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Promotor-lacZ-construct

NESTELBACHER Reinhard reinhard at wst.edvz.sbg.ac.at
Sat Dec 4 12:08:51 EST 1993


Dear netters, Please help me!

I have a really big problem.
I am working with a promotor-lacZ costruct to search for transcription factors
 for a Aminoacyl tRNA-synthetase,or for factors that influence the expression of 
"my" aaTS-gene. With a Gal1-promotor-aaTS costruct inserted, we did EMS Mutagenesis
searching for colonies growing on Gal-Medium, not growing on YPD. Additionally we 
have the promotor-lacZ costruct to have a 2nd kind of selection.

Now I have 60 mutants selected with the Gal/YPD Selection, but this is too unspecifical
so I need the selection on X-Gal. Said mutations do not switch off the promotor but rather
lead to spectrum of quantitative different expression patterns.As of yet I am not able to
design out a test that is sensitive enough and also allows to select many colonies from 
tetrade analyses - all colonies are just blue.I observe different intensities when growing
mutants on X-Gal medium ( current protocols ) but in tetrade analyses this seems not 
sensitive enough.


Who works with promotor-lacZ constructs and also has/had problems?
Who knows a relevant test that allows testing many colonies?
Who knows of problems with such a construction?
Who works with a reporter construct other than lac-Z, or knows literature about it?

I promise to  write a summary of all the answers I get.

Reinhard NESTELBACHER
Reinhard at dgen10.sbg.ac.at
University of Salzburg
Institut fuer Genetik



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