PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (852740)

Clipboard (0)
None

Related Articles

1.  Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum. 
Molecular and Cellular Biology  1996;16(3):968-976.
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.
PMCID: PMC231079  PMID: 8622700
2.  Early activated replication origins within the cell cycle-regulated histone H4 genes in Physarum. 
Nucleic Acids Research  1999;27(10):2091-2098.
It was previously shown that the two members of the cell cycle-regulated histone H4 gene family, H4-1 and H4-2, are replicated at the onset of S phase in the naturally synchronous plasmodium of Physarum polycephalum, suggesting that they are flanked by replication origins. It was further shown that a DNA fragment upstream of the H4-1 gene is able to confer autonomous replication of a plasmid in the budding yeast. In this paper, we re-investigated replication of the unlinked Physarum histone H4 genes by mapping the replication origin of these two loci using alkaline agarose gel and neutral/neutral 2-dimensional agarose gel electrophoreses. We showed that the two replicons containing the H4 genes are simultaneously activated at the onset of S phase and we mapped an efficient, bidirectional replication origin in the vicinity of each gene. Our data demonstrated that the Physarum sequence that functions as an ARS in yeast is not the site of replication initiation at the H4-1 locus. We also observed a stalling of the rightward moving replication fork downstream of the H4-1 gene, in a region where transient topoisomerase II sites were previously mapped. Our results further extend the concept of replication/transcription coupling in Physarum to cell cycle-regulated genes.
PMCID: PMC148428  PMID: 10219081
3.  Low rate of replication fork progression lengthens the replication timing of a locus containing an early firing origin 
Nucleic Acids Research  2007;35(17):5763-5774.
Invariance of temporal order of genome replication in eukaryotic cells and its correlation with gene activity has been well-documented. However, recent data suggest a relax control of replication timing. To evaluate replication schedule accuracy, we detailed the replicational organization of the developmentally regulated php locus that we previously found to be lately replicated, even though php gene is highly transcribed in naturally synchronous plasmodia of Physarum. Unexpectedly, bi-dimensional agarose gel electrophoreses of DNA samples prepared at specific time points of S phase showed that replication of the locus actually begins at the onset of S phase but it proceeds through the first half of S phase, so that complete replication of php-containing DNA fragments occurs in late S phase. Origin mapping located replication initiation upstream php coding region. This proximity and rapid fork progression through the coding region result in an early replication of php gene. We demonstrated that afterwards an unusually low fork rate and unidirectional fork pausing prolong complete replication of php locus, and we excluded random replication timing. Importantly, we evidenced that the origin linked to php gene in plasmodium is not fired in amoebae when php expression dramatically reduced, further illustrating replication-transcription coupling in Physarum.
doi:10.1093/nar/gkm586
PMCID: PMC2034475  PMID: 17717000
4.  Mapping of a Physarum chromosomal origin of replication tightly linked to a developmentally-regulated profilin gene. 
Nucleic Acids Research  1992;20(13):3309-3315.
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.
Images
PMCID: PMC312482  PMID: 1630902
5.  Replicational organization of three weakly expressed loci in Physarum polycephalum 
Nucleic Acids Research  2002;30(11):2261-2269.
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.
PMCID: PMC117180  PMID: 12034812
6.  Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. 
Journal of Bacteriology  1992;174(24):8119-8132.
The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.
Images
PMCID: PMC207551  PMID: 1459960
7.  Comparison of Insertional RNA Editing in Myxomycetes 
PLoS Computational Biology  2012;8(2):e1002400.
RNA editing describes the process in which individual or short stretches of nucleotides in a messenger or structural RNA are inserted, deleted, or substituted. A high level of RNA editing has been observed in the mitochondrial genome of Physarum polycephalum. The most frequent editing type in Physarum is the insertion of individual Cs. RNA editing is extremely accurate in Physarum; however, little is known about its mechanism. Here, we demonstrate how analyzing two organisms from the Myxomycetes, namely Physarum polycephalum and Didymium iridis, allows us to test hypotheses about the editing mechanism that can not be tested from a single organism alone. First, we show that using the recently determined full transcriptome information of Physarum dramatically improves the accuracy of computational editing site prediction in Didymium. We use this approach to predict genes in the mitochondrial genome of Didymium and identify six new edited genes as well as one new gene that appears unedited. Next we investigate sequence conservation in the vicinity of editing sites between the two organisms in order to identify sites that harbor the information for the location of editing sites based on increased conservation. Our results imply that the information contained within only nine or ten nucleotides on either side of the editing site (a distance previously suggested through experiments) is not enough to locate the editing sites. Finally, we show that the codon position bias in C insertional RNA editing of these two organisms is correlated with the selection pressure on the respective genes thereby directly testing an evolutionary theory on the origin of this codon bias. Beyond revealing interesting properties of insertional RNA editing in Myxomycetes, our work suggests possible approaches to be used when finding sequence motifs for any biological process fails.
Author Summary
RNA is an important biomolecule that is deeply involved in all aspects of molecular biology, such as protein production, gene regulation, and viral replication. However, many significant aspects such as the mechanism of RNA editing are not well understood. RNA editing is the process in which an organism's RNA is modified through the insertion, deletion, or substitution of single or short stretches of nucleotides. The slime mold Physarum polycephalum is a model organism for the study of RNA editing; however, hardly anything is known about its editing machinery. We show that the combination of two organisms (Physarum polycephalum and Didymium iridis) can provide a better understanding of insertional RNA editing than one organism alone. We predict several new edited genes in Didymium. By comparing the sequences of the two organisms in the vicinity of the editing sites we establish minimal requirements for the location of the information by which these editing sites are recognized. Lastly, we directly verify a theory for one of the most striking features of the editing sites, namely their codon bias.
doi:10.1371/journal.pcbi.1002400
PMCID: PMC3285571  PMID: 22383871
8.  GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome 
Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progression
In mitotic division, cells duplicate their DNA in S phase to ensure that the proper genetic material is passed on to their progeny. This process of DNA replication is initiated from several hundred specific sites, termed origins of replication, spaced across the genome. It is essential for replication to begin only after G1 and finish before the initiation of anaphase (Blow and Dutta, 2005; Machida et al, 2005). To ensure proper timing, the beginning stages of DNA replication are tightly coupled to the cell cycle through the activity of cyclin-dependent kinases (Nguyen et al, 2001; Masumoto et al, 2002; Sclafani and Holzen, 2007), which promote the accumulation of the pre-RC at the origins and initiate replication. Replication fork movement occurs subsequent to the firing of origins on recruitment of the replicative helicase and the other fork-associated proteins as the cell enters S phase (Diffley, 2004). The replication machinery itself (polymerases, PCNA, etc.) trails behind the helicase, copying the newly unwound DNA in the wake of the replication fork.
One component of the pre-RC, the GINS complex, consists of a highly conserved set of paralogous proteins (Psf1, Psf2, Psf3 and Sld5 (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003)). Previous work suggests that the GINS complex is an integral component of the replication fork and that its interaction with the genome correlates directly to the movement of the fork (reviewed in Labib and Gambus, 2007). Here, we used the GINS complex as a surrogate to measure features of the dynamics of replication—that is, to determine which origins in the genome are active, the timing of their firing and the rates of replication fork progression.
The timing of origin firing and the rates of fork progression have also been investigated by monitoring nascent DNA synthesis (Raghuraman et al, 2001; Yabuki et al, 2002). Origin firing was observed to occur as early as 14 min into the cell cycle and as late as 44 min (Raghuraman et al, 2001). A wide range of nucleotide incorporation rates (0.5–11 kb/min) were observed, with a mean of 2.9 kb/min (Raghuraman et al, 2001), whereas a second study reported a comparable mean rate of DNA duplication of 2.8±1.0 kb/min (Yabuki et al, 2002). In addition to these observations, replication has been inferred to progress asymmetrically from certain origins (Raghuraman et al, 2001). These data have been interpreted to mean that the dynamics of replication fork progression are strongly affected by local chromatin structure or architecture, and perhaps by interaction with the machineries controlling transcription, repair and epigenetic maintenance (Deshpande and Newlon, 1996; Rothstein et al, 2000; Raghuraman et al, 2001; Ivessa et al, 2003). In this study, we adopted a complementary ChIP-chip approach for assaying replication dynamics, in which we followed GINS complexes as they traverse the genome during the cell cycle (Figure 1). These data reveal that GINS binds to active replication origins and spreads bi-directionally and symmetrically as S phase progresses (Figure 3). The majority of origins appear to fire in the first ∼15 min of S phase. A small fraction (∼10%) of the origins to which GINS binds show no evidence of spreading (category 3 origins), although it remains possible that these peaks represent passively fired origins (Shirahige et al, 1998). Once an active origin fires, the GINS complex moves at an almost constant rate of 1.6±0.3 kb/min. Its movement through the inter-origin regions is consistent with that of a protein complex associated with a smoothly moving replication fork. This progression rate is considerably lower and more tightly distributed than those inferred from previous genome-wide measurements assayed through nascent DNA production (Raghuraman et al, 2001; Yabuki et al, 2002). Our study leads us to a different view of replication fork dynamics wherein fork progression is highly uniform in rate and little affected by genomic location.
In this work, we also observe a large number of low-intensity persistent features at sites of high transcriptional activity (e.g. tRNA genes). We were able to accurately simulate these features by assuming they are the result of low probability arrest of replication forks at these sites, rather than fork pausing (Deshpande and Newlon, 1996). The extremely low frequency of these events in wild-type cells suggests they are due to low probability stochastic occurrences during the replication process. It is hoped that future studies will resolve whether these persistent features indeed represent rare instances of fork arrest, or are the result of some alternative process. These may include, for example, the deposition of GINS complexes (or perhaps more specifically Psf2) once a pause has been resolved.
In this work, we have made extensive use of modeling to test a number of different hypotheses and assumptions. In particular, iterative modeling allowed us to infer that GINS progression is uniform and smooth throughout the genome. We have also demonstrated the potential of simulations for estimating firing efficiencies. In the future, extending such firing efficiency simulations to the whole genome should allow us to make correlations with chromosomal features such as nucleosome occupancy. Such correlations may help in determining factors that govern the probability of replication initiation throughout the genome.
Previous studies have led to a picture wherein the replication of DNA progresses at variable rates over different parts of the budding yeast genome. These prior experiments, focused on production of nascent DNA, have been interpreted to imply that the dynamics of replication fork progression are strongly affected by local chromatin structure/architecture, and by interaction with machineries controlling transcription, repair and epigenetic maintenance. Here, we adopted a complementary approach for assaying replication dynamics using whole genome time-resolved chromatin immunoprecipitation combined with microarray analysis of the GINS complex, an integral member of the replication fork. Surprisingly, our data show that this complex progresses at highly uniform rates regardless of genomic location, revealing that replication fork dynamics in yeast is simpler and more uniform than previously envisaged. In addition, we show how the synergistic use of experiment and modeling leads to novel biological insights. In particular, a parsimonious model allowed us to accurately simulate fork movement throughout the genome and also revealed a subtle phenomenon, which we interpret as arising from low-frequency fork arrest.
doi:10.1038/msb.2010.8
PMCID: PMC2858444  PMID: 20212525
cell cycle; ChIP-chip; DNA replication; replication fork; simulation
9.  Site-specific initiation of DNA replication within the non-transcribed spacer of Physarum rDNA. 
Nucleic Acids Research  1995;23(9):1447-1453.
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.
Images
PMCID: PMC306881  PMID: 7784195
10.  Selective initiation of replication at origin sequences of the rDNA molecule of Physarum polycephalum using synchronous plasmodial extracts. 
Nucleic Acids Research  1989;17(20):8343-8362.
A cell-free system using synchronous plasmodial extracts initiates replication selectively on the 60 kb rDNA palindrome of Physarum polycephalum. Preferential labeling of rDNA fragments by nuclear extracts, in which elongation is limited, indicates that initiation occurs at two positions corresponding to in vivo origins of replication estimated by electron microscopy. Both nuclear and whole plasmodial extracts initiate selectively within a plasmid, pPHR21, containing one of these origins. In this plasmid bubbles expand bidirectionally and generate DpnI-resistant DNA. Extracts made at prophase or early S phase, times when the nucleolus is disorganized, are most active in pPHR21 replication. Mapping positions of replication bubbles locates the initiation point in a 3.2 kb BstEII fragment at the upstream border of a series of 31 bp repeats 2.4 kb from the initiation point for ribosomal gene transcription.
Images
PMCID: PMC334968  PMID: 2813064
11.  USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator 
PLoS Biology  2012;10(3):e1001277.
A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.
The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the β-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.
Author Summary
All eukaryotic organisms must duplicate their genome precisely once before cell division. This occurs according to an established temporal program during S-phase (when DNA synthesis takes place) of the cell cycle. In vertebrates, this program is regulated at the level of large chromosomal domains ranging from 200 kb to 2 Mb, but the molecular mechanisms that control the temporal firing order of animal replication origins are not clearly understood. Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cis-regulatory DNA elements that is able to shift the timing of a naturally “mid-late” replicated region to “mid-early.” This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein. The additional presence of a strongly transcribed gene shifted the region towards an even earlier replication time, suggesting cooperation between cis-elements when establishing temporal programs of replication. We speculate that USF binding sequences cooperate with sites of replication initiation and transcribed genes to promote the establishment of early replicating domains along vertebrate genomes.
doi:10.1371/journal.pbio.1001277
PMCID: PMC3295818  PMID: 22412349
12.  Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences. 
Journal of Bacteriology  1993;175(15):4843-4850.
The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously. The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli. Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E. coli and only slightly affects the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system. Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively. The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins. We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids. It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating.
Images
PMCID: PMC204937  PMID: 8393008
13.  Asynchronous Replication, Mono-Allelic Expression, and Long Range Cis-Effects of ASAR6 
PLoS Genetics  2013;9(4):e1003423.
Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.
Author Summary
Mammalian chromosomes are duplicated every cell cycle during a precise temporal DNA replication program. Thus, every chromosome contains regions that are replicated early and other regions that are replicated late during each S phase. Most of the genes, present in two copies on homologous chromosomes, replicate synchronously during each S phase. Exceptions to this rule are genes located on X chromosomes, genetically imprinted genes, and genes subject to allelic exclusion. Thus, all mono-allelically expressed genes are subject to asynchronous replication, where one allele replicates before the other. Perhaps the best-studied example of asynchronous replication in mammals occurs during X inactivation in female cells. A large non-coding RNA gene called XIST, located within the X inactivation center, controls the transcriptional silencing and late replication of the inactive X chromosome. We have identified a locus on human chromosome 6 that shares many characteristics with XIST. This chromosome 6 locus encodes a large intergenic non-coding RNA gene, ASAR6, which displays random mono-allelic expression, asynchronous replication, and controls the mono-allelic expression of other genes on chromosome 6. Our work supports a model in which all mammalian chromosomes contain similar cis-acting loci that function to ensure proper chromosome replication, mitotic condensation, mono-allelic expression, and stability of individual chromosomes.
doi:10.1371/journal.pgen.1003423
PMCID: PMC3617217  PMID: 23593023
14.  Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids. 
Journal of Bacteriology  1994;176(16):5022-5032.
The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregulation with replication and transfer genes indicates that they are likely to be important for RK2. In this report, we describe molecular and genetic studies on kilC. We determined the nucleotide sequence of the kilC region, which is located between the origin of vegetative replication (oriV) and transposon Tn1 on RK2. Primer extension analysis identified the transcriptional start site and showed that a sequence corresponding to a strong sigma 70 promoter is functional. The abundance of RNA initiated from the kilC promoter is reduced in the presence of korA and korC, as predicted from genetic analysis of kilC regulation. The first gene of the kilC operon (klcA) is sufficient to express the host-lethal phenotype of the kilC determinant in the absence of korA and korC. By comparing RK2 to the related IncP alpha plasmids pUZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determinant is normally part of an autoregulated operon of three genes: klcA, klcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide that is related to the ArdB antirestriction protein of the IncN plasmid pKM101, suggesting a role for klcA in the broad host ranges of IncP alpha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant similarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp upstream of the kilC promoter is a 10th copy of the 17-bp oriV iteron sequence in inverted orientation relative to that of the other nine iterons of oriV. Iteron 10 is identical to the "orphan" iteron 1, and both have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise the possibility that these iterons promote the formation of a DNA loop via protein-protein interactions by bound TrfA and lead us to propose that they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon in wild-type IncP alpha plasmids is saturated by the kilC, kilE, and kilA loci arranged in four kor-regulated operons encoding a total of 12 genes.
Images
PMCID: PMC196341  PMID: 7519596
15.  Multiple Functional Elements Comprise a Mammalian Chromosomal Replicator 
Molecular and Cellular Biology  2003;23(5):1832-1842.
The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5′ to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.
doi:10.1128/MCB.23.5.1832-1842.2003
PMCID: PMC151694  PMID: 12589000
16.  Replication Fork Reversal after Replication–Transcription Collision 
PLoS Genetics  2012;8(4):e1002622.
Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.
Author Summary
Genomes are duplicated prior to cell division by DNA replication, and in all organisms replication impairment leads to chromosome instability. In bacteria, replication and transcription take place simultaneously, and in eukaryotes house-keeping genes are expressed during the S-phase; consequently, transcription is susceptible to impair replication progression. Here, we increase head-on replication–transcription collisions on the bacterial chromosome by inversion of a ribosomal operon (rrn). We show that only one recombination protein is required for growth when the rrn genes are highly expressed: the RecBCD complex, an exonuclease/recombinase that promotes degradation and RecA-dependent homologous recombination of linear DNA. In the absence of RecBCD, we observe linear DNA that ends in the collision region. This linear DNA is composed of only the origin-proximal region of the inverted rrn operon, indicating that it results from fork breakage. It is partly RuvABC-dependent (i.e. produced by the E. coli Holliday junction resolvase), indicating that blocked forks are reversed. The linear DNA ends up at the inverted rrn locus only if the RecJ exonuclease is inactivated; otherwise it is degraded, with major products ending in other upstream rrn operons, indicating that DNA degradation is slowed down by ribosomal operon sequences.
doi:10.1371/journal.pgen.1002622
PMCID: PMC3320595  PMID: 22496668
17.  5-Methyldeoxycytidine in the Physarum minichromosome containing the ribosomal RNA genes. 
Nucleic Acids Research  1984;12(3):1501-1515.
5-Methyldeoxycytidine (5MC) was analyzed by high pressure liquid chromatography (HPLC) and by restriction enzyme digestion in rDNA isolated from Physarum polycephalum. rDNA from Physarum M3C strain microplasmodia has a significant 5MC content (about half that of the whole genomic DNA). This rDNA contains many C5MCGG sites because it is clearly digested further by Msp I than by Hpa II. However, most 5MC is in other sites. In particular, alternating CG sequences appear to be highly methylated. HPLC of deoxyribonucleosides shows tha most of the transcribed regions contain little or no 5MC. Restriction digestion indicates that there is little or no 5MC in any of the transcribed regions including the transcription origin and adjacent sequences. Over 90% of the total 5MC is in or near the central nontranscribed spacer and most methylated restriction sites are in inverted repeats of this spacer. rDNA is very heterogeneous with respect to 5MC. The 5MC pattern doesn't appear to change with inactivation of the rRNA genes during reversible differentiation from microplasmodia (growing) to microsclerotia (dormant), showing that inactivation is due to changes in other chromatin variables. The 5MC pattern is different between Physarum strains. The possible involvement of this 5MC in rDNA chromatin structure and in cruciform and Z-DNA formation is discussed.
Images
PMCID: PMC318592  PMID: 6322108
18.  Transcriptomic changes arising during light-induced sporulation in Physarum polycephalum 
BMC Genomics  2010;11:115.
Background
Physarum polycephalum is a free-living amoebozoan protist displaying a complex life cycle, including alternation between single- and multinucleate stages through sporulation, a simple form of cell differentiation. Sporulation in Physarum can be experimentally induced by several external factors, and Physarum displays many biochemical features typical for metazoan cells, including metazoan-type signaling pathways, which makes this organism a model to study cell cycle, cell differentiation and cellular reprogramming.
Results
In order to identify the genes associated to the light-induced sporulation in Physarum, especially those related to signal transduction, we isolated RNA before and after photoinduction from sporulation- competent cells, and used these RNAs to synthesize cDNAs, which were then analyzed using the 454 sequencing technology. We obtained 16,669 cDNAs that were annotated at every computational level. 13,169 transcripts included hit count data, from which 2,772 displayed significant differential expression (upregulated: 1,623; downregulated: 1,149). Transcripts with valid annotations and significant differential expression were later integrated into putative networks using interaction information from orthologs.
Conclusions
Gene ontology analysis suggested that most significantly downregulated genes are linked to DNA repair, cell division, inhibition of cell migration, and calcium release, while highly upregulated genes were involved in cell death, cell polarization, maintenance of integrity, and differentiation. In addition, cell death- associated transcripts were overrepresented between the upregulated transcripts. These changes are associated to a network of actin-binding proteins encoded by genes that are differentially regulated before and after light induction.
doi:10.1186/1471-2164-11-115
PMCID: PMC2837032  PMID: 20163733
19.  DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons. 
Nucleic Acids Research  1978;5(3):713-721.
The direction of replication of DNA within replicons of Physarum polycephalum was studied by pulse-labelling with 5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) -photolysis and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within replicons at all stages of completion is split in two equal halves upon UV irradiation when BrdUrd was given at the time of initiation of DNA synthesis. This shows that replication within replicons of Physarum polycephalum starts at an origin located in the center of each unit, proceeding bidirectionally from this origin.
PMCID: PMC342018  PMID: 565510
20.  Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein. 
Journal of Bacteriology  1996;178(5):1420-1429.
The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.
PMCID: PMC177817  PMID: 8631720
21.  Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix. 
Nucleic Acids Research  1983;11(4):1181-1195.
We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.
PMCID: PMC325785  PMID: 6828380
22.  Protein tightly bound near the termini of the Physarum extrachromosomal rDNA palindrome 
The Journal of Cell Biology  1981;91(1):309-314.
The genes coding for ribosomal RNa in plasmodia of Physarum polycephalum are arranged palindromically on extrachromosomal rDNA molecules of 61 kb (kilobasepairs). Incubation of mildly extracted rDNA with the 125I Bolton-Hunter reagent results in incorporation of label not removed by SDS, CsCl, or various organic solvents. Labeled protein is preferentially associated with terminal rDNA restriction fragments, as detected after gel electrophoresis of the DNA. Antibody reaction with dinitrophenylated protein-rDNA complexes allows visualization of protein located from 1 to 2 kb from the termini, in a region containing multiple inverted repeat sequences and single-strand gaps. DNase I treatment of either rDNA or rDNA termini releases primarily two labeled protein bands of 5,000 and 13,000 daltons as well as less prominent bands of higher molecular weight. We discuss mechanisms for involvement of terminal protein in replication of 3' ends and chromosomal integration of the rDNA.
PMCID: PMC2111919  PMID: 7298725
23.  Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle. 
Molecular and Cellular Biology  1987;7(5):1933-1937.
During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.
Images
PMCID: PMC365298  PMID: 3600651
24.  Inheritance of extrachromosomal rDNA in Physarum polycephalum. 
Molecular and Cellular Biology  1983;3(4):635-642.
In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.
Images
PMCID: PMC368579  PMID: 6855770
25.  Initiation of DNA replication at the human β-globin 3′ enhancer 
Nucleic Acids Research  2005;33(14):4412-4424.
The origin of DNA replication in the human β-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the β-globin 3′ enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3′ enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3′ enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3′ enhancer also cooperates with elements in an expressing HS3β/γ-globin construct to initiate replication. These data indicate that the β-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.
doi:10.1093/nar/gki747
PMCID: PMC1183104  PMID: 16085752

Results 1-25 (852740)