Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line.
Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.
Lawrence
J B
JB
Department of Anatomy, University of Massachusetts Medical School, Worcester 01605.
Villnave
C A
CA
Singer
R H
RH
D001483:Base Sequence; D002051:Burkitt Lymphoma; D002467:Cell Nucleus; D002843:Chromatin / Q000032:analysis*; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007399:Interphase; D008677:Metaphase; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004279:DNA, Viral