BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 cluster in certain parts of the genome, the 44 uORFs were placed on the D. melanogaster cytological map (Table 1) and compared to a random distribution (Methods). uORF frequencies were not statistically different from a ran- domly generated dataset except for a cluster of 6 uORFs residing on band 57 (p-value = 10-5), five of which fall on a much smaller segment of the chromosome, band 57F. Upon closer examination, some of these uORFs may have arisen as a result of tandem duplications; one uORF found in the CG30290 transcript as well as two uORFs found in the CG9865 transcript (uORF1 and uORF3) all contain twin CX9C motifs. Interestingly, the observed clustering is not dependent upon the putative duplication events of CX9C motif-containing uORFs. Eliminating the duplica- tion bias by collapsing CX9C-containing uORFs to one representative, clustering is still statistically significant, with 4 uORFs on cytological band 57 (p-value = 0.004) and 3 uORFs on band 57F (p-value = 0.0002). Therefore, the data suggest that there is a preponderance of both clus- tering and duplicate retention of uORFs on band 57. Clus- tering at this region could be an indicator of chromatin interactions at this site that could mediate CPuORF regu- lation. The twin CX9C motif is an integral part of coiled-coil helix, coiled-coil helix (CHCH) domains, a domain previ- ously implicated in uORF-mORF associations in group 8 plant uORFs [19]. In fact, the group 8-like Drosophila uORF member described in the plant study is uORF3 of CG9865. It is interesting to note that the plant group 8 uORF has consistently lost its duplicate copy during both recent and ancient polyploidy events whereas the Dro- sophila group 8 putative homologue may be retaining its duplicates. Different duplication retention histories could indicate that twin CX9C motif-containing ORFs play dif- ferent roles in plants and animals. CPuORF-mORF pair duplicate retention is low within Drosophila melanogaster To determine whether there has been retention of uORF- mORF pair duplicates within the Drosophila genome itself, the 41 mORFs with strongly conserved uORFs were com- pared to the D. melanogaster transcriptome. A single gene, CG17325 showed evidence of a duplicate copy, CG10570, in which the uORF-mORF pair is conserved (See Additional File 3). CG10570 was not detected by our program due to the short length of its mORF (< 100 amino acids), therefore this gene was added to our list of CPuORFs following our duplication analysis (Tables 1 and 2). CG17325 and CG10570 reside adjacent to one another on chromosome 2, band 37A4-A5, and are tran- scribed on opposite strands away from one another. The close proximity of the genes suggests a segmental duplica- tion gave rise to the two genes, both of which are con- served throughout the Drosophila lineage and exhibit a Ka/ Ks ratio < 0.28 (Table 1). This duplication presumably occurred more than 40 Mya since both loci are present in D. melanogaster, D. grimshawi, and D. virilis. Unlike the extensive uORF-mORF duplication retention history of the Arabidopsis genome, CG17325 and CG10570 were the only example of gene duplicate retention in Dro- sophila. GO term and protein domain analysis suggest a link between CPuORFs and both mitochondrial proteins and methyltransferases Further differences between plant and insect CPuORFs were observed following gene ontology (GO) term analy- sis. GO term frequencies in the D. melanogaster genome were compared to frequencies in the insect uORF dataset to look for overrepresentation of terms. P-values were determined using the Bonferroni correction method, a method that accounts for multiple comparisons and cal- culates a conservative p-value. Also, the recent tandem duplicate (see above) was not included in the analysis to eliminate bias from recent duplication events. Because GO terms have been assigned to all ORFs found in bicis- tronic transcripts, GO terms were extracted for both uORF and mORF gene identifiers, designated hereafter as the uORF dataset (41 mORFs and 7 uORFs). This analysis dif- fers from previous analyses in plants; it not only identifies 1) classes of mORF proteins that tend to associate with CPuORFs, but it also identifies 2) ORFs that preferentially associate with other ORFs on a single transcript. In plants, a large proportion of CPuORFs associate with mORFs encoding transcription factors, however this trend was not observed in insects. Instead, mORF proteins showing evi- dence of N-methyltransferase activity (GO term for CG9666 and CG9960 mORFs; Table 3) tend to associate with CPuORFs (p = 0.02). This methyltransferase activity may act on DNA or RNA, since both types of Interpro domains are overrepresented in these two genes. Additionally, overrepresentation of GO term 'protein import into the mitochondrial inner membrane' is driven by two proteins in the Drosophila uORF dataset, CG9878 (Translocase of inner membrane 10, Tim10) and CG17767 (Tim9b), which contain the Interpro Zn-finger Tim10/ DDP-type domain (p = 0.01). Unlike the overrepresented methyltransferase domain, the Tim10/DDP-type domain is not limited to the mORFs, but appears in either the uORF or mORF, demonstrating that these ORFs show a preference for associating with other ORFs in a transcript. Specifically, Tim10 is encoded by the mORF of its tran- script while Tim9b is encoded by the uORF. This does not imply that Tim9b does not act as a regulatory uORF, how- ever. Tim9b may act both as a chaperone in the intermem- brane space, as well as a regulatory element controlling the translation of its associated mORF. Page 7 of 14 (page number not for citation purposes)