A NOVEL CLASS OF SUPERCOIL-INDEPENDENT NUCLEASE HYPERSENSITIVE SITE IS COMPRISED OF ALTERNATIVE DNA STRUCTURES THAT FLANK EUKARYOTIC GENES

The cell makes a fundamental distinction between genes and non-gene se quences, which mechanistically underlies the process of gene regulatio n. Here, we describe the properties of a novel class of genetic sites that reproducibly flank and delineate the coding regions of the eukary otic genes tested. Defined in vitro reaction conditions that include a ltered solvation and elevated temperature rendered the sites hypersens itive to nuclease cleavage. Consequently, the complete coding regions of the Drosophila genes tested were quantitatively excised from genomi c DNA or genomic clones by this treatment. Identical reaction products were generated from linear or supercoiled DNA substrates. Chemical mo dification and fine-structure analysis oi several cleavage sites flank ing Drosophila genes showed that the cleavage sites were stable nuclei c acid structures that contained specific arrangements of paired and u npaired nucleotides. The locations and properties of the cleavage site s did not correspond to previously known nuclease hypersensitive sites nor to known alternative DNA structures. Thus, they appear to represe nt a new class of genetic site. Ln a deletion analysis, the minimal se quence information necessary to direct in vitro nuclease cleavage 3' t o the Drosophila GART gene colocalized with the signal required for te rmination of transcription in vitro. The data suggest that a novel cla ss of DNA site with distinct structural properties encodes biological information by marking the boundaries of at least some gene expression units in organisms as diverse as Plasmodium and Drosophila. (C) 1998 Academic Press Limited.