BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 Drosophila mitochondrial DNA primers. Template prepa- rations were performed as described by Dias-Neto and colleagues [12] with some minor modifications as fol- lows: 15 ng of purified mRNAs were used for cDNA syn- thesis and amplification, using RT-PCR beads (Amershan- Pharmacia Biotech, USA) and a set of randomly selected oligonucleotide primers (15 to 20 mers). ORESTES pro- files were generated after a cDNA synthesis step at 42°C for 60 mins immediately followed by cDNA denaturation at 75°C and amplification by PCR using a multiple annealing step. The annealing was performed for 10 secs at each temperature and the temperatures varied from 66°C to 44°C (with progressive reductions of 2°C), within each cycle. Primer extension was performed at 72°C for 1 min and denaturing at 95°C for 45 secs in 40 cycles. A final extension step at 72°C for 7 mins was undertaken. Profiles composed of a DNA smear were size selected in order to separate amplification products of dis- tinct size ranges, varying from 0.3 to 1.5 kb. The fragments were ligated into pUC18 using Sureclone (Amersham- Pharmacia) and the recombinant plasmids used for bacte- rial (DH5α E.coli) transformation. The resulting colonies were grown overnight in liquid media and used as tem- plates for PCR using vector primers. One microliter of the resulting PCR product was used for DNA sequencing using standard protocols of the ThermoSequenase II dye terminator cycle sequencing kit (Amershan-Pharmacia Biotech) and the reactions run on a MegaBACE 1000 automated sequencer. Computational analysis A set of 10,092 ORESTES from different developmental stages of Drosophila melanogaster were generated. These sequences were submitted to an automated protocol for checking sequence quality, trimming to exclude vector and primer sequences, removing mtDNA, rRNA, bacterial and yeast and masking repetitive elements resulting in a total of 9,081 ORESTES (GeneBank_Accn EG974084 to EG974151 and ES688489 to ES697501): 360 from embryos at various stages (DE), 2,207 from larvae plus prepupae and pupae (DL), 4,490 from adults (DA) and 2,024 derived from a RNA mixture from embryos, larvae, pre-pupae, pupa and adults (DP). To assess the quality values of the sequences we used phred [36,37]. "N" nucle- otides were trimmed by using a PERL script (cleanN). Sequences corresponding to pUC18 and primers were identified by crossmatch (cleanup_vector). rRNA and mtDNA sequences were identified using a program based on FASTA3 [38] and bacterial and yeast DNA sequences were identified by BLAST [39]. The databases for rRNA, mtDNA, bacterial DNA (E.coli) and yeast were compiled from GenBank. These ORESTES were processed with the assembly tool Cap3 [40]. Clustering of the 9,081 ORESTES resulted in 1,303 non-redundant clusters: 575 contigs plus 728 singletons. The 1,303 obtained clusters were submitted to automated BLASTn search against the release 1 annotated genes [1] resulting in the identifica- tion of 176 unannotated ORESTES clusters. Clustering of the ORESTES generated from each library resulted in 113 non-redundant clusters from DE, 430 from DL, 887 from DA and 729 from DP libraries. RNA extraction and northern blot analysis Drosophila total RNA was extracted from embryo, larvae plus prepupae and pupae or adults. After homogenization in lysis buffer (10 mM Tris-HCl, 2% SDS, 50 mM EDTA, 5% ethanol, pH 9.0), total RNA was extracted by adding 10 vol of Trizol, following the manufacter's instructions (Invitrogen). RNA PolyA+ was obtained using the Oligotex kit (Qiagen). The RNA was fractionated in 1% agarose for- maldehyde-denaturing agarose gels and blotted to nylon membranes (Hybond N, Amershan, UK). RT-PCR, clon- ing, northern blotting, probe labelling, hybridization and post hybridization washes were performed essentially as described in Sambrook et al. [41]. The final washes were performed at 65°C in the presence of 0.1× SSC and 0.2% SDS. Primers used for SP212 cloning: Foward-exon1-1: 5' TCA GTC TTA TTT GCC CAC CG 3'; Foward-exon1-2: 5' GTG TCG CTA ATC GCC TTG G 3'; Foward-exon2-1: 5' GCC TCC GCC GTG GGT TCC 3'; Reverse-exon2-1: 5' CCG ATC CTG TAA GCT GTC G 3'; Reverse-exon3- ORESTES: 5' TGC CAC GGG ATG AGG TAG G 3'; Reverse- exon4-1: 5' ACA GGG TCC AGA TCC ATC G 3'; Reverse- exon4-2: 5' CTG ATT AAG CTG GCA GGT GC 3'. Infecting experiments The insects were challenged by pricking with sharpened needles, which had been previously dipped into concen- trated cultures of microorganisms. In the asceptic pricking the needles were first disinfected by ethanol. Microorgan- isms. E.coli (Gram-) was cultured in LB medium and Sta- phylococcus aureus(Gram+) was grown in BHI medium (a gift from P.S.R. Coelho). Aspergillus fumigatus was grown on Sabouraud-agar medium. Spores and hyphae were har- vested in saline (a gift from Dr. C. Maffei). Authors' contributions RMM participated in ORESTES cloning and sequencing, carried out part of the in silico analysis and performed the molecular experiments, VV participated in ORESTES gen- eration and in silico analysis and helped to prepare the manuscript, EDN, EME, NM, RGRP participated in gener- ation of ORESTES profiles, JFS, DDA helped to perform the ORESTES cloning and sequencing, MAVC, WASJr and SJS developed bioinformatic tools for the ORESTES data- bank analysis, AJGS and MAZ conceived the study, MLPL participated in the design and coordination of the study and prepared the manuscript. All authors read and approved the final manuscript. Page 8 of 9 (page number not for citation purposes)