MODULAR STRUCTURAL ELEMENTS IN THE REPLICATION ORIGIN REGION OF TETRAHYMENA RDNA

Computer analyses of the DNA replication origin region in the amplifie d rRNA genes of Tetrahymena thermophila identified a potential initiat ion zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids R es. 22, 2479-2489], This region consists of a putative DNA unwinding e lement (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions, In this study, two mung bean nu clease-hypersensitive sites in supercoiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels, Restriction fragme nts containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the repli cation origin region with the nuclear matrix in vivo, The direct demon stration in a protozoan origin region of elements previously identifie d in Drosophila, chick and mammalian origin regions suggests that clus ters of modular structural elements may be a conserved feature of euka ryotic chromosomal origins of replication.