The sano gene annotation, CG12758, contains a highly conserved 111 codon open reading frame in the 5' UTR in addition to the downstream 778 residue polypeptide encoded (FBrf0202906). As the upstream and downstream ORFs are in frame in D. melanogaster, the possibility that sano encodes a polypeptide with stop codon readthrough was assessed. With the exception of D. elegans, the upstream and downstream ORFs are in frame in all Drosophila species analyzed and the intervening polypeptide sequence is invariant.

CLUSTAL 2.1 multiple sequence alignment of the region between the upstream ORF and the conventional sano polypeptide. The stop codon of the upstream ORF is indicated by 'X'. The conventional N-terminus of the sano polypeptide is included (MAAT...)

Dmir GEQLRQ---PVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 40

Dper GEQLRQ---PVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 40

Dpse GEQLRQ---PVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 40

Dmoj PEREPQ---PVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 40

Dmel GEQLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dkik GEQLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Drho GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dyak GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dfic GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dere GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Deug GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dsim GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dsec GEHLHP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dbia GEHLQP--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dbip GEHLPQ--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dwil GEHLPTP-QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 42

Dana GEHLPQ--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dtak GEHQHPHPQPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 43

Dgri SSQSAP--HPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

Dvir NSNESC--QPVQ X NLLSFNVQLARSKLLSFFNRD MAATDGQIIL 41

.. *** * ********************* **********

Additionally, analysis of the USCS 12 genomes alignment (http://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=347748995&c=chr2R&g=multiz15way) indicates the presence of substantial synonomous nucleotide substitution (see the associated sano_readthrough.2013.9.23.png file).

Because of the conservation of the intervening polypeptide sequence, and the high degree of synonomous nucleotide substitution indicative of protein coding sequence, the sano-RF transcript was annotated to represent a candidate stop codon readthrough polypeptide that comprises these two distinct ORFs.