cloning DNA-binding proteins

John Cho pladypus at sympatico.ca
Sat Jul 13 21:54:50 EST 2002


wouldn't you want to check the databases for TF consensus sequences in your
promoter first? that could give you a clue about what to look for and then
see if they are there on a band-shift experiment.

yeast one hybrid experiments looking for TFs to a promoter do not generally
work. i have only ever seen a handful of papers describing such experiments
and in those cases, they were lucky enough not to have too much background
caused by spurious, low level interaction. MOST one hybrid screens utilize
short repetitive DNA sequence to isolate a single unknown transcription
factor which may bind to that site.

i've thrown in some 300 bp promoter sequences into a yeast vector and
attempted to do a one hybrid but every time, no matter how dead the promoter
looks in silicio, it was damn lively enough to turn on the reporter genes.

hope this rant helps

j

"taskan4 rey" <taskan4 at yahoo.es> wrote in message
news:20020711115216.36242.qmail at web13607.mail.yahoo.com...
> Dear cloning gurus,
>
> I'm thinking about cloning genes that code for
> proteins able to bind to my favourite fungal promoter.
> I can only think of two possible strategies:
> One-hybrid: screen a one-hybrid cDNA library in yeast.
> Southwestern: screen a cDNA expression library with my
> promoter.
>
> Any other possibility? Any good, or bad, experience
> with these two?
>
> taskan
>
>
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