc.edu
Thu May 4 08:39:08 EST 1995
Dear Qing Lin,
This problem is occasionally seen in two hybrid systems. It is
more or less protein-specific: ie, some DNA-binding domain fusions
will give absolutely consistent results, colony to colony, transformation
to transformation; while others will be extremely heterogenous. The
most frequent class of variability that I've seen out of inspecting >>
200 LexA-fusion proteins is that a high percentage of individual colonies
will produce either -no- beta-galactosidase activity or very little
betagal activity versus seemingly similar colonies that produce moderate
to high levels of activity. In at least some cases, the heterogenous
phenotype correlates with a protein that is somewhat toxic to yeast.
In a lot of cases, if you look by Western blot to check levels of the
DNA-binding domain fusion in the high versus low beta-gal clones, the
low beta-gal clones are making 1. little protein 2. no discernible
protein or 3. anomalously migrating protein. In these no-betagal clones,
you can recover perfectly normal plasmid, retransform it, and again
produce the heterogenous phenotype: so how the shutoff of the fusion
is occurring is anybody's guess. Again, from experience, once you've
identified individual clones that are making protein and have appreciable
beta-gal levels, they tend to be quite stable.
If you are doing the experiment using a dual selection (ie, with
the DNA binding domain also driving expression of the LEU2 or HIS3
gene), it is generally informative to correlate your betagal and
growth phenotypes - you get a lot of information that way.
C572794 at MIZZOU1.MISSOURI.EDU (Qing Lin) wrote:
> Dear Netters:
> Hi, everyone. I got inconsistent results of B-galactosidase activity in
> both liquid and filter assays. The difference were among different banches of
> transformation with same two-hybrid plasmids and among different transformants
> of a single transformation. Is it a rare or common problem? Most important,
> how to eliminate the problem? Every suggestion is welcomed and well appreciated
> You can mail to me or to the network. THANKS.
> Qing Lin
> Div. Bio. Sci.
> Univ. of Missouri, Columbia
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