[BiO BB] reading frame, putative protein

[Ketil Malde]

"Dr. Christoph Gille" <christoph.gille at charite.de> writes:

> I want to verify or reject the hypothesis that there is a yet unknown
> putative reading frame in a known coding viral gene.

One caveat: I haven't worked with viral sequences.

> The translated amino acid sequence does not yield any blast or prosite
> hits.

> Question: how can I verify by computational methods, that this could
> be indeed an additional coding reading frame, resulting in an amino
> acid sequence for which no similar sequence exist in todays databases.

> I would first check in related sequences that the reading frame is
> indeed open.

What related sequences do you have?  If you have other strains of the
virus, conservation of the amino sequence (Ka/Ks ratio) might indicate a
translated sequence.

> Then I could look for irregularities in codon usage resulting from the
> two overlapping reading frames.

Is there enough codon bias that this gives useful evidence?

> Then I might observe absense of base wobbling in the third position
> due to the other reading frame.

> Can you recommend any search for functional sites?

> All methods based on sequence similarity will fail since there
> is no similar sequence so far.

I think that if you have conserved functional sites, they would show up
as short BLAST hits as well.  Perhaps HMM-based methods can be more
sensitive. 

I don't think any purely computational method is going to be a solid
indicator for this, is it an option to try to capture the putative
transcript with PCR?  Or use some other in vitro method?

-k
-- 
If I haven't seen further, it is by standing in the footprints of giants