| Summary: | A novel class of structures termed "microbubbles" has been detected in Physarum nuclear DNA molecules. Microbubbles are specifically associated with replicating DNA, and are present in a clustered arrangement. Microbubbles range from about 100-5,000 nucleotides in length, with a number average length of 510 nucleotides and an average number of 11 microbubbles per cluster. The average centre to centre distance between clusters of microbubbles closely resembles the average length of a Physarum replicon, consistent with the hypothesis that microbubble clusters represent regions of multiple initiation within a single replicon. Small fragments of Physarum DNA have been cloned into the plasmid pUR 322 and the resultant cloned segments of DNA assayed to determine their repetition frequency in the genome. Individual clones representing both repetitive and low-copy number sequences have been characterised by the Southern blot technique and have been used to measure the relative concentrations of repeated sequences present in nascent DNA during S-phase, and to examine the topology of replication. Sequences homologous to 6 cloned segments of DNA containing inverted-repeat sequences are present throughout S-phase in relative concentrations which do not undergo major fluctuations. The topology of replication for a repeated and a low-copy number sequence during the first two thirds of S-phase is qualitatively different. In the case of the repeated sequence the observations are consistent with replication of the sequence at a constant rate during this interval. These results suggest that the movement of replication forks is random with respect to repeatedDNA sequences during S-phase. In vitro 5' end-labelling of replicating DNA I has revealed the existence of a discrete species of single-stranded DNA molecules of approximately 150 nucleotides in length. Molecules smaller than this were not detected under the experimental conditions employed, nor were discrete sized polymers of the 150 nucleotide species. It is suggested that this discrete species is the product of discontinuous replication of Physarum chromatin.
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