Identification of cis-regulatory sequences in ascidian ribosomal DNA using a rapid filter-binding assay

Using 32P-labelled random primed ribosomal DNA (rDNA) from the ascidian, Herdmania momus, multiple and large-scale filter-binding assays were performed to identify cis-regulatory sequences interacting with H. momus oocyte germinal vesicle protein. A vacublot apparatus was used to isolate DNA-protein complexes, providing a means of filtering multiple binding reactions simultaneously and for isolating sufficient amounts of bound DNA for further investigations. DNA bound to the filter was used to identify unknown cis-elements in the rDNA by Southern-blot analysis. The trapped rDNA hybridized specifically to the intergenic spacer, a region which contains cis-regulatory sequences that interact with rDNA transcription factors in several other species. Gel shift analysis of intergenic spacer fragments and native Southwestern blots confirmed that cis-elements were localized in the rDNA intergenic spacer. In principle, this method allows for the rapid identification of cis-regulatory sequences within any large, cloned DNA fragment which interact with nuclear extract.