Supplementary MaterialsFigure S1: Absence of wild-type episome in the re-infected cell clone. These repeated sequences (IRs, TR, and FR) consist of unique sequences within loci made up of varied numbers of repeats. The FR consists of multiple copies of 30-bp repeat models . Each repeat unit contains a binding site for viral protein EBNA1, which plays JTC-801 inhibitor important functions in EBV latent contamination . Several crucial roles have been assigned to the FR for the maintenance of EBV latent contamination , . The binding of TSPAN4 the EBNA1 protein to the FR sequence enables EBV genomes to be maintained as episomes, and it enhances the activity of viral latent promoters, which drive the expression of viral transforming gene products . Although the copy number of the FR repeats varies substantially in different EBV strains, the length of the FR within each EBV strain is usually stably maintained throughout long-term passage , . The B95-8 strain of EBV  contains 29 copies of FR repeats , , each copy 30-bp long, although it was formerly thought that this strain contained only 20 copies JTC-801 inhibitor , , . On the other hand, the Burkitt’s lymphoma-derived Akata strain of EBV  contains 32 copies of the FR sequence . It has been some time since bacterial artificial chromosome (BAC) systems were introduced into the field of EBV virology . The EBV-BAC system enables precise and rapid engineering of the large EBV genome by means of very efficient homologous recombination in is used as a bacterial host. By contrast, the full length FR of the B95-8 strain EBV was stably maintained in a BAC vector, and during recombinant computer virus production and B cell transformation as well. Investigation of primary DNA sequence of the Akata strain FR provides clues to how it becomes unstable in region (Fig. 1A). The restriction enzyme map of regions of the B95-8 strain EBV and Akata strain EBV are schematically drawn in Fig. 1B. The FR sequence of the B95-8 strain of EBV has a 128-bp palindromic sequence in its 3 end , and the 252-bp sequence made up of this palindromic sequence tends to be deleted when subcloned in region (spanning the FR region), the regions , and the region deleted in the B95-8-strain EBV (spanning the region of IR4 and the right regions. The 252-bp sequence that is missing in the B95-8 sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”V01555″,”term_id”:”94734074″,”term_text”:”V01555″V01555) is usually indicated in gray JTC-801 inhibitor within the B95-8 with genomic DNA of EBV-positive Akata cells harboring both wild-type episomes and targeted episomes  (Fig. 2A). We developed a novel experimental strategy to compare the restriction enzyme-digested fragments of AK-BAC and those of wild-type Akata strain EBV genome from which it is derived. In this experimental strategy, a pool of BamHI-digested restriction fragments of AK-BAC was labeled and used as a probe for Southern blot analysis. We found that the BamHI C fragment of AK-BAC was slightly shorter than that of wild-type Akata EBV genomes (Fig. 3A), which was overlooked by the restriction enzyme mapping in our study . All the other restriction fragments were found to be intact sizes, except for those that had been generated by the insertion of transgenes (a BAC vector and a neomycin resistance gene). Open in a separate window Physique 2 A lineage map of BAC clones of the Akata strain EBV.Latently-infected EBV episomes are schematically drawn as small circles in cell nuclei. Transgenes (BAC: BAC vector sequence, neor: neomycin resistance gene, and GFP) are JTC-801 inhibitor indicated as small boxes (either black or gray) around the circles. The estimated FR lengths of various BAC clones are also indicated. Open in a separate window Physique 3 The FR instability of the Akata strain EBV-BAC clones.(A) in Fig. 1B). The bands representing the full-length.