BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 1 Electrophoresis and Southern blot analysis of PEVE Line M – marker, 1 kb ladder (Gibco BRL); Line 1 – denatur- ing Southern blot hybridization of asexual planarian DNA with ORF2S as a probe. Equivalent data were obtained for all other PEVE clones; Line 2 – denaturing Southern blot hybrid- ization of sexual planarian DNA with ORF2S as a probe. Equivalent data were obtained for all other PEVE clones; Line 3 – non-denaturing Southern blot hybridization of asexual planarian DNA, using the ORF1L fragment as a probe. Similar results were observed with all probes from the Ul PEVE region; Line 4 – non-denaturing Southern blot hybridization of asexual planarian DNA with ORF2S as a probe. Compara- ble results were obtained with the ORF1S probe; Line 5 – partially purified PEVE DNA on an agarose gel stained with ethidium bromide. vealed four variants of orientation of Ul and Us, relative to one another (Figure 2B). To purify PEVE DNA, the plasmid DNA alkaline lysis method was used. Partially purified PEVE DNA was visu- alized on an agarose gel, following ethidium bromide staining (Figure 1). Two bands corresponding to two ex- trachromosomal DNA fragments were detected. Southern blot hybridization with different PEVE-specific probes revealed that each fragment contained the entire PEVE sequence (Figure 1). Using non-denaturing South- ern blot hybridization on planarian genomic DNA, we es- tablished that the PEVE Ul sequences hybridized to the lighter fragment, whereas Us probes stained the less mo- bile fragment (Figure 1). These results imply that the lighter fragment contains a single-stranded (ss) Ul region and double-stranded (ds) Us region, while the less mobile fragment is composed of ss Us and ds Ul sequences (Figure 2C). Sequence analysis BLAST analyses of identified ORFs showed that two were significantly similar to known viral proteins characteristic of distant viral phyla. Other ORFs displayed no significant homology to known proteins. The protein encoded by ORF2S (designated PEVE E1) is homologous to the helicase domain of superfamily 3 (SF3) viral helicases, such as E1 proteins of papillomavi- ruses (Figure 3) [18–20]. Similar to SF3 helicases, the PEVE E1 domain contains a P-loop (motif A) and two conserved motifs (B and C) that determine helicase func- tion [18,19]. However, unlike SF3 helicases, PEVE E1 con- tains only the helicase domain with no additional protein domains. The sequence of the ORF4L product (denoted PEVE Rep) is homologous to circoviral replication initiator (Rep) proteins (Figure 4). Rep proteins of circoviruses, nanovi- ruses and geminiviruses consist of two domains, specifi- cally, a N-terminal nicking domain (with conserved motifs 1, 2 and 3) and a C-terminal SF3 helicase domain (conserved motifs A, B and C) [21,22]. Conserved se- quence motifs 1–3 are characteristic of Rep proteins that utilize the rolling circle mechanism of replication em- ployed by different groups of viruses (circoviruses, nano- viruses, geminiviruses, bacteriophage φ ×-174) [21,23– 25]. However, replication-associated proteins of animal parvoviruses, which utilize the rolling hairpin mechanism of replication, only contain motifs 2 and 3 [23]. The PEVE Rep protein is considerably similar to circoviral Rep proteins and comprises two domains. The C-terminus of PEVE Rep contains all three conserved motifs (A, B and C), signifying helicase activity. However, like animal par- vovirus Rep proteins, the N-terminal part of PEVE Rep contains only conserved motifs 2 and 3, suggesting repli- cation by the rolling hairpin mechanism. Distribution of the PEVE element in the planarian body For analysis of PEVE distribution in planarian body, a sin- gle planarian was cut transversely into six zones as depict- ed in Figure 5A, and six corresponding DNA samples were prepared. PCR data from these samples showed that PEVE is distributed unevenly along the worm's body (Figure 5). The anterior zones (zones 1 and 2) contain lower concen- trations of the element than the posterior part of the worm. We analyzed the PEVE expression using Northern blot analysis and RT-PCR (Figure 5). ORF2S and ORF1L probes were used for Northern blot. The ORF1L probe hy- Page 3 of 9 (page number not for citation purposes)