BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 2 PEVE genome organization (A) The structure of Ul and Us regions that constitute the PEVE genome. ORFs are specified by open arrows, and their orientations by the direction of the arrows. Arrow numbers signify the length of the putative pro- teins encoded by ORFs. Small dark arrows indicate flanking inverted repeats. Simple arrows specify the primers used for PCR analysis shown in (B). M – marker, 100 bp ladder (Gibco BRL). positions of primer used for PCR were as follow: pr1 (Us region) 286–305; pr2 (Us region) 1518–1541; pr3 (Ul1 region) 461–482; pr4 (Ul1 region) 5430–5452; pr5 (Ul1 region) 1765– 1786; pr6 (Ul1 region) 2985–3005. (C) Putative PEVE genome variants. bridized to an about 1000 base poly(A)+ message. The major transcript revealed by ORF2S probe hybridization was about 850 base. For each gene, transcript length ob- tained using Northern blot is consistent with the cDNA length plus a poly(A) tail, assuming the putative promoter (positions 1133–1094 for ORF1L and 944–983 for ORF2S) and potential polyadenylation signal sequence are utilized. RT-PCR studies showed that all PEVE ORFs are expressed in planarian body with the same pattern. PEVE expression was not detected in the head region, and increased from zone 2 to the tail. Whole-mount in situ hybridization with PEVE RNA probes confirmed the above data. PEVE-containing cells were not detected in the head region, and increased in number from zone 2 to the tail. Cells containing PEVE were sparse in unstained parenchyma (Figure 6A,6B). For ORF1L and ORF2S, both sense and antisense ribo- probes displayed visible staining (figure 6C,6D), while in situ hybridization with sense ORF4L probe yielded no sig- nal (data not shown). We proposed that sense probes can hybridize with PEVE ss-DNA, while antisense probes can hybridize with both RNA and ss-DNA. If so, there is one polarity of the ss DNA regions within PEVE. ORF1L and ORF2S are present in the antisense orientation and their sense probes hybridize with PEVE ss-DNA stretches. ORF4L exists in the opposite (sense) orientation within the ssDNA region of PEVE and ORF4L antisense probe hy- bridizes with both PEVE RNA and ss-DNA. Examination of PEVE in Spanish asexual planaria We performed PEVE analyses in asexual planaria from an independent Spanish population. PCR with PEVE-specific primers of genomic DNA and cDNA, and Southern blot hybridization on genomic DNA did not reveal PEVE in planaria nucleic acid samples (data not shown). This indi- Page 4 of 9 (page number not for citation purposes)