BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 6 Whole-mount in situ hybridization with ORF1L ribo- probes performed on intact planaria (A) Whole-mount in situ hybridization with antisense ORF1L riboprobe of planarian head. (B) Whole-mount in situ hybridization with antisense ORF1L riboprobe of planarian central body region. Bar, 20 µ m (C) A larger magnification of the results of whole-mount in situ hybridization with the antisense ORF1L riboprobe. (D) Whole-mount in situ hybridization with the sense ORF1L riboprobe. formed with the Advantage 2 PCR Kit (CLONTECH). PCR with oligonucleotide primers for Dth-2 [31] was used to confirm the uniformity of the samples. Whole mount in situ hybridization The cDNA clones of ORF1L, ORF4L and ORF2S were used as templates to produce DIG-labeled RNA probes for in situ hybridization. Whole-mount in situ hybridization was performed, as described previously [13]. Authors' contributions DR carried out the subtractive hybridization of planarian genomes and PEVE cloning. MB carried out whole mount in situ hybridization and PCR and RT-PCR analysis. EB carried out Southern blot analysis, participated in the se- quence alignment and analysis, and drafted the manu- script. LV carried out Northern blot analysis. SL conceived of the study, and participated in its design and coordina- tion. List of abbreviations #SSH, Suppression Subtractive Hybridization; MOS, Mir- ror Orientation Selection; PEVE, Planarian extrachromo- somal virus-like element; Rep, replication initiator protein; ss, single-stranded; ds, double-stranded; super- family 3, SF3; RT-PCR, reverse transcription polymerase chain reaction. Acknowledgements We thank Prof. V.I. Agol and Prof. A.E. Gorbalenya for helpful discussions. We are additionally grateful to Dr. M.V. Matz for advice, and Dr. I.M Shei- man for providing the animals used in this study. Dr. E. 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