Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 Page 6 of 14 Figure 3 The diversity of the PPT/3’LTR junction of classic Ty1/ copia retrotransposons. Alignment of the conserved Sirevirus PPT/ 3’LTR junction to the respective region of classic Ty1/copia retrotransposons. Underlined bases represent the 3’ terminus of the IR left arm, and nucleotides shaded in black indicate the beginning of the 3’LTR. The location of the IR left arm is universally conserved in all Pseudoviridae. the 3’LTR. Each element contains highly similar arms (on average 89% identity, 28 bp length), however the sequence conservation is higher in the region surround- ing the loop and decreases rapidly towards the periphery of the motif (Figure S2 in Additional file 2). The upstream PPTs lack an IR structure, which suggests that any putative function of the motif is available only through the natural PPT/3’LTR junction. We discovered that the IR also occurs in almost half classic retrotransposons (Figure 2c), indicating that this structure may serve a fundamental role in the life cycle of the Pseudoviridae. In all cases the left arm extends down- stream till the beginning of the predicted PPT, which occupies the 5’ border of the loop sequence (Figure 3). This precise arrangement offers great support to the pro- posed PPT for classic retrotransposons. The IR length within each element is considerably smaller compared to Sireviruses (on average 88.6% identity, 19 bp length) (Fig- ure S3 in Additional file 2), whilst the right arm starts on average 18 bp inside the 3’LTR. Characteristically, in ele- ments with very short (RIRE1, Tpv2, Mosqcopia) or absent (Art1) linker the left arm resides partially or completely in the coding domain (nucleotides in bold, Figure 2c). A novel type of repeated motif (RM) resides in the Sirevirus LTRs and the region upstream of the ENV-gene We identified in Sireviruses a highly repeated motif with distinctive characteristics in two key areas of the genome of LTR retrotransposons [2], which may indicate its invol- vement in the regulation of Sirevirus activity. The RM is found in 13 elements and is a Sirevirus specific feature, as it is absent from the genome of classic retrotransposons (Table 3). Besides being abundant in the LTRs, the exis- tence of the ENV-gene in nine elements is intriguingly concomitant to the presence of additional RM copies in the linker domain (Table 3, Figure 4b and Figure S4 in Additional file 2). In fact, the distribution of these copies contradicts the multiple PPT topology as the vast majority (77/79, 97%) are preferentially located upstream of the ENV-gene (Table 2 and 3). It appears, therefore, that for these copies the ENV-gene is the determining factor. Moreover, in Osr10 and HOPIE there are extra copies at the beginning of the ENV-gene, near the junction with the linker domain. In contrast, the four Sireviruses without the ENV-gene are simultaneously devoid of linker-based copies. The distribution of the RMs in the Sirevirus 3’LTR is also non-random. Most of the copies (117/127, 92%) cluster within the upper half of each LTR, as they start 200-400 bp after the PPT/3’LTR junction and extend to the following 300-500 bp. As much as 32 units can occur in one element, while all Sireviruses contain at least eight copies of their RM. The overall nucleotide conservation within most elements is remarkable, commonly displaying more than 85% identity (Table 3). The sequence of all RMs is palindromic with dyad symmetry structure (Table 3), which often characterizes retrotransposon cis-acting transcriptional activators [14]. Besides Tnd-1, the RMs of the other Sireviruses contain a CGG-CCG signature that forms their core structure and generates the palindromy (Figure 4a). The highly conserved signature comprises the absolute base of homology, irrespective of the presence of intervening nucleotides between the two symmetrical subunits, or the additional sequence that participates in the symme- try. As an example, Osr10, Osr8, Vitis and Inga share nearly identical RMs, although they have very low sequence homology and reside in evolutionary distant monocot and eudicot species. When we compared the RMs against known plant cis-acting regulatory sequences using the PLACE database [27], we mainly recovered stress-related transcription factor recognition