lacZ may be dangerous!
FATIMA at AIMP.UNA.AC.AT
Sat Dec 4 11:21:59 EST 1993
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
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