BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 conservation of the amino acid sequence, and therefore are good indicators of translation. Another indicator of translation is start codon context. Based on nucleotide frequencies of sequences surround- ing mORFs, it is predicted that the Drosophila optimal con- sensus sequence is CAaaAUGg [2,30], but no functional experiments have been conducted in insects to validate the strength of this initation context. Therefore, although the predominant CPuORF start context (AAaaAUGa) seems to be weaker than the predominant mORF context, it remains to be determined whether ribosomes initiate efficiently at the uORF AUG. It is also quite likely that ini- tiation of some CPuORFs is dependent upon cellular con- ditions, as has been shown in various genes [6,31], leading to regulation of mORF protein levels. A number of uORF-mORF pairs were used as positive con- trols for the modified uORF-Finder program. In a previous study, CG9865 was shown to contain a putative uORF- mORF pair that has been conserved among distantly related insect species [19]. This gene was identified by our analysis, therefore validating our approach. Drosophila Tat-like (DTL), a gene containing a uORF with amino acid similarity in D. melanogaster and D. pseudoobscura [24] was also found by the uORF-Finder program. A third gene identified by our analysis, CG10238, is a bicistronic tran- script encoding the small and large subunit of Molybdop- terin synthase 2 (MOCS2) [23]. It is well conserved across distantly related eukaryotic species (see Additional File 2). In addition, 5 of the 31 dicistronic genes described by Misra et al [22] were shown to contain CPuORFs (Table 2; denoted by Misra and colleagues as CG33071ORFA- CG33071ORFB, Tim9b-CG12788, CG33009ORFA- CG33009ORFB, CG33005ORFA-CG33005ORFB, and snapin-CG9960, but subsequently renamed CG33713- CG33714, CG12788-CG17767, CG33671-CG33672, CG33786-CG33785, and CG9960-CG9958, respec- tively). Many of the dicistronic transcripts identified by Misra et al [22] are transcripts with ORF pairs that are not well conserved among the Drosophila species. For exam- ple, the mei217-mei218uAUG is not conserved in any of the 11 other sequenced Drosophila genomes (UCSC D. melanogaster genome browser) [32], therefore it is not sur- prising that a number of the dicistronic genes were not identified by the uORF-Finder program. Additionally, it is likely that neither the D. melanogaster annotations nor the dipteran ESTs are representative of the complete transcript population within each species due to the incomplete annotation of 5' transcription start sites [33], and incom- plete coverage of the genomes by ESTs. Initially, 41 genes and 43 uORFs showed evidence of mild to strong purifying selection (Ka/Ks ratio significantly < 1), and an additional gene with one uORF was detected dur- ing subsequent duplication analysis (see below). The pro- portion of genes in the Drosophila genome showing evidence of CPuORFs is approximately 0.3% (42 genes out of 14,040 genes), which is similar to the frequency predicted for the Arabidopsis genome (0.4–0.5%) [19]. The present study likely underestimates the prevalence of CPuORFs due to incomplete EST resources and poten- tially misannotated 5' regions in D. melanogaster. Consistent with calculated Ka/Ksvalues, the majority of CPuORFs with a low Ka/Ks ratio are present in lineages beyond the Drosophilidae (Table 1) and therefore have been conserved more than 40 My (Assembly/Alignment/ Annotation of 12 Drosophila species) [28,29]. Those uORFs that exhibit a low Ka/Ks ratio but are only found within Drosophila species may represent uORFs that have recently emerged within the Drosophila lineage but are nonetheless under mild to strong selection pressures. Insect CPuORFs are longer in average length than plant CPuORFs Two studies have shown that the length of a uORF can influence the ability of a ribosome to reinitiate scanning and translation initiation at a mORF [34,35]. The plant and mammalian cell systems used in these studies show that reinitiation at a downstream AUG is generally more efficient in the presence of shorter uORFs, and in plant protoplasts reinitiation drops sharply in constructs con- taining uORFs longer than 34 amino acids. Both studies were carried out using viral components, and as such it is not clear whether these observations extend to mRNAs in a native eukaryotic cellular environment. Nonetheless, uORF length could play an important role in the regula- tion of mORFs, therefore we analyzed Drosophila CPuORFs in terms of their amino acid lengths. Initial characterization of the 44 putative CPuORFs under nega- tive selection reveals a wide distribution of lengths, rang- ing from 15 to 179 amino acids (Table 2, Figure 1A). To date, most, if not all, functionally characterized uORFs are smaller than 100 amino acids, but more than one fourth (12/44) of D. melanogaster CPuORFs are above this size. In general, Drosophila CPuORFs seem to be larger than those found in plants. While 83% of Arabidopsis CPuORFs are between 21 and 60 amino acids in length (mean of 40 amino acids ± 16 standard deviation; Figure 1B), the Drosophila uORF length distribution peaks between 41 and 80 amino acids (mean of 76 amino acids ± 44; Figure 1A). These plant and insect datasets were not generated by comparing species with the same evolution- ary distance but a more convincing comparison can be made by analyzing uORFs that have been conserved over more than 200 My: between Arabidopsis and rice, and between Drosophila and non-Brachycera lineages (e.g. Anopheles). The Arabidopsis distribution peak remains Page 4 of 14 (page number not for citation purposes)