BMC Genomics 2007, 8:42 http://www.biomedcentral.com/1471-2164/8/42 IFnitgeur-rsepe4cies alignment of exon-intron boundaries of the exon 0 of CNGA3 Inter-species alignment of exon-intron boundaries of the exon 0 of CNGA3. Conserved nucleotides are labelled with colours and with the star in the bottom those conserved in all the analysed species (human, mouse, rat, rabbit, dog cow, elephant and tenrec). Arrow highlights the splice donor site. gene annotation In most cases the RefSeq was incomplete. Transcripts were missing part of the first exon or even complete exons at the 5' end. This experimental determi- nation of TSSs shows that the current gene annotation was in most cases obtained from data sources that are not strictly selective for the complete transcribed form, and need to be updated. This procedure led us to discover sev- eral transcript isoforms that were un-annotated and to locate retina specific TSSs. Proteins encoded by these genes are essential for retina function and stability. A mutation in the cis-regulatory elements may influence the level of transcription and have a strong effect due to sen- sitivity of photoreceptors for high level transcription of genes involved in phototransduction. The new described cis-regulatory regions and untranslated exons are possible targets for mutation studies in retinal disorders. Therefore new isoforms give a more complete picture of alternative start sites use in retina genes. The 5' untranslated region may contain important transcriptional and post transcrip- tional regulatory sites [33-35] and therefore only the com- plete 5' UTR provides the opportunity to study the potential regulatory role of these non-coding sequences. New reported TSSs contribute to the identification of reg- ulatory elements active in tissue specific gene regulation [36,37]. Moreover, bioinformatics tools that identify common regulatory elements rely on the correct determi- nation of TSSs within a particular tissue [38], therefore these computational approaches will only be effective after experimental validation of the 5' end of transcripts. Conclusion We herein report the TSSs for 54 retina expressed genes. Our results define new and more precise locations of TSSs for 76% of the analysed genes; moreover in 15% of the genes we found new exons in the 5' end of the transcripts. Thus, this analysis of TSSs in human retina was essential to define the complex pattern of transcripts present in this tissue. Our results highlight the importance of applying a tissue specific approach with a systematic program of Cap-finder RACE using the known gene structures as a starting point and/or gene predictions to complete the existing gene annotation. The new TSSs and transcribed sequences pro- vide crucial information for further exploration of the promoter and other cis-regulatory sequences at the 5'end of the gene, and in particular for the study those elements that are functionally active in human retina. Methods In silico analysis We used available public database information (RefSeq, NCBI and Ensemble predictions covered by at least one EST, Unigene EST database) to perform in silico assembly and analysis of 5'transcript termini. The procedure involves downloading the sequences from public data- bases, clustering them to obtain a consensus and design- ing a gene model with the most complete 5' transcript termini. Sequences were downloaded and assembled using the SeqMan program (DNAstar). In our analysis we consider retina ESTs as well as non-retinal ESTs to obtain the most complete information about the different alter- native start sites already mapped. Additionally, the infor- mation about putative TSS was assessed using Promoser [39], Eukaryotic Promoter Database Current Release 87 [40], and Database of Transcriptional Start Sites DBTSS [41]. First exon boundaries were determined by aligning the predicted sequence to the genome using BLAT [42]. The gene model was then deduced considering the cluster- ing of all the collected sequences giving the priority to the most accurate database in the 5' termini (EPD and DBTSS), but considering also gene prediction (GENS- Page 10 of 14 (page number not for citation purposes)