DNA replication in Physarun polycephalum: electron microscopic and autoradiographic analysis of replicating DNA from defined stages of the S-period

Funderud, Steinar; Andreassen, Rolf; Haugli, Finn

doi:10.1093/nar/6.4.1417

Cited by 37 publications

(21 citation statements)

References 18 publications

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“…This finding suggests that these replicons are representative of the early activated replicons in the plasmodium, a proposition that is also sustained by their kinetics of bidirectional elongation. It has long been known that the replicons activated at the onset of S phase in P. polycephalum are growing bidirectionally at a rate of 1.2 kb/ min per replicon, with an equal rate for the divergent forks (21,22). For the first time, we confirm these results for a defined replicon, by analyzing the respective positions of the replication forks of the ardC replicon at different time points (Fig.…”

supporting

confidence: 76%

“…Considering the known elongation rate of 0.6 kb/min per fork in the plasmodium (21,22), this finding suggests that these two genes are at most 6 kb away from a replication origin. In this study, we establish by neutral-neutral 2D gel mapping (9) and detection of nascent single-stranded replication intermediates (RIs) on alkaline gels (3) that these two abundantly transcribed actin genes are replicated at the onset of S phase from an efficient replication origin that is located within the promoter region of each gene.…”

mentioning

confidence: 80%

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Mapping of a Replication Origin within the Promoter Region of Two Unlinked, Abundantly Transcribed Actin Genes of Physarum polycephalum

Bénard

Lagnel

Pallotta

et al. 1996

Molecular and Cellular Biology

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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.

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supporting

confidence: 76%

mentioning

confidence: 80%

Mapping of a Replication Origin within the Promoter Region of Two Unlinked, Abundantly Transcribed Actin Genes of Physarum polycephalum

Bénard

Lagnel

Pallotta

et al. 1996

Molecular and Cellular Biology

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“…The extended replication schedule of the altA locus seems not to be solely a matter of the larger restriction fragments with which it has been studied. Assuming a fork rate of 600 bp/min (21,22), the 7-kb altA2 fragment would be involved in replication 10 min longer than the 1-kb altB2 fragment would be. We observed, however, that the altA2 fragment changed in abundance over an interval of 25 + 5 min longer than the altB2 fragment.…”

Section: Discussionmentioning

confidence: 99%

“…The average rate of replication fork movement in P. polycephalum has been estimated to be 600 bp/min (6,21,22). At this rate, a 12-to 13-kb fragment would require only about 20 min to complete replication.…”

Section: Methodsmentioning

confidence: 99%

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

Cunningham

Dove

1993

Mol. Cell. Biol.

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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

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“…According to this view, the Ter region might include a terminus of a normal replicon or might include a site that functions as a termination site for replication only when several replication forks, rather than one, move toward it. It should be noted in this regard that the existence of sequence elements that function as termination sites for replication has been documented in bacterial chromosomes and plasmids (2,5,15,17 rate-limiting step in chromatin replication indicate that arrest sites for replication exist in eucaryotic chromatin (9,14,16). (3,4,12,13,25,28), particularly in rodents (H. Manor and R.G.…”

mentioning

confidence: 99%

Unusual sequence element found at the end of an amplicon.

Baran¹,

Lapidot²,

Manor³

1987

Mol. Cell. Biol.

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In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as-the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed. (6,18,21).We have previously reported experiments in which the extent of the amplified region (amplicon) was determined by a Southern blot analysis of DNAs from uninduced and induced LPT cells (1). The hybridization probe used for the analysis was a cloned 0.95-kilobase (kb) fragment of cell DNA flanking one of the two virus-cell DNA junctions, defined as the left junction. These studies indicated that the amplification is arrested within a specific segment of the cell DNA that maps beyond the 0.95-kb probe (1).Here, we report the results of a Southern blot analysis of LPT DNA, in which two recently cloned hybridization probes derived from more distal regions of the flanking cell DNA were used in addition to the previously available probe. These experiments provided more rigorous proof of the existence of a sharp boundary between the flanking cell DNA, which is strongly amplified, and the more distal cell DNA, which is only slightly amplified. We also report the sequencing of a fragment extending through the boundary. We discovered in this fragment an unusual sequence element consisting of two components, a potential cruciform structure and a d(G-A)27 -d(T-C)27 tract. These components are contiguous, and both map within the boundary region. The possibility that this sequence element plays a role in the arrest of the amplification process is discussed. To induce amplification of the integrated Py DNA and flanking chromosomal sequences in LPT cells, the cells were treated with mitomycin C (MMC), which is the most effective inducing agent in this system (7). To map the amplified flanking sequences, high-molecular-weight chromosomal DNAs were purified from the MMC-treated cells and from LPT cells that were not exposed to MMC. Samples of the two DNAs were digested with various restriction enzymes, and the digests were analyzed by Southern blotting (27). The blots were hybridized with the three probes mentioned above.A physical map of the left portion of the LPT amplicon, the map position of the previously available 0.95-kb probe, * Corresponding author. designated SCL2-1 (1, 22), and the map positions of the two new probes, designated SCL7-2 and SCL7-3, are shown in Fig...

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DNA replication in Physarun polycephalum: electron microscopic and autoradiographic analysis of replicating DNA from defined stages of the S-period

Cited by 37 publications

References 18 publications

Mapping of a Replication Origin within the Promoter Region of Two Unlinked, Abundantly Transcribed Actin Genes of Physarum polycephalum

Mapping of a Replication Origin within the Promoter Region of Two Unlinked, Abundantly Transcribed Actin Genes of Physarum polycephalum

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

Unusual sequence element found at the end of an amplicon.

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