Hi wormers!
I wanted to define the promoter region for a gene.
the promoter for the ortholog of the same gene in any other system is not defined yet.
what is the correct bioinformatic approach to take in this scenario and any specific tool or program that can be used.
Thanks
To paraphrase Steve, what is the point of your experiments? More info always yields a more specific answer. Do you want to clone the promoter? Compare the regulatory sequence across species? A bit more experimental detail is needed to answer the question properly.
Jordan,
I want to define the regulatory sequence across species .
the gene of my interest is 1. Not a transcription factor
2. but is a transcription co-regulator and the regulation is through chromatin remodelling.
3. to see what other T.F interact with this gene or what other proteins do so, I need to know about the promoter region.
4. since the orthologs across species do not have any such report, I will have to start on my own .
5. how do I begin.
Thanks
Will offer the following suggestions with the caveat that I haven’t needed to do such an analysis for myself. It looks like CisRed (http://www.cisred.org) would be able to provide the info that you need, but it only has ~3800 promoter regions in the database. The advantage is that it compares promoter sequences from C. elegans to seven other nematodes, including several highly diverged parasites. If you are not lucky enough to have your sequence in that database, you could do what the authors in this paper do (http://www.sciencedirect.com/science/article/pii/S0012160604006347):
“We took the C. elegans and C. briggsae egl-5 genomic sequence as the input of a NCBI Blast 2 Sequences search using the default parameters.”
Then take the conserved regions and look for motifs. This is a quick and easy method.
Another more involved approach is described here: http://www.sciencedirect.com/science/article/pii/S0012160608012396
Genomic sequence and bioinformatics
The Cre-lin-11 genomic sequence was retrieved from the Washington University St. Louis Genome Sequencing Center web server (http://genomeold.wustl.edu/blast/client.pl). In the case of Cbn-lin-11, a fosmid clone containing lin-11 5′ region and part of the open reading frame was sequenced (John DeModena and Paul W. Sternberg, personal communication). The C. elegans and C. briggsae lin-11 sequences were obtained from the Wormbase (http://www.wormbase.org).
The MultiPipMaker (web-based) (http://pipmaker.bx.psu.edu/pipmaker/) and Mussa (standalone version, stable release 1.0.0) (http://woldlab.caltech.edu/cgi-bin/mussa) alignments were carried out using 5.0 kb Cel-lin-11, 4.5 kb Cbr-lin-11, 4.9 kb Cre-lin-11, and 3.9 kb Cbn-lin-11 5′ regulatory sequences. For MultiPipMaker, Cel-lin-11 was chosen as a reference sequence for alignments. Mussa requires two user-defined parameters, a sliding window length and the minimum desired conservation (within each window), to search for the conserved blocks in input sequences. Mussa alignments were carried out using 67% identity in the overlapping blocks of 30 nucleotides.
I’m sure that there are many other approaches, but this should get you started.
cheers,
Jordan