BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Figure 4 Multiple sequence alignment of the complete sequences of PEVE and circovirus Rep proteins supplemented with the N-terminal region of nanoviral Rep and the C-terminal region of caliciviral 2C-proteins Strictly con- served residues are displayed as white letters on a black background. BCV – Bovine circovirus; PCV – Porcine circovirus; BFDV1 – Beak and feather disease virus; BBTV – Banana bunchy top virus; CFDV – Coconut foliar decay virus; NV – Norwalk virus; FCV – Feline calicivirus. Materials and Methods Species Experiments were conducted on both sexual and asexual strains of the planarian Girardia (formerly Dugesia) tigrina (Platyhelminthes, Turbellaria, Tricladida). Animals used in subtractive hybridization (from both sexual and asexu- al strains) were generously provided by Dr. Sheiman I.M. (Institute of Biophysics, Pushino). Genomic DNA and to- tal RNA from asexual planaria of the Spanish population were supplied by Dr. Salo E. (University of Barcelona). DNA and RNA preparation, first-strand cDNA synthesis Genomic DNA was isolated from planaria using a rapid extraction method [27]. DNA was further treated with RNase A for RNA degradation. Total RNA was purified by homogenization with guanidine isothiocyanate and phe- nol/chloroform extraction [28]. All procedures was per- formed at neutral pH. RNA was precipitated with 96% ethanol, dissolved and then re-precipitated with 12M lith- ium chloride. RNA was further treated with bovine DNAse I (Sigma) to degrade impurity DNA. The Advantage RT- for-PCR Kit (CLONTECH) was used for cDNA synthesis. SSH and MOS Planarian genomic DNA of 10 animals from each strain was used for subtractive hybridization. SSH was per- formed as described in the CLONTECH PCR-Select™ Bac- terial Genome Subtraction protocol. MOS and differential screening were performed, as described earlier [9]. PEVE DNA purification Total genomic DNA (100 mg) from planaria was dis- solved in 100 µ l TN buffer (10 mM Tris-HCl, pH 8.0; 5 mM NaCl), and mixed with two volumes of 0.2 M NaOH with 1% SDS, followed by 1.5 volumes of 5 M potassium acetate (pH 4.8). The mixture was incubated for 10 min at 4°C, and centrifuged at 14000 × g for 10 min. Following transfer of the supernatant into another tube, PEVE DNA Page 6 of 9 (page number not for citation purposes)