BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 uORF start and stop codons [32]. Any putative uORF sequences that showed evidence of in-frame fusion with the mORF on the UCSC browser (in an alternative splice form, for example) were not included in the final list of CPuORF-containing transcripts. Calculation of Ka/Ks The Ka/Ks ratio was determined using pairwise_kaks.PLS (version 1.7) [54] and is derived from the highest scoring BLAST homolog in the D. melanogaster-dipteran high scor- ing pairs. Both the approximate method (option -kaks yn00) and the maximum likelihood method (-kaks codeml) were used. Only the approximate method calcu- lation is reported in Table 1 due to the typically short evo- lutionary distance between the organisms found in the highest scoring BLAST pairs. The Nei-Gojobori p-distance model was used to test for purifying selection (Null hypo- thesis Ka = Ks; alternate hypothesis Ka <Ks). MEGA4 default settings were used to run codon-based Z-test analyses [55] on highest scoring BLAST homologs. Cytological distribution of uORFs To determine whether the 44 uORFs were randomly dis- tributed along the Drosophila chromosomes relative to annotated transcript positions, a perl script was written to generate a random distribution of 44 positions along the chromosomes. Cytological positions for each CG gene identifier were extracted from D. melanogaster release 4.3 gene annotations [51], from which 44 positions were ran- domly chosen. This ensured that clustering would not simply reflect gene rich regions. The number of 'hits' within a given cytological band were tallied, and the entire process was iterated 30,000 times, providing a random distribution of 'hits' at any given band when 44 positions were picked across the entire genome. The random distri- butions were then used to provide a p-value for the observed number of uORFs within a given cytological band. Gene Ontology, Pfam domain, and Interpro domain retrieval and analysis Over- and under-representation of Gene Ontology (GO) terms in the uORF dataset (41 mORFs and 7 uORFs with associated GO terms) versus the D. melanogaster genome was determined using Genemerge v.1.2 [56], a program which provides a Bonferroni-corrected p-value. Associa- tion files were derived from Gene Ontology website files (D. melanogaster annotation received from Flybase March 13, 2007) [57], and from the BioMart website [58] (Ensembl Gene ID, Pfam ID, and Interpro ID numbers obtained; downloaded files are based on D. melanogaster genome release 4.3). Description files were derived from GO term files [59] (gene_ontology.obo.zip), and from Interpro files [60]. Abbreviations Upstream open reading frame (uORF), Major open read- ing frame (mORF), Upstream start codon (uAUG), Con- served peptide upstream open reading frame (CPuORF), General Control Nondepressible 4 (GCN4), Throm- bopoietin (TPO), Expressed sequence tag (EST), Dro- sophila Tat-like (DTL),Molybdopterin synthase 2 (MOCS2), Gene ontology (GO), Translocase of inner membrane 10 (Tim10), Translocase of inner membrane 9b (Tim9b), Uncoupling protein 2 (UCP2), B-cell lym- phoma 2 (BCL-2), p8 mature T-cell prolifera- tion(p8MTCP1), Mitochondrial Ribosomal Protein 10 (Mrp10p), Cytochrome Oxidase 19 (Cox19p), Cyto- chrome Oxidase 17 (Cox17p), Mitochondrial intermem- brane space import and assembly 40 (Mia40p) Authors' contributions CAH and GB conceived and designed the experiments. CAH carried out the analysis and drafted the manuscript. GB provided critical feedback for the final version. Both authors have read and approved the final manuscript ver- sion. Additional material Additional file 1 Conserved peptide uORF sequences. Conserved peptide uORF and associ- ated mORF amino acid sequences in Drosophila melanogaster. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-61-S1.txt] Additional file 2 uORF and mORF sequences and alignments. Amino acid sequences and alignment of insect conserved peptide uORFs and associated mORFs. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-61-S2.txt] Additional file 3 CG17325/CG10570 sequences and alignment. Amino acid sequences and alignment of putatively duplicated D. melanogaster genes, CG17325 and CG10570. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-61-S3.txt] Acknowledgements This research was funded by an NIH IRACDA fellowship to CAH (Grant #GM000708), and an NIH R01 grant to GB (GM069462). References 1. Cavener DR, Cavener BA: Translation start sites and mRNA leaders. In An Atlas of Drosophila genes Edited by: Maroni G. New York, Oxford University Press; 1993:359-377. 2. Rogozin IB, Kochetov AV, Kondrashov FA, Koonin EV, Milanesi L: Presence of ATG triplets in 5' untranslated regions of Page 11 of 14 (page number not for citation purposes)