MAPPING OF A REPLICATION ORIGIN WITHIN THE PROMOTER REGION OF 2 UNLINKED, ABUNDANTLY TRANSCRIBED ACTIN GENES OF PHYSARUM-POLYCEPHALUM

We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmo dium of the slime mold Physarum polycephalum. Detection and size measu rements of single-stranded nascent replication intermediates (Rls) dem onstrate that these two genes are concomitantly replicated at the onse t of the 3-h S phase and tightly linked to replication origins. Appear ance of RIs on neutral-neutral two-dimensional gels at specific time p oints in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, si te-specific, bidirectional origin of replication is localized within t he promoter region of the gene, We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. F inally, taking advantage of a restriction fragment length polymorphism , we studied allelic replicons and demonstrate similar localizations a nd a simultaneous firing of allelic replication origins. Computer sear ch revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the ye ast autonomously replicating sequence consensus sequence in these Phys arum origin regions. Our results with the ardB and ardC actin genes su pport the model of early replicating origins located within the promot er regions of abundantly transcribed genes in P. polycephalum.