BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 5 Analysis of PEVE distribution and expression in planarian body (A) Six fragments of the planarian body presented in the scheme were used to prepare first-strand cDNA and genomic DNA samples, which were subsequently employed for PCR with oligonucleotide primers for PEVE ORFs. Lines 1–6 correspond to the tissue samples taken as shown at scheme. PCR was performed with oligonucleotide primers for ORF2S (5'-caacgatagtcaccggaatgtca-3' and 5'-gcggctcctgtcttgagtcc-3') and ORF4L: (5'-ttttcatcagcatgtccgttcg-3' and 5'-cctcggttcaggcatctgtttc-3') Each PCR cycle included 95°C for 10 s, 64°C for 15 s and 72°C for 40 s. twenty one cycles were performed in the case of genomic DNA samples and twenty four cycles in the case of cDNA sam- ples. Line 7 – negative control. RNA sample from zone 4 was used for RT-PCR without prior first-strand cDNA synthesis. (B) Northern blot analysis of the ORF2S (line 2) and ORF1L (line 3) transcripts. Line 1 – negative control. Hybridization with ORF2S probe was performed on RNA sample pre-treated with RNase A. M – marker, 0,16–1.77 kb RNA ladder (Gibco BRL). was precipitated with isopropanol. Purified PEVE DNA was dissolved in TN buffer and detected on an agarose gel, following ethidium bromide staining. Sequence analysis Sequences were analyzed with the DNASTAR 4.00 and GeneRunner 3.00 software. Promoters were predicted by "Neural Network Promoter Prediction" software [http:// www.fruitfly.org/seq_tools/promoter.html] Southern blot hybridization Approximately 300 ng undigested planarian genomic DNA from each strain was resolved on agarose gels and transferred to Hybond-N membranes (Amersham). A standard protocol was used for denaturing Southern blot hybridization [29], while for non-denaturing hybridiza- tion, DNA was transferred to membranes under non-de- naturing conditions. Hybridization was performed using an optimized low-background protocol [30]. Northern blot analysis Total RNA from planarian (10 µ g per line) were fraction- ated by 1,1% formaldehyde-agarose denaturing gel elec- trophoresis and transferred to the Hybond-N membrane that was used for hybridization with [α-32P]-labeled probes. Gel-electrophoresis and transfer of RNA to the membrane were carried out in accordance with the stand- ard protocol [29]. The hybridization was carried out ac- cording to the optimized low-background protocol [30]. PCR amplified DNA fragment for probe preparation was purified using Wizard PCR Preps Columns (Promega) and labeled using Prime-a-Gene Labeling System (Promega). For negative control, the RNA sample was treated with RNase A before fractionation. PCR and RT-PCR analysis Genomic DNA and first-strand cDNA prepared from dif- ferent planarian body regions were used in PCR analyses to detect PEVE distribution and expression. PCR was per- Page 7 of 9 (page number not for citation purposes)