Titering Gal1 promoter

Arle Kruckeberg arle at biovax.leeds.ac.uk
Fri Sep 1 12:16:55 EST 1995


In article <jjschm-310895165456 at jjschm.monsanto.com>
jjschm at ccmail.monsanto.com (Jon Schmuke) writes:

> In article <424qdb$opj at swsu65.swmed.edu>, Mark Walberg
> <walberg at simmons.swmed.edu> wrote:
> 
> > Regarding titering the GAL1 promoter with mixtures of glucose and 
> > galactose:  I think that this is going to be very difficult to do in a 
> > controlled way.  In such mixtures I would expect a diauxic growth 
> > pattern with consumption of glucose first, then as glucose concentration 
> > falls the GAL1 promoter will become activated.
> 
> I suspected this would be a problem.  In the first experiment I used 4%
> raffinose in the presence of increasing amounts of galactose.  Invitrogen
> suggested using raffinose as a non-repressor/inducer source of carbon. 
> Since the gene is essential, the increasing level of galactose won't
> enhance growth unless the enzyme level is increased via the GAL1 promoter. 
> The cells wil not grow in raffinose alone but grow at the same rate with
> 0.2% or 2% galactose.  If the enzyme titer need only be a few copies or if
> the GAL1 is an "all or nothing" promoter this might make sense.  I was
> hoping that perhaps I could titer it better by repressing with glucose, but
> perhaps switching to another promoter would be better.  CUP1 was one
> suggestion.

Non-metabolizable analogs of glucose or galactose could act as
gratuitous repressors or inducers, respectively, and give you control
over the gene that is independent of the assimilation of the compound.

Non-metabolizable glucose analogs include: 
2-deoxyglucose, which is generally toxic to the cell, and 

5-thio-glucose (reference Egilsson, V., V. Gudnason, A. Jonasdottir, S.
   Ingvarsson, and V. Andresdottir. 1986. Catabolite repressive effects
of 5-thio-D-glucose on Saccharomyces cerevisiae. J. Gen. Microbiol.
3309-3313.

Non-metabolizable galactose analogs include:
D-fucose and L-arabinose (these are gratuitous inducers of the
galactose transport system in some cerevisiae strains; whether they
work on the Gal1 promoter I don't know, but galactose transporter
expression is Gal2-dependent). Reference Cirillo, V.P. 1968. Galactose
transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as
substrates and inducers of the galactose transport system. J.
Bacteriol. 95:1727-1731.

0.2% galactose might in fact be a lot of sugar for this application;
since the Km of these non-metabolizable analogs for repression or
induction is probably a lot higher than glucose or galactose, and they
are not consumed, it should be possible to control the transcriptional
response more precisely with them.

Also, raffinose is hydrolyzed to fructose + melibiose (and in a Mel+
strain, melibiose is cleaved to glucose + galactose); mannose or
maltose can be good alternatives.

Al Brown (Univ. Aberdeen, Scotland) has successfully used an estrogen
response element promoter/estrogen receptor system to modulate PYK1
gene expression by varying [estradiol]; interestingly, the expression
level never exceeded that from the native promoter. And yes, the CUP1
promoter looks ideal for this kind of thing; I'm trying it too.

Good luck!

Arle
*************************************************
a.k.a. Dr. Arthur L. Kruckeberg
 Department of Biochemistry and Molecular Biology
University of Leeds        phone  +44 +113 2333172
Leeds LS2 9JT              FAX    +44 +113 2333167
Great Britain              arle at biovax.leeds.ac.uk
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