BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 levels in the cell [9]. For example, the yeast General Control Nondepressible 4 (GCN4) transcript contains multiple uORFs that differentially regulate the protein level of a transcription factor-encoding mORF under starvation and non-starvation conditions. In turn, the protein produced from the mORF, the GCN4 protein, is essential to the transcriptional activation of some 40 genes involved in amino acid biosynthesis [6]. Because uORFs influence the levels of mORF protein, it is not surprising that disruption of the uAUG can lead to human disease such as thrombo- cythemia [10], a disease which is thought to arise as a result of increased mORF protein product, thrombopoie- tin (TPO). In addition, uAUGs occur in transcripts coding for oncogenes more frequently than other mammalian transcripts [11]. Indeed, oncogenes Mdm2 [12], her-2 [13], MYEOV [14], Bcl-2 [15], and SCL [16], all contain uORFs that affect the level of oncoproteins produced. Potentially thousands of genes are regulated via uORFs, but there are no demonstrated examples of uORFs affect- ing mORF protein production in Drosophila or other insect species. Several uORF-containing genes have been well studied in fungi, plants, and mammals [17] and genome- wide searches of conserved uORFs have been conducted using fungal, mammalian and plant transcripts [4,18-21]. Given the examples found in other eukaryotic species, it is plausible that uORFs fill a regulatory role in the arthropod lineage as well. There is some evidence that regulatory uORFs may occur in insect species. Firstly, a Drosophila gene coding for a putative mannosyl transferase contains a uORF-mORF pair that seems to be evolutionarily conserved in insects [19]. Secondly, there are several examples of Drosophila dicistronic transcripts in which the first open reading frame could be regulatory to the second [22-24]. How- ever, polycistronic transcripts do not all code for putative uORFs; many transcripts defined as polycistronic are ini- tially transcribed as pre-mRNA with two or more ORFs, but are subsequently processed into separate monocis- tronic transcripts [25]. For this reason, we prefer to use the terminology 'uORF' to refer to an ORF (a) which is upstream of a mORF on a single mature mRNA, and (b) which is itself translated as a polypeptide distinct from protein translated from a mORF. In addition, polycis- tronic transcripts that are not processed into separate mRNA molecules are at times part of this uORF/mORF classification. The computational identification of dicis- tronic transcripts by Misra et al [22] resulted in the rean- notation of 31 gene models, some of which may contain conserved uORF-mORF pairs. However, their search was limited to polycistronic transcripts with ORFs greater than 50 a.a., and it is known that uORF peptides as short as 6 a.a. can regulate mORF translation in mammals [26]. Their analysis also discarded overlapping ORFs, some of which are important for the regulation of mORFs [27]. To identify transcripts with uORFs that are likely to be translated, we took a comparative genomics approach using D. melanogaster transcript annotations, Anopheles gambiae transcript annotations, and dipteran expressed sequence tags (ESTs). Using this approach, we determined the prevalence, diversity, and genomic clustering of CPuORFs under negative selection in dipteran genomes and compared these findings to those reported for the plant lineage. Results and Discussion Identification of conserved peptide uORFs in D. melanogaster To determine the prevalence of uORFs most likely to be translated, Drosophila melanogaster release 4.3 transcript sequences (19,389) were used to identify the largest, or major, ORF (mORF). Of these, 13,746 contain unique Flybase gene numbers, 5,851 of which contain one or more AUGs upstream of the mORF. This suggests that 43% of Drosophila mORF proteins could be affected in their expression level by translated uORFs. Our calculated percentage is slightly lower than previously reported Dro- sophila uAUG frequencies [2], but this discrepancy can be explained by the smaller dataset used in the previous study. Putative dipteran homologs were found by comparing D. melanogaster mORFs to 666,153 NCBI ESTs using tBLASTn. Many of the EST sequences contained truncated uORF and mORF sequences, therefore the search was lim- ited to species that diverged from D. melanogaster more than 15 Mya (non-melanogaster group species; AAA: 12 Drosophila Genomes Website) [28,29], to increase detec- tion of negative selection acting on short protein sequences. For each pair of homologs, global alignment of uORFs identified candidate CPuORFs and Ka/Ks ratios were used to further verify evolutionary conservation of the uORF amino acid sequence. In addition, Flybase tran- script annotations were used to discard any genes in which the putative CPuORF was fused to the mORF in any given transcript splice variant. Ka/Ks ratios < 1 indicate that a sequence is under negative selection, Ka/Ks ratios close to 1 imply that the sequence is undergoing drift, and Ka/Ksratios > 1 suggest that the sequence is under positive selection. We found a total of 44 CPuORFs with a Ka/Ksratio significantly less than one (Table 1; Additional File 1). Importantly, our Ka/Ks ratio analysis distinguishes between high-scoring amino acid alignments that reflect conservation of nucleotide sequences versus alignments that reflect true evolutionary Page 2 of 14 (page number not for citation purposes)