help in cloning the promoter region of a gene - (Sep/17/2006 )
I am having some problems with the promoter region in my gene. the ATG start site that I downloaded from the ref seq @ NCBI a year ago has been altered by NCBI. the new ATG start site is now abt 180 bp upstream of the old start site. I cloned the promoter region up to the old ATG but the experiments turned out unremarkable.
What I'd like to ask is
- when doing promoter activity assays, how much of the promoter does one normally clone?
- is it OK to include the ATG start site as part of the promoter insert I am testing or should I design another primer just upstream of the new ATG start site ?
I would reclone. But before that, I would be in touch with the person who is updating the Genbank entry, who presumably knows more about this issue. The things he/she knows may not be published yet.
thanks for the advice. I'll do that. I wrote to NCBI but I was directed to a web page listing the history of the reference sequences i.e current live seq and previous dead sequences but it didn't state why there was a change in the ref seq. Strangely enough, the EMBL database has the old ref seq.
For some promoter work only the initiator region or tata box region (plus start site) need to be included. This is not always the case. This tends to work in TATA-less genes.
I have used promoters of up to 1200 bp cloned into reporter plasmids and have based work on papers in which multiple deletions are made of the same promoter. Chopping of every 100 bp until something changes, etc.
You can also include a significant amount of sequence downstream of the initiation site. By this I mean the transcription initiation site NOT the ATG start codon.
This maybe where you are going wrong. In some genes the promoter finishes several hundred bp upstream of the ATG codon. You need to find where transcription starts and where the cis and trans acting regulatory elements are. This will give you a good idea of how much promoter to include. This may range from 100-4000 bp.