Page 7 of 14 92.7 88.1 80.1 66.5 86.8 87.4 85.2 87.4 76.1 100 95.5 85.4 100 Element Osr10† Osr9 Sorghum Citrus HOPIE† Vitis Barbara Inga Usier OPIE-2 PREM-2 Osr8 Tnd-1 22 (+1) 20 9 12 21 (+4) 12 26 32 18 9 8 9 8 Y Y Y Y Y Y Y Y Y N N N N Linker bef. ENV aft. ENV 12 0 7 0 4 2 8 0 7 0 2 0 13 0 18 0 6 0 0 0 0 0 10 13 3 4 14 10 13 14 12 9 8 9 8 CGGTCTGACCG TTTTCGGACWTRTCCGAAAA AGGGCGGCAGTGCCGCCCT AATCCGGCTWRCCGGATT CGGACCGTCCG CGGTCGACCG TCGGTCYCACCGA AGCGGTASTACCGCT GTCGGACGTCCGAC CACCGGACTGTCCGGTG CACCGGACTGTCCGGTG CGGTCAGACCG ACAATCGATTGT Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 Table 3 Sequence, copy number and nucleotide identity of the 13 novel RMs Total copy number ENV 3’LTR Consensus sequence†† Nucleotide identity % † The parenthesis includes the additional copies found at the beginning of the ENV-gene. †† The symmetrical subunits of each RM are underlined, revealing the length and composition of the intervening bases. sites (Table S2 in Additional file 3). Possibly then, stress-related stimuli may regulate the Sirevirus activa- tion, although experimental confirmation like RM:pro- moter:GUS fusion analysis is required to correlate the RMs to any regulatory function. Clusters of RMs in the LTRs and upstream of the ENV- gene define the borders of predicted CpG islands CpG islands are known to be strongly associated with genes, as they typically occur within or close to their 5’ terminus near the transcription start site and have been found both in vertebrates [28] and plants [29]. Their unmethylated status has been involved with the regula- tion of gene expression [30]. The Sirevirus genome seems to contain CpG islands, which are preferentially found in the LTRs and the region upstream of or within the 5’ domain of the ENV-gene. Of the 21 elements, Tnd-1, Sorghum and ToRTL1 lack CpG islands, while only twice (in pol of Osr9 and Osr8) does a predicted island occur in a region other than the LTRs or the ENV. Among the RM-containing Sireviruses, the RM clusters of HOPIE, OPIE-2, PREM-2, Vitis, Barbara, Inga and Usier are the only CpG islands in their gen- ome, whilst one or two additional islands are present in Osr10, Osr9, Citrus and Osr8 (Figure 4b and Figure S4 in Additional file 2). Presumably, the conserved and high copy number CGG-CCG signature is the key motif of the islands. In most occasions the first and last units Figure 4 Sequence composition and distribution of the novel Sirevirus RMs. (A) Alignment of the Sirevirus palindromic RM boxes (refer to Table 3 for the intervening bases of each RM), (B) and their organization (pink boxes) in four elements at the 5’ side of the ENV gene (only HOPIE and Inga), and upstream of the TATA box (blue circle) in the 3’LTR. All or a section of the RM clusters define the borders of CpG islands (orange bar) (all elements are shown in Figure S4 in Additional file 2)