Promotor-lacZ costruct / XGal

NESTELBACHER Reinhard reinhard at
Tue Dec 7 14:22:34 EST 1993

Dear netters, please help me!!

I am working with a promotor-lacz construct to search for transcription factors 
for a Aminoacyl tRNA-synthetase, or for factors that influence the expression of
"my" aaTS-gene in YEAST. With a GAL1-Promotor-aaTS construct inserted, we did EMS-
mutagenesis, searching for colonies growing on Gal-Medium, not (less) growing on
Glucose. Additionally we have the prom.-lacZ to have a 2nd kind of selection.

Now I have 60 mutants selected with Gal/Glu selection, but now it is too unspecifical
so I need the selection on X-Gal. Said mutants do not switch off the promotor but
rather lead to spectrum of quantitative expression patterns (more or less blue).
As of yet I am not able to design out a test, that is sensitive enough AND also
allows to select many colonies.

My questions:	Who works with promotor-lacZ and also have/had problems?
		Who knows a relevant test that allows testing many colonies?
		Who knows of problems with such a construction (literature)?
		Who works with a reporter construct other than lac-Z. (literature)?
		What's about measuring the lacZ-RNA?

I promise to write a summary of the relevant answers, if there are a lot!

Reinhard at
University of Salzburg/Austria
Department of genetics

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