Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae

Abstract

The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a Tm of 80.6 degrees C and the second phase has a Tm of 87.3 degrees C, corresponding to GC contents of 28 and 44%, respectively. The Tm of the nonribosomal nuclear DNA, called alpha DNA, is 85.7 degrees C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6 X 10(6) dalton rDNA SmaI restruction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions. A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.