Mapping of a Physarum chromosomal origin of replication tightly linked to a developmentally-regulated profilin gene.

We compared the pattern of replication of two cell-type specific profilin genes in one developmental stage of the slime mold Physarum polycephalum. Taking advantage of the natural synchrony of S-phase within the plasmodium, we established that the actively transcribed profilin P gene is tightly linked to a chromosomal replication origin and is replicated at the onset of S-phase. In contrast, the inactive profilin A gene is not associated with a replication origin and it is duplicated in mid S-phase. Mapping by two-dimensional gel electrophoresis defines a short DNA fragment in the proximal upstream region of the profilin P gene from which bidirectional replication is initiated. We further provide an estimate of the kinetics of elongation of the replicon and demonstrate that the 2 alleles of the profilin P gene are coordinately replicated. All these results were obtained on total DNA preparations extracted from untreated cells. They provide a strong evidence for site specific initiation of DNA replication in Physarum.     

Structure and identity of a late-replicating and transcriptionally active gene

Eukaryotic genes are usually replicated early during S-phase in the cell lineages in which they are expressed. Using partially characterized cDNA probes, we recently established two exceptions to this rule in the slime mold Physarum polycephalum. In this paper, we analyzed the structure and the identity of one of these two genes. By genomic cloning and Southern analysis we demonstrate that it is a single-copy gene and decipher the structure of the two alleles by taking advantage of a restriction fragment length polymorphism. By cDNA cloning and sequencing, we deduced the amino acid coding capacity of the mRNA. Finally, we confirmed the late replication of this abundantly expressed gene by "gene dosage" analysis, an experiment that did not require any drug treatment of the cell. Our results provide for the characterization and the structure of the first developmentally regulated gene known to be replicated late in S-phase and abundantly expressed within a eukaryotic cell.     

Premature replication of late S period DNA regions in early S nuclei transferred to late S cytoplasm by fusion in Physarum polycephalum.

Fusion of a late S period plasmodium of Physarum polycephalum to an early S period plasmodium causes premature replication of late S replicating regions in the nuclei of the early S plasmodium. The extent of ahead-of-schedule replication of late S replicating regions in early S period nuclei increases to a plateau of 16-20% for fusions with 40-70 min of phase difference, then declines for larger phase differences. The stimulatory factors for late S replicative units are present only in late S plasmodia and appear to act only on late S regions. Once replicated, early S replicating regions are not stimulated to replicate again by fusion to a plasmodium entering the S period. Our data do not discriminate between anti-termination of replication by factors of stop sites on long replicons, and a sequential initiation of replication on new, possibly non-adjacent regions, but does provide evidence that the stimulatory factors are distinct from one another and specific for certain target replicative units.     

Physical relationship between a gene and its origin of replication in Physarum polycephalum

Taking advantage of the natural synchrony of the S-phase within the plasmodium of Physarum polycephalum, we extracted highly synchronous DNA samples at precise time points in early S-phase. We then separated, by electrophoresis under denaturating conditions, the newly synthesized DNA strands of the nascent chromosomal replicons from the parental DNA template. Using the cDNA clone of the early-replicating LAV1-2 gene as a probe, we could establish by filter hybridization that the elongation rate of the replicon which encompasses this gene is constant, at a rate of 1 kb/min during the first 30 min of S-phase. The smallest replication intermediate (RI) that we have detected by probing with the LAV1-2 cDNA was 5 kb long, suggesting that the LAV1-2 gene and its origin of replication are closely associated within the chromosome. This procedure should facilitate the mapping of replication origins within the genome of Physarum.     

Physical relationship between a gene and its origin of replication in Physarum polycephalum

Taking advantage of the natural synchrony of the S-phase within the plasmodium of Physarum polycephalum, we extracted highly synchronous DNA samples at precise time points in early S-phase. We then separated, by electrophoresis under denaturating conditions, the newly synthesized DNA strands of the nascent chromosomal replicons from the parental DNA template. Using the cDNA clone of the early-replicating LAV1-2 gene as a probe, we could establish by filter hybridization that the elongation rate of the replicon which encompasses this gene is constant, at a rate of 1 kb/min during the first 30 min of S-phase. The smallest replication intermediate (RI) that we have detected by probing with the LAV1-2 cDNA was 5 kb long, suggesting that the LAV1-2 gene and its origin of replication are closely associated within the chromosome. This procedure should facilitate the mapping of replication origins within the genome of Physarum.     

Site-specific initiation of DNA replication within the non-transcribed spacer of Physarum rDNA.

Physarum polycephalum rRNA genes are found on extrachromosomal 60 kb linear palindromic DNA molecules. Previous work using electron microscope visualization suggested that these molecules are duplicated from one of four potential replication origins located in the 24 kb central non-transcribed spacer [Vogt and Braun (1977) Eur. J. Biochem., 80, 557-566]. Considering the controversy on the nature of the replication origins in eukaryotic cells, where both site-specific or delocalized initiations have been described, we study here Physarum rDNA replication by two dimensional agarose gel electrophoresis and compare the results to those obtained by electron microscopy. Without the need of cell treatment or enrichment in replication intermediates, we detect hybridization signals corresponding to replicating rDNA fragments throughout the cell cycle, confirming that the synthesis of rDNA molecules is not under the control of S-phase. The patterns of replication intermediates along rDNA minichromosomes are consistent with the existence of four site-specific replication origins, whose localization in the central non-transcribed spacer is in agreement with the electron microscope mapping. It is also shown that, on a few molecules, at least two origins are active simultaneously.     

Mapping of a replication origin within the promoter region of two 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 plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.     

Replication timing control can be maintained in extrachromosomally amplified genes.

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.     

Preferential binding of S-phase proteins to temporally-characteristic units of replication in Physarum polycephalum.

Synchronous plasmodia of Physarum polycephalum were pulse-labeled with 3H-thymidine in early or late portions of the S-phase, and the binding capacity of the replicated DNA for isochronous S-phase plasmodial proteins assessed by nitrocellulose filter binding assay. Replication units replicating during the first one-third of the S-phase preferentially bind cytosol proteins present in plasmodia engaged in early S DNA replication, while late S replicating DNA exhibits a corresponding preferential binding of plasmodial proteins present only in late S plasmodia. Temporally-characteristic nascent replication units were isolated by Hydroxylapatite column chromatography and were found to contain binding sites for isochronous proteins.     

Coordinate replication of dispersed repetitive sequences in Physarum polycephalum

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.     

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences. Received: 7 July 1997; in revised form: 1 October 1997; Accepted: 1 October 1997     

Replicational organization of three weakly expressed loci in Physarum polycephalum

We previously mapped early-activated replication origins in the promoter regions of five abundantly transcribed genes in the slime mold Physarum polycephalum. This physical linkage between origins and genes is congruent with the preferential early replication of the active genes in mammalian cells. To determine how general this replicational organization is in the synchronous plasmodium of Physarum, we analyzed the replication of three weakly expressed genes. Bromodeoxyuridine (BrdUrd) density-shift and gene dosage experiments indicated that the redB (regulated in development) and redE genes replicate early, whereas redA replicates in mid-S phase. Bi-dimensional gel electrophoresis revealed that redA coincides with an origin that appears to be activated within a large temporal window in S phase so that the replication of the gene is not well defined temporally. The early replication of the redB and redE genes is due to the simultaneous activation of flanking origins at the onset of S phase. As a result, these two genes correspond to termination sites of DNA replication. Our data demonstrate that not all the Physarum promoters are preferred sites of initiation but, so far, all the expressed genes analyzed in detail either coincide with a replication origin or are embedded into a cluster of early firing replicons.     

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.