Promotor-lacZ construct /XGal

NESTELBACHER Reinhard reinhard at wst.edvz.sbg.ac.at
Tue Dec 7 14:40:14 EST 1993



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?
__________________________________________________
Fatima answered in 305

Dear Reinhard (reinhard at dgen10.sbg.ac.at),
don't be so unhappy! May be there's no reason to worry and your mutants are
O.K. after all. There is a lot of (moreless anecdotical) reports and 
rumors indicating that lacZ itself contains sequences which can affect
transcriptional regulation. 60 mutants are not too many (of course, it depends
how many colonies did you screen to begin with...). So, what I would recommend:
1. Make sure that your mutants are not in the structural aaTS gene itself
(by plasmid transformation).
2. Try to sort the mutants into complementation groups to reduce the number
of strains for initial characterization. You can still use the YEPD lethality
to score both complementation and Mendelian segregation of the mutations
in crosses to the parental strain.
3. Rather than measuring betagal activity, look at the levels of endogenous 
aaTS RNA on Northerns. (I know, it's a lot of work, but I think it may be
worth it).

_______________________________________________________________________

Dear all! (especially dear FATIMA!! Thanks for answer)

1.Does somebody know more about the idea that lac-Z can affect transcriptional
  regulation? (literature?)

2.My mutants are not in the structural aaTS gene itself (tested)

3.I did sort into complementation groups a few mutants (But I am not shure that
  did it right anymore (Does somebody know GOOD literature of it (I have enough bad ones)

4. the endogenous aaTS RNA is very low, what's about measuring the RNA of lacZ??


Reinhard NESTELBACHER
reinhard at dgen10.sbg.ac.at
University of Salzburg/Austria
Department of genetics



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