Eukaryotic genomes are organized into multiple replicons that fire only once per cell cycle and at a specific time in S phase, resulting in a complete replication of the chromosomes and a defined temporal order of replication of the genes (1
Despite rapid progress in our understanding of the regulation of DNA replication as a once per cell cycle event (2
), difficulties in defining replication origins genetically have precluded a detailed analysis of the replicons in metazoa (6
). Although it is apparent in mammalian cells that the temporal order of replication of the genes is closely linked to their pattern of expression (8
), the molecular basis for early replication in S phase of the active genes is undefined and our understanding of the functional interactions between replication and transcription is rudimentary.
It is only in the yeast Saccharomyces cerevisiae
that the replicational organization of a complete genome has been elucidated (10
). Furthermore, replication origins have been precisely defined only in this organism, leading to the identification of trans
-acting factors, implicated in the once per cell cycle activation of the chromosomal origins (12
). Importantly, these trans
-acting factors are conserved and play a similar role in DNA replication in metazoan cells (14
However, in S.cerevisiae
, the early and late replicating portions of the genome are transcribed to the same extent (10
), and the replication origins appear to be distributed randomly with respect to expressed genes. This organization of replication origins has been recently questioned in Schizosaccharomyces pombe
. In both yeasts, the proportion of convergent, divergent or tandemly oriented genes is similar, and very close to what can be expected by chance (25, 25 and 50%; 17
). Gomez and Antequera (18
), by localizing a subset of S.pombe
origins, produced evidence for a pronounced bias for initiation sites of DNA replication located within divergent promoter regions rather than in convergent 3′ ends of genes.
A biased distribution of replication origins would be consistent with the observation in mammalian cells that promoter-associated CpG islands are enriched within isolated newly synthesized DNA strands (19
). Similarly, CpG dinucleotide runs are preferentially found in cross-linked DNA fragments immunoprecipitated with antibodies against human Orc1 and Orc2 proteins (20
). However, the presence of mammalian replication origins has been reported within intergenic regions of convergently transcribed genes, such as the hamster DHFR and GNAI3 loci, as well as within promoter regions, such as the human lamin B2-ppv1 and PRKDC-MCM4 loci (20
). Therefore, it remains uncertain whether replication origins are preferentially associated with transcriptional promoters in eukaryotic cells.
We previously studied the replicational organization of five abundantly transcribed genes within the slime mold Physarum polycephalum
, and found an efficient bidirectional replication origin within the promoter region of each gene (24
). To determine whether these results are biased by the fact that only abundantly transcribed genes have been studied so far (two actin genes, two histone H4 genes and a profilin gene), we analyze the pattern of replication of three single copy genes transcribed at low levels within the plasmodium (27
The cDNA clones for the three red
genes examined in this study (redA
: regulated in development), were isolated from a subtracted cDNA library prepared from developing cultures of P.polycephalum
). Northern blot analysis indicated that all three genes are maximally expressed during the development of uninucleate amoebae into multinucleate plasmodia; none of the genes are expressed in amoebae and their mRNAs are of low abundance in total RNA from macroplasmodia. The sequences of the cDNA clones do not give any clues to the functions of these genes during development, although redB
is related to invertebrate sarcoplasmic calcium-binding proteins (27
We found that one of these three genes (redA) contains a replication origin in its 5′ proximal region. The firing of this origin takes place in mid-S phase, in a broad temporal window so that, despite being tightly linked to an origin, the replication of redA is not precisely defined temporally. The two other genes (redB and redE) are replicated very early in S phase, yet instead of containing an origin, they coincide with termination sites of DNA replication. Thus, not all the promoters correspond to replication origins in Physarum.