BMC Genomics 2006, 7:282 http://www.biomedcentral.com/1471-2164/7/282 ated from six sequences, all of which had laboratory evi- dence of RT activity in vitro, using the EMBOSS:cons software from the European Bioinformatics Institute [32] with the default scoring matrix. The RT regions of the fol- lowing six sequences were used: mitochondrial group II intron cobI1 (X54421), chloroplast group II intron psbA (AY290861), HIV1-RT (AAY66384), T. thermophila telom- erase reverse transcriptase TtTERT (AF062652), LINE ele- ment DONG (L08889) and D. melanogaster telomere associated retrotransposon DmTART (U14101). Acces- sion numbers are those in GenBank. This initial consen- sus sequence was refined manually in the following way: i) Residues that were determined by crystal structure of a retrovirus RT to have sidechain or mainchain contact to either nucleic acid, dNTPs or Mg2+ cofactor [29] and that were present in at least one of the six retroelements above were inserted in the consensus sequence. ii) Amino acid residues that have been demonstrated to be essential for RT activity in vitro [30] were inserted in the consensus sequence. iii) A ClustalW alignment [33] (software available at [32]) with default parameters of the six sequences used to gen- erate the consensus sequence was analysed. Where the same residue was present more than once in the same position in the alignment, it was considered significant due to the diversity of the alignment and was included in the consensus. If there was more than one residue repre- sented more than once, both residues were included as alternatives at that position. Table 3 shows the final RT consensus sequence used in the analysis. Genome mining MGE domains in InterPro and Pfam were investigated for P. falciparum and P. y. yoelii entries. The chloroplast and mitochondrial group II intron HMMs were used to search the P. falciparum organellar genome in PlasmoDB release 4.4 using the software package HMMER 2.3.2. Pfam_fs HMMs were extracted from the database and used to search the P. vivax genome in TIGR. An E value of 1 and the individual trusted cutoff value for each Pfam entry were used in mining the P. vivax genome so that results could be compared. Since not all InterPro entries have a corresponding Pfam entry, P. falciparum and P. y. yoelii sequences located only in InterPro were used to search for orthologues in P. vivax using WashU-BLASTP at TIGR. P. falciparum was cultured in human blood and P. y. yoelii and P. vivax were cultured in their respective host organ- isms in the sequencing projects [8,9,34]. MGE signature domains that represented contaminating vertebrate host DNA were therefore excluded from this study. For P. y. yoe- lii this was done by a BLASTP search of the M. musculus genome. The complete genome of S. boliviensis boliviensis (P. vivax host) has not been sequenced. We therefore screened the available S. b. boliviensis sequences as well as the complete genome of a related primate, Mucaca mulatta, for any of the P. vivax MGE domains identified. The P. falciparum MGE domains were screened against the human genome. The genomes of M. musculus, M. mulatta and H. sapiens are available at [35]. Authors' contributions PD conceptualized and designed the study, carried out the analysis and drafted the manuscript. AO assisted with and critically reviewed the bioinformatic methods. TC concep- tualized the inclusion of P. vivax in the study, helped draft the manuscript and gave considerable intellectual input in analyzing the results. All authors read and approved the final manuscript. Acknowledgements Funding received from the University of the Witwatersrand Endowment Fund and National Health Laboratory Service, Johannesburg and the National Research Foundation, South Africa under GUN 2051859. Table 3: The six conserved domains used in this study to represent the RT sequence of RT-encoding MGEs. Domain Sequence RT region Fingers Fingers Palm Palm Palm Palm 1 2 A B' C D II1XXL1[KG]S[GY]P1E1N[FP]K1XG1[RP][IV]XXI2,3[PL]K2,3[PK]D1[GK][GS] XR2,3P2[LI]2,3[TS]XXS3XXXK2,3IXQ1XV[IL]X[IK]XL2XXXXEXK K1XXX[WS]X[TV]XL1D2,4I1K2K1,3CF2,3D2S2I2P1H2D1KL2I1XXL2XXY R1XXXXXXG2XP1Q2,3G2,3SX3XS2P2,3XL1F[NS]IYL1XXL2XE1XLXNXX [YL]XR1Y2,3A1,3D2,4D2,4I[IL]V1X[VS]XXXKN KXXLDXXTXFCXXIG2L1T1VN1XXK2T1K[IT] Alternative amino acid residues are in brackets. 'X' is any residue. 1 – residue present in three or more of the six aligned sequences; 2 – residue present in four or more of the six aligned sequences; 3 – residue in contact with nucleic acid template, dNTP or Mg2+ ; 4 – point mutations of this residue abolish catalytic activity. Page 9 of 10 (page number not for citation purposes)