The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ard, ardC, ardO) are duplicated laVy, corresponding to the first 10% of the genome. The fourth locus (ard4) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various tinmes during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.It has been clearly demonstrated, both by cytological methods and density-labeling, that certain segments -of the eukaryotic genome replicate at defined time intervals in S phase of the cell cycle (1, 2) in lower eukaryotes like Physarum (3) as well as in human cells (4). Heterochromatin is generally, found to replicate late in S phase (5-7), whereas certain potentially active genes (8, 9) replicate early. Employing electron microscopic spreads of chromatin, we have recently visualized actively transcribing genes in early-replicated DNA of Physarum. Typically, both coding strands of a newly replicated locus are transcribed in early S phase (10). A relationship between the onset of DNA replication in S phase and transcription has been documented for yeast (11), Drosophila qmbryos (17), and tissue culture cells (13,14) (16,18), the evidence for the temporal order of DNA synthesis (3), and tfhe preferential transcription of newly replicated DNA in early S phase (10, 15), prompted us to determine the chronology of replication of the four actin loci.In this communication we demonstrate that three of the four actin loci are replicated at the time when only 5-10% of the genome has replicated. The synchrony of DNA replication at the gene level is such that two allelic DNA sequences of one actin locus are coordinately replicated. The fourth actin locus is replicated when DNA synthesis is 75% completed. Moreover, this temporal order of replication remains invariant, when monitored either through two consecutive S phases in a single macroplasmodium or over a period of at least 400 generation times in suspension culture. The significance of this fixed order of replication is discussed in light of the replication-transcription-coupling hypothesis.
MATERIALS AND METHODSCultures. The strain Tu 291, provided by F. Haugli (Tromso University, Norway), was used throughput this investigation. Obtained by crossing stains RSD4 by'RSD8 amoebae, it is 'a diploid derivative of the Wis 1 natural isolate (19). The synchronous cultures (macroplasm...