A study of inverted repeat and repetitive sequences in nuclear DNA from Physarum polycephalum
Foldback stem duplexes were isolated from the Physarum genome by digestion of briefly-annealed DNA with single-strand specific nucleases. Electrophoresis of the resulting fragments provided some evidence for the existence of discrete size classes within the foldback stem population in Physarum; however, the technique could not be used to extend the results previously obtained by electron microscopy. Self-driven reassociation kinetic analysis of isolated inverted repeat duplexes revealed the presence of three components within this fraction of the genome; a "snapback" component (40% of the total), a repetitive component (30% of the total) and a low copy number component (also 30% of the total). The complexity of the repetitive component was estimated to be 1125bp, which implies that only a small number of sequence families make up the majority of this fraction of the Physarum genome. The averagee reiteration frequency of these sequences was estimated to be 5 232. Individual families of inverted repeat sequences were identified following digestion of the DNA with selected restriction endonucleases. The resulting fragments were denatured, annealed briefly and separated by gel electrophoresis. The most intense foldback DNA bands were observed following digestion with the enzymes TaqI and HaeIII. Investigation of the properties of these bands suggested that they arose from the same sequence component of Physarum DNA which comprises at least 2% of the total genome. A possible structure for this component is proposed, based on evidence derived from electrophoresis in neutral and denaturing gels, enzymic treatment and hybridisation analyses. Selected cloned fragments of DNA were used as probes to clone libraries of Physarum sequences. The results of these experiments suggested that the repetitive component of the Physarum genome is dominated by a relatively small number of sequence families. In addition, the very similar hybridisation patterns shown by a total nuclear DNA probe and a probe derived only from inverted repeat sequences to the clone libraries suggested that the middle-repetitive and inverted repetitive components of the genome are dominated by the same sequence families. Elements from these families appear to occur in clusters at the same genomic locations, with their arrangement "scrambled" between clusters.