Properties of an abundant transposon-like element in the genome of Physarum polycephalum
The genome of the eukaryotic slime mould Physarum polycephalum contains highly methylated (M+) regions, 20-50kb long, which are resistant to cleavage by methylation sensitive restriction endonucleases. The major sequence comprising the M+ component is an abundant family of repetitive DNA sequences: Tpl elements. Members of the Tpl family are around 8.6Kb in length and have a number of structural characteristics in common with eukaryotic retrotransposons. The direct long terminal repeat (LTR) sequences defining the ends of Tpl elements are a typical feature of well-characterised eukaryotic transposons. The similarity extends to the ends of the LTRs, which are terminated by short inverted repeats. The present study describes some of the properties of the transposon-like Tpl family. Sequence analysis of cloned segments of copies of Tpl has allowed partial characterisation of the element at the nucleotide sequence level. In addition, novel sequence arrangements are described which may represent variant forms of Tpl or could indicate the presence of other, previously unidentified, repetitive sequence families in the Physarum genome. Tpl elements are arranged in scrambled clusters which are believed to have arisen by transpositional insertion of the element into copies of its own sequence. Several putative target sites for transposition have been identified and found to be clustered in specific regions of the Tpl sequence. These sites bear some resemblence to the Chi sequences found in bacteriophage lambda, thus suggesting an alternative mechanism for the rearrangement of Tpl sequences, i.e. homologous recombination. A study of the replication and transcription of Tpl elements was also initiated. The timing of replication during the cell cycle was determined using synchronous plasmodial cultures. Tpl elements were found to replicate at between 60 and 90 min into S-phase. Thus, they are contained within the relatively late-replicating fraction of the genome. Tpl elements were found to be transcribed in amoebae and plasmodia and could be the first examples of late-replicating Physarum sequences which are transcriptionally active. Some evidence of developmental regulation of expression of Tpl elements was also found. The similarity with retrotransposons suggested that an RNA intermediate may be involved in transposition. However, no full-length transcript was detected in this study.