BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 3 Multiple sequence alignment of the C-terminal regions (for HPV-16, from aa 378 to 600) of several SF3 heli- cases and full-length PEVE E1 protein Strictly conserved residues are displayed as white letters on a black background. E1 – E1 protein; NS1-NS1 protein; HPV16-human papillomavirus type 16; BPV1 – bovine papillomavirus type 1; Tag_SV40 – UL T antigen of SV40 virus; AAV2 – adeno-associated virus type 2; BPAV – bovine parvovirus. cates that only our asexual planarian population is infect- ed by PEVE. gion. In accordance with in situ hybridization data, PEVE is detected in certain differentiated parenchyma cells. Discussion Using the subtractive hybridization technique, we identi- fied a novel planarian virus-like element, PEVE. Examina- tion of PEVE genome organization and structure reveals that this virus-like element cannot be assigned to any known group of viruses or plasmids, and thus represents a novel type of viral element that encodes two helicases in one genome. Both putative helicases may be involved in the initiation of DNA replication. The proposed scheme of PEVE genomic organization is presented in Figure 2C. Based on Southern blot and in situ hybridization data, we hypothesize that PEVE exists as two variant forms with different compositions of ss and ds DNA stretches, corresponding to the Ul and Us regions. The ss stretches are present in a unique orientation and therefore have only one type of polarity. The ss Ul region displays sense orientation for ORF4L and antisense for ORF1L, while the Us region comprises a strand with all encoded ORFs in an antisense orientation. These ss re- gions are flanked by ds sequences in both orientations. PEVE is distributed unevenly along the worm's body. Our PCR data reveal very low amounts of the viral-like element in the head region. The PEVE expression was also detected in central and tail regions and was not found in head re- PEVE is the first viral element identified in free-living flat- worms. This novel element is not widespread in planarian populations, and has been detected in only one planarian asexual strain to date. However, it is possible that similar virus-like elements and viruses are present in other planar- ian strains. Although asexual strains of planaria exist independently for a long time, they display substantial genetic resem- blance. In particular, the identities of five Antennapedia- like homeobox genes have been disclosed from both our and the Spanish asexual strains [11,26]. These findings imply that PEVE may affect other asexual strains of planaria, thus paving the way for the development of transgenic planaria using the planarian viral sequences as vectors for introduction of exogenous DNA into planarian cells. Conclusions We have identified a novel extrachromosomal viral-ele- ment (PEVE) in the asexual strain of planarian G. tigrina. PEVE is the first viral element identified in planaria, and comprises two potential helicases that are homologous to proteins from distant viral phyla. This element cannot be assigned to any known group of viruses or plasmids. PEVE is unevenly distributed in the worm body, and is detected in differentiated parenchyma cells. Page 5 of 9 (page number not for citation purposes)