BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 iSFnPigg2ue1v2roewlu4atsiopnrobfaSbPlyfaomrilgyinated through gene duplication dur- SP212 was probably originated through gene duplica- tion during evolution of SP family. The phylogram is based on multiple alignment of the amino acid sequences of the catalytic domain of SP212 and other 3 SPs and 1 SPH of Drosophila. These sequences are from proteins that pre- sented the highest similarity with SP212 catalytic domain sequence (e-value < -20) in searches against the NCBI data- bank, using Blastx. The alignment was performed using Clus- talw (20). SP55, SPH144 and SP60, as well SP212, localize in the same region (88A12-B1) of chromosome 2R, whereas SP186 is located in chromosome X. simpler structure, with a short amino-terminal activation peptide connected to a catalytic domain. Additional domains in larger enzymes allow protein-protein interac- tions, which are usually needed for regulating their activ- ity and specific localization. The presence of a disulfide- bridge structure, called the clip domain, for example, is characteristic of arthropod SPs and SPHs that are involved in defense response and embryonic development [26]. Interestingly, data of high-density oligonucleotide micro- arrays assaying nearly every Drosophila gene indicated that the SP55, SP60 and SPH144 encoding genes, mapped at the locus 88A12-B1 (CG9649, CG31326 and CG9631), are all upregulated upon septic injury with a mixture of Gram-negative (E.coli) and Gram-positive (M.luteus) bac- teria, or to natural infection with the entomopathogenic fungus B.bassiana [27]. Additionally, there are evidences that CG9631 (SP60) is controlled by Toll and CG31326 (SPH144) by both Toll and Imd signalling pathways [28], the major regulators of immune response in D. mela- nogaster. These observations lead to the suggestion that SP212, which is localized in the same genomic cluster, could also be involved in defense responses. To test this hypothesis we analysed the effect of aseptic injury and septic injuries with gram-positive (S. aureus) and gram- negative (E. coli) bacteria and fungi (A.fumigatus) on the SP212 levels of expression at different time points after the injuries. As shown in figure 5, we observed that the amounts of SP212 mRNA increase upon infection, with the highest levels being detected 3 hours after injuries with either bacteria or fungi. The increase of SP212 mRNA amounts is also observed after aseptic pricking, albeit in lower amounts than in the animals challenged with microorganisms, similarly to what has been previously described for antimicrobial peptide genes [29]. Therefore, the SP212 gene and the genes encoding the other three serine proteases mapped in the locus 88A12-B1 seem to be co-regulated in response to injury and probably have a role in D. melanosgaster defense against pathogens. Recent genetic [30,31] and in vivo RNAi studies [32-34] have pro- vided information about the role of ten Drosophila SP/SPH in septic injury. To our knowledge no further functional studies about the SP/SPH clustered in the 88A12-B1 chro- mosome region have been reported so far. It will be important to characterize the injury-response elements involved in this locus activation as well as to determine the function of these new enzymes in the defense response. Conclusion The analysis of a relatively small set of ORESTES allowed the validation of 17 novel exons present in low abun- dance transcripts, and led to the cloning of a new serine peptidase that is induced during the defense response. These results illustrate the importance of PCR-based approaches as complementary tools for the identification of transcribed regions in sequenced genomes. The final determination of any transcriptome is not a trivial task and one might envisage the occurrence of rare transcripts that will be missed by conventional cloning. Methods Biological samples, ORESTES preparation and sequencing Dechorionated embryos, larvae plus prepupae and pupae as well as adult flies were collected from an isogenic y, w1118 stock of Drosophila melanogaster, immediately frozen in liquid nitrogen and stored at -80 C until use. Total RNA was extracted with TRIZOL and poly(A)+ RNA was isolated (MiniMacs; Miltenyi Biotec). The RNA quality was assessed by northern blot hybridization using a Drosophila α1-tubulin probe [35]. High quality total RNA prepara- tions were further treated with DNase I (Promega). The absence of DNA contaminants was assessed by Southern blot hybridization of PCR amplification products using IFwniidgtuhucrdteioffe5nroefnthmeicSrPo2o1r2gmanRisNmAs in adult flies after infection Induction of the SP212 mRNA in adult flies after infection with different microorganisms. Northern blot analysis of total RNA extracted from adult flies at different times (3, 24 and 48 hours) after challenging by pricking with a needle dipped into 109 cells/ml cultures of either Gram+ (S.aureus) or Gram- (E.coli) bacteria or fungi (A.fumigattus). Note that asceptic pricking by itself triggers the induction of the SP212, albeit at a lower level. Page 7 of 9 (page number not for citation purposes)