Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

Abstract

The replication timing of a pair of natural alleles was compared at two ac-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altAl allele completes replication at between 20 and 40 min of S phase. The same region of the a4tA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both aWl and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than alt4 message on Northern (RNA) blots. The A recurring theme within the variety of recent advances in the investigation of eukaryotic DNA replication is that chromosomal context is a fundamental determinant of origin activity (20,23,44,64). It is reasonable to assume that not only origin activity but also replication fork progression and termination are sensitive to the chromosomal context. This position effect may be a reflection of the longitudinal heterogeneity of a chromosome with regard to protein composition, nucleotide base composition, transcriptional activity, higher-order structure, and association with the nuclear matrix. With this in mind, we have sought to analyze

Low rate of replication fork progression lengthens the replication timing of a locus containing an early firing origin

“…Altogether, these results suggest a delayed activation of the origin in a small part of the nuclei contained within a plasmodium or they reflect different replication patterns of the two alleles contained within each nucleus. Although our previous studies have clearly shown a concerted activation of allelic origins at other loci ( 28 , 32 ), such a different replication pattern between two alleles has been already described in Physarum ( 47 ). From a gene dosage analysis, the authors found that the 2 allelic alt B1 and alt B2 alpha-tubulin loci replicate synchronously in early S phase, while alt A locus replicates later.…”

Genetic Variation and Dna Replication Timing, or Why Is There Late Replicating Dna?

“…Interestingly, the checkpoint-constrained replication origins locate to telomeric heterochromatin regions that are enriched in histone H3K9 methylation [Correction made here after initial online publication]. A relaxation of replication timing control in late replicating heterochromatin in Physarum polycephalum (C-value 0.3 pg) has also been suggested (Cunningham and Dove 1993;Diller and Sauer 1993;Bénard et al 1996), which would be consistent with a weaker inhibition of late-firing replication origins in organisms with small genomes.…”

Replicational organization of three weakly expressed loci in Physarum polycephalum

“…In G 2 phase, all three genes are replicated, providing a standard for normalizing hybridization values in a DNA sample containing two copies of each gene (2/2/2). At one time point in S phase, if one gene replicates earlier than the others, the relative copy number is changed (1/2/1) and the abundance ratio of the hybridization signals varies (31,35). Using this principle, we determined that redB replicates in the interval 0-30 min of S phase, like the early-replicating proP gene, whereas redA replicates mainly during the 60-90 min interval (Fig.…”

“…It has been previously shown by gene dosage experiments that within the same plasmodium, one allele of the weakly expressed α-tubulin altA gene completed replication in a large interval between 40 and 80 min in S phase, when the other allele was found to replicate earlier and more rapidly between 20 and 40 min in S phase (35). It was hypothesized that the more relaxed time schedule of replication of one of these alleles might result from a long distance between the gene and its replication origin and a random variation in the fork rate movement along the template.…”