A study of cloned, repetitive nuclear DNA from Physarum polycephalum
Attempts to clone Physarum DNA, performed using E. coli RR1 as host and pBR322 as vector, resulted in the elimination of the Physarum sequences leaving only a very short segment of inserted DNA in the plasmid. When E. coli, HB101 was used as host with the same vector, Physarum DNA segments of up to a few thousand nucleotide pairs were detected. By the use of a rapid electron microscopy assay, clones containing relatively large recombinant plasmids were screened for the presence of Physarum inverted repeat DNA sequences. On the basis of this assay six clones were selected for further analysis. Examination of these six recombinant plasmids revealed that in at least two cases deletion of Physarum sequences had occurred. The position of the inverted repeat sequences in the recombinant plasmid DNA molecules was mapped with respect to the BamH1 and HindIII restriction sites which marked the limits of the Physarum DNA insert. All the plasmids were shown to contain repetitive DNA sequences which appeared to be principally interspersed with other DANA sequences in the Physarum genome. The presence of homologies between some of the six recombinant plasmids were observed using a filter hybridisation procedure. The exact nature of these homologies appears to be complex as indicated by electron microscopic examination, showing that in the case of at least two of the plasmids, the DNA contains regions of internal repetition. Restriction mapping has been performed on two recombinant plasmids and the position of the major repeat in the DNA of the plasmid, pPH29, has been tentatively assigned to be positioned between the two foci which can form hairpin structures in single-stranded DNA.