BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 strands of the longest EST representing each one of the 17 validated ORESTES clusters were sequenced. Alignment of these complete sequences with the Drosophila genome show that 8 of them map to intergenic regions, 6 map to introns of annotated genes, two align in intron/exon boundaries and another partially overlaps the last exon of an annotated gene (Table 1). With the exception of ORE- 13, that lies more than 14 kb from the closest annotated gene, the other ORESTES mapping in intergenic regions (ORE-2, -3, -4, -10, -11, -12, -17) are located less than 1 kb of the last exon of the nearest gene, and could represent a novel alternate 3'UTR of these genes. If this were the case, the novel isoforms would be larger than the annotated transcripts since their sizes are much shorter than the detected mRNAs. ORE-16 overlaps the last exon of the CG13165, whose CDS is incomplete, and therefore could represent an extension of this gene. Additionally, ORE-16 detected three transcripts longer than the CG13165 pre- dicted transcript, indicating that this gene might express different mRNA isoforms. Of the five ORESTES located in introns, four (ORE-7, -8, -9, -15) detected mRNAs shorter than the transcripts mapped in the same region and could represent exons of other isoforms. ORE-5 and ORE-6 map to an exon-intron boundary of electronically predicted genes and could either represent a different isoform or the correct structure of the transcription units. However, since ORESTES are not strand-specific, they can also represent exons of unannotated genes encoded in the opposite strand of the same genomic region. Interestingly, all the new annotated exons belong to low abundance messages confirming the ability of ORESTES in identifying rare new transcripts [13]. Cloning a new D. melanogaster serine-protease – SP212 Although representing partial transcripts, ORESTES infor- mation can be useful in the process of transcriptome fin- ishing [20]. Here we illustrate this potential by cloning a cDNA, which we could not isolate after several attempts of screening cDNA libraries. The chosen transcript was first detected by ORE-5 that maps in an intron/exon boundary of a computationally predicted gene (CG33329). Based on the sequence of ORE-5, together with the exons and the genomic sequences flanking this genomic region, we designed primers with the aim of amplifying fragments covering at least the full coding sequence (CDS) of the gene (Figure 3A). We have generated 3 overlapping frag- ments, derived from the same transcript, that were com- pletely sequenced producing a consensus sequence of 1,664 nt (figure 3B). This transcript presents 4 exons, as for the CG33329 predicted transcript, but the third exon is longer in the cloned cDNA (Figure 3). Despite several attempts using mRNA from animals at all developmental stages we could not confirm the existence of the predicted CG33329 and we suggest that the cloned sequence prob- ably represents the correct transcription unit of CG33329. This consensus has an open reading frame of 1.584 nt encoding a 528 amino acids polypeptide (figure 3B). The deduced protein has an estimated molecular weight of 57.8 kDa and an isoeletric point of 6.41. Using BLASTx tool we found that this polypeptide presents significant Table 1: Genomic mapping of the validated ORESTES and sizes of the respective transcripts ORE GeneBank_Accn Cytogenetic map Exon/Intron/ Intergenic Transcriptαsizes (kb) Closest annotated gene Predicted transcript sizesβ of the closest gene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 EG974100 EG974085 EG974101 EG974134• EG974091• EG974139 EG974151 EG974118 EG974120 EG974125 EG974117 EG974128 EG974088• EG974149 EG974105• EG974142• EG974111 7D3–5 88D1 25A3 32A2 88A12 66A22-B3 87E8 91C6-D1 9F2–4 33A1–2 98F10 74E4–5 25D6-E1 74D4 33A1–2 48F6 90D1-E1 Intron Intergenic Intergenic Intergenic Exon/Intron Exon/Intron Intron Intron Intron Intergenic Intergenic Intergenic Intergenic Intron Intron Exon/Intergenic Intergenic 10.0 8.4 2.1; 4.5 4.5 1.5 1.8 0.6 0.9 2.0 11.5 10.2 11.0 3.5 9.8 1.8; 5.0; 9.8 2.2; 3.1; 4.0 8.5 fs(1)h CG33967 CG11928 CG7329 CG33329 nmo (CG7892) CG9813 CG7720 Imp (CG1691) CG18265 CG11874 TORC nompC (CG11020-RB) CycT (CG6292) crol (CG14938) CG13165 cpo (CG31243) 4.0 4.9 0.3 1.6 1.2 Isoforms 2.2–3.1 Isoforms 2.1–2.8 Isoforms 2.4–2.5 Isoforms 2.8–3.9 6.4 3.2 3.0 5.1 Isoforms 3.2–4.3 Isoforms 6.2–7.1 1.5 Isoforms 2.8–6.2 α sizes of the transcripts detected by ORESTES; βsizes of the predicted transcripts encoded by genes located closest to the ORESTES mapping site, • do not match any EST/cDNA sequence available up to October 2006 in the NCBI database Page 5 of 9 (page number not for citation purposes)