HIGH-RESOLUTION FISH OF THE ENTIRE INTEGRATED EPSTEIN-BARR-VIRUS GENOME ON EXTENDED HUMAN DNA

Here we report a high-resolution fluorescence in situ hybridization (F ISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chr omosomes, decondensed interphase nuclear chromatin, and linearly exten ded chromatin fibers. We analyzed the EBV DNA integrated into the huma n genome in the well-characterized Burkitt's lymphoma cell line Namalw a, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromo some band 1p35. Using the biotinylated BamHIW fragment of the viral DN A, we observed distinct pairs of signals or small nuclear RNA ''tracks '' within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and, therefore, enables analysis of the structural or ganization of DNA. Application of this methodology to linearly extende d chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length m easurements of 30 images showed a good correlation between the mean ph ysical length of hybridized EBV DNA of 52.8 mu m (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of in tegration processes.