minimal promoter - (Jan/19/2006 )
i have inserted a putative promter of a gene (including UTR, exon1, intron1) into luciferase reporter vector. However it turn out that, the signal generated is way LOWER than the vector without insert (ie. 10 fold+). could it be the product of exon1 of my gene fused/interrupt the luciferase protein ??
secondly, is most of the minimal promoter present ard 150bp upstream of transciption start site? IF not, how do u guys normally search for it ??
am I understanding this correctly? your insert only goes to -150? I would use a larger piece
you may have encountered a portion of the promoter used for negative regulation; if you took a larger piece of the promoter and saw good expression I would wonder if perhaps there is a repressor motif of some sort in the -150 region
is your gene prokaryotic? eukaryotic? archaeaic?
I'd go with your first thought. Having an exon and an intron there is likely to disrupt the luciferase protein (sructure, processing, frame shift -all are possible)
I don't think that the concept of 'minimal promoter' can be applied generically. I agree with aimikins that you should extend your fragment 5' (can't say how far) and see what you get.
my insert is 2.4kb long including 400+ bp of exon1 and intron1 at its 3' end.... It's mouse gene. I included the exon and intron because i suspected it contain some regulatory element on it...
sigh. think now i shld elongate more to 5' end and get rid of the exon and intron fragment....
any more suggestion before i proceed... sad~~
So your insert is mainly promoter. I don't think to need to extend the sequence 5' as it's fairly large. I still think that the intron and exon might be a problem, especially as the signal is lower than the promoter-less control. I feel that it is unlikely to be due to a genuine repressor element and is more likely to be due to some sort of disruption.
Do you have a +ve control e.g. viral driven? How does that look?
I do a lot of promoter work but it is restricted to only a few genes, so I'm not so good on the general stuff. Hopefully some else will have some input