PARTITION OF UNIT-COPY MINIPLASMIDS TO DAUGHTER CELLS .3. THE DNA-SEQUENCE AND FUNCTIONAL-ORGANIZATION OF THE P1-PARTITION REGION

The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 .times. 103 base-pairs of DNA. The DNA sequence of the region revealed two large open reading frames that could encode proteins of Mr 44,000 and Mr 38,000. Both would be read in the same direction. The first open reading frame corresponds to the parA gene, the Mr 44,000 protein product of which was shown to be trans acting and essential for partition. The second open reading frame (parB) follows closely and may be cotranscribed with parA. The codon usage frequency for parB is consistent with its producing a protein product. The ParB protein was identified in cell extracts as a product with an apparent Mr of 45,000, suggesting that it behaves anomolously on gel electrophoresis. Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus. Subcloning of this site showed it to consist of a maximum of 112 base-pairs. The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat. Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis. The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes. It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Echerichia coli host.