Gene or accession: CG5785
Gene annotation error
I have new supporting evidence or functional assignment for gene CG5785.
Comments: Dear Madam or Sir,
I would like to present evidence that the protein product encoded by the gene
CG5785 (thr) is larger at its N-terminus than appears in FlyBase.
The FlyBase (FBpp0001383) and the GenBank entries (AAB60210) correspond to a
D. melanogaster THR protein of 1209 aa in length. In fact, the thr reading frame
can be extended in the 5' direction, so that a protein of 1379 aa is encoded.
There is a full length EST entry (SD03660, accession number AY128502) that
supports this extension. Unfortunately, this sequence contains a frame shift,
thus resulting again in a protein that is too small. There is a run of seven
adenosines at nt positions 2045-2051 of AY128502. The deletion of one adenosine
would correct the frame shift, which then results in an ORF long enough to
encode a protein of 1379 aa. This deviation in the length of the adenosine track
is obvious when performing alignments using AY128502. All genomic sequences as
well as the ESTs LP01826 (accession no. AI258595) and LD22604 (accession no.
AA817348) have six instead of seven adenosines at the respective position.
Indeed, when using the FlyBase Apollo Genome Annotation tool, the SD03660
sequence that can be extracted has the coding potential for a 1379 aa protein.
At last but not at least, we have experimental data supporting a 1379 aa long D.
melanogaster THR protein: First, western blot analyses reveal that the
endogenous THR protein runs at a higher molecular weight than the 1209 aa
version expressed from a transgene. Second, the heat shock induced expression of
the 1379 aa version of THR rescues the thr mutant phenotype in the embryo, while
the same experiment with the 1209 aa version failed to rescue. Also, we have
recently undertaken a comparative analysis of thr genes from various Drosophila
species (Jaeger et al. Structure Predictions and Interaction Studies Indicate
Homology of Separase N-Terminal Regulatory Domains and Drosophila THR. Cell
Cycle 2004. in press). The similarity, particularly when the N-terminal
extension of D. melanogaster THR is compared with the N-terminal region of the
proposed D. pseudoobscura THR protein (AY352649) suggests functional
conservation of these sequences. Finally, we have produced a series of genomic
THR deletion
constructs fused at their C-terminus with an epitope tag (Jaeger et al.
Drosophila
separase is required for sister chromatid separation and binds to PIM and THR.
Genes Dev. 2001.  15:2572-2584 ). The migration behaviour in a gel of those
epitope-tagged products containing the THR N-terminus is consistent with an
N-terminally extended, 1379 aa version of THR.
Taken together, all our data clearly support a 1379 aa protein encoded by
the D. melanogaster thr gene. Therefore, we believe that the annotation of thr
in Flybase should be corrected to take these observations into account.
Please don't hesitate to contact me in case you need further information.
Sincerely,
Stefan Heidmann