Developmentally regulated usage of Physarum DNA replication origins

Abstract

To determine the extent to which eukaryotic replication origins are developmentally regulated in transcriptionally competent cells, we compared origin use in untreated growing amoebae and plasmodia of Physarum polycephalum. At loci that contain genes transcribed in both developmental stages, such as the ribosomal RNA genes and two unlinked actin genes, we show that there is a similar replicational organization, with the same origins used with comparable efficiencies, as shown by two-dimensional agarose-gel electrophoresis. By contrast, we found cell-type-specific replication patterns for the homologous, unlinked profilin A (proA) and profilin P (proP) genes. proA is replicated from a promoter-proximal origin in amoebae, in which it is highly expressed, and is replicated passively in the plasmodium, in which it is not expressed. Conversely, proP is replicated passively and is not expressed in amoebae, but coincides with an efficient origin when highly expressed in the plasmodium. Our results show a reprogramming of S phase that is linked to the reprogramming of transcription during Physarum cell differentiation. This is achieved by the use of two classes of promoter-associated replication origins: those that are constitutively active and those that are developmentally regulated. This suggests that replication origins, like genes, are under epigenetic control associated with cellular differentiation.

Figure 1

Ribosomal DNA replication in two proliferative stages of Physarum polycephalum. A map of a palindromic ribosomal DNA molecule is shown (top panel). Arrows indicate the transcriptional polarity of the ribosomal RNA genes. Plasmodial replication origins o₁ and o₂ and their mirror counterparts o′₁ and o′₂ are shown within the central non-transcribed spacer. The restriction fragments A (7 kb; PstI–BamHI), B (6.4 kb; HincII) and C (5 kb; HindIII) were analysed by two-dimensional gel electrophoresis using total DNA from untreated amoebae and untreated plasmodia. A bubble arc is only observed in the A (and A′) fragment in both cell types. It is associated with a strong Y arc, because A can be passively replicated from initiation in A′, and vice versa. Furthermore, both fragments are replicated passively when initiation takes place at o₂ and o′₂. Electron microscopy analysis has previously revealed that o₂ is approximately twice as active as o₁ (Vogt & Braun, 1977). Taking these facts into consideration, a 1:9 bubble-arc to Y-arc ratio was expected in the A + A′ pattern (Bénard et al., 1995). Here, an approximately 1:10 ratio is defined experimentally for both amoebae and plasmodia. Analysis of the B (and B′) inter-origin fragment reveals a strong Y arc and a weak termination signal with amoebal and plasmodial DNAs, indicating a low frequency of simultaneous firing of adjacent origins in both cell types. The intragenic C (and C′) fragment is replicated passively in both cell types, as shown by an arc of simple Ys. Our results show invariant origin usage in the rRNA genes of Physarum.

Figure 2

Origin use at two unlinked actin loci of Physarum polycephalum. Synchronous early S phase plasmodial DNA and total nuclear DNA from asynchronous amoebae was purified. Replication patterns of EcoRI fragments encompassing the ardC and ardB promoters (see maps below micrographs) were determined using specific probes (Bénard et al., 1996). For the ardB locus, amoebal replication intermediates were purified further by BND (benzoylated naphthaylated DEAE)–cellulose chromatography. Prominent, fully extended bubble arcs show that origins are active at both loci in both cell types (red arrows). The similarity of the two-dimensional gel patterns shows that the same chromosomal origins are used to replicate these two abundantly transcribed actin genes during the amoebal and plasmodial cell cycles (see maps below micrographs). In the diploid plasmodium, because of a polymorphism in the downstream restriction sites (*), the allelic replication intermediates are separated, giving rise to two bubble arcs and two Y arcs (Bénard et al., 1996).

Figure 3

The origin of the ardC gene is site-specific in amoebae. Overlapping restriction fragments of the ardC locus were analysed by two-dimensional gel electrophoresis. Total amoebal DNA (EcoRI fragment B) or BND-enriched amoebal DNAs (HindIII fragments A and C) were used. Bubble structures (arrow) are only seen in the B fragment, restricting initiations to the centre of this fragment in the amoebae, as previously shown in the plasmodium (Bénard et al., 1996). BND, benzoylated naphthaylated DEAE.

Figure 4

Cell-type-specific DNA replication origins in Physarum polycephalum. The replication and transcription of two profilin genes were analysed in growing amoebae and plasmodia. Northern blot experiments were carried out using 10 μg of total RNA from each developmental stage to measure messenger RNA levels. Overnight PhosphorImager exposures are shown (N). proA gives rise to two abundant mRNAs of 600 nucleotides and 500 nucleotides in amoebae (upper left panel), which are absent from plasmodia (upper right panel; Binette et al., 1990). Quantitation indicates that proA mRNAs are at least 1,000 times more abundant in amoebae than in plasmodia. A similar bias in favour of proP expression in the plasmodium was seen (lower panels; Binette et al., 1990). Replication (2D) was analysed using total amoebal DNA for proA and BND-enriched amoebal DNA for proP. Early (5 min) and mid (60 min)s-phase plasmodial DNA samples were used for proP and proA, respectively. Bubble arcs (see red arrows) indicate that proA and proP are coupled to origins when transcribed. By contrast, these genes are replicated passively when they are not expressed (see maps below micrographs), showing that developmentally regulated origins exist in Physarum. BND, benzoylated naphthaylated DEAE.