Early activated replication origins within the cell cycle-regulated histone H4 genes in Physarum

It was previously shown that the two members of the cell cycle-regulated hi stone H4 gene family, H4-1 and H4-2, are replicated at the onset of S phase in the naturally synchronous plasmodium of Physarum polycephalum, suggesti ng that they are flanked by replication origins. It was further shown that a DNA fragment upstream of the H4-1 gene is able to confer autonomous repli cation of a plasmid in the budding yeast. In this paper, were-investigated replication of the unlinked Physarum histone H4 genes by mapping the replic ation origin of these two loci using alkaline agarose gel and neutral/neutr al 2-dimensional agarose gel electrophoreses, We showed that the two replic ons containing the H4 genes are simultaneously activated at the onset of S phase and we mapped an efficient, bidirectional replication origin in the v icinity of each gene. Our data demonstrated that the Physarum sequence that functions as an ARS in yeast is not the site of replication initiation at the H4-1 locus, We also observed a stalling of the rightward moving replica tion fork downstream of the H4-1 gene, in a region where transient topoisom erase II sites were previously mapped. Our results further extend the conce pt of replication/transcription coupling in Physarum to cell cycle-regulate d genes.