TnPhoA mutagenesis question

Richard E. Showalter show at agouron.com
Tue Sep 5 12:23:44 EST 1995


In article <cliff.11.3042B554 at molbiol.uct.ac.za>, cliff at molbiol.uct.ac.za wrote:

> Hi all
> 
> I have an E.coli mutant which was created by TnPhoA mutagenesis. The 
> transposon jumped into the 3-phosphoglycerate kinase (pgk) gene in the
> 
>                 gapB - pgk - fda
> 
> glycolytic operon. My question is - does this mean that the fda gene 
> downstream of pgk is automatically inactive (polar mutation) OR does 
> TnphoA (or Tn5 for that matter) have a known endogenous promoter in its 
> inverted repeats ie. will the fda gene be transcribed from within the 
> transposon ? The gene would have to be transcribed from within the right 
> inverted repeat.
> 
> The literature (that I have read thus far) is not very helpful in this 
> regard.
> 
> Look forward to hearing from you
> Cliff Dominy

Hello Cliff,

TnphoA does cause polar mutations preventing downstream transcription. 
This is the case with every transposon that I have worked with.  (TN5,
TN5Lac, TNphoA, Minu-MuLux).  You can verify that mRNA transcription is
terminated by the transposon using a fda(DNA) probe against a Northern
blot of mutated vs unmutated E. coli RNA.  Your pgk:TNphoA mutant should
not (under normal circumstances) be making any RNA that can bind to fda
specific DNA.  Hope this helps.

-- 
Richard E. Showalter             "Hey Hey Hey Hey it was
Senior Associate Scientist        the DNA.  Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc.     that made me this way."
show at agouron.com                  Queen-Shear Heart Attack



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