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
> Look forward to hearing from you
> Cliff Dominy
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."
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