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PubMed1984

Transposition and transposition immunity of transposon Tn3 derivatives having different ends.

Novel Tn1/3 derivatives that contained either two left- or two right-hand ends of the transposon were constructed in a small plasmid. Both transposed at reasonable frequencies to give normal transposition products, suggesting that only the 38-bp inverted repeats of Tn3 are essential for transposition. Plasmids containing transposon derivatives with only one end (either left or right) undergo transposase-dependent transposition between replicons at much lower frequencies, resulting in co-integrate molecules in which there is no substantial duplication of transposon DNA and that appear to be simple fusions of the two plasmids. Both the right and left halves of the transposon are separately able to confer transposition immunity to the plasmid, this immunity being inseparably linked to transposition proficiency and specificity.

DNA Transposable Elements↗
PubMed1991

Expression of the replication protein Arp of phasyl shows dual regulation by an antisense promoter.

Phasyl is the smallest naturally occurring replicon found so far in Escherichia coli. It encodes a protein which is essential for autonomous replication (Arp). The transcriptional start of the arp gene was mapped. A strong antisense promoter was found in close proximity to the arp promoter. The inactivation of this promoter led in cis to a strong increase of the transcription of the arp gene and to the inactivation of autonomous replication of phasyl. The product of the antisense promoter is an 83 nt RNA molecule, which is not translated. The antisense RNA led in trans to the inhibition of the translation of the arp mRNA, presumably mediated by the formation of an RNA-RNA hybrid in which the Shine-Dalgarno sequence of the arp transcript is sequestered. The expression of the arp gene is thus controlled by two negatively acting mechanisms: it is subject to a transcriptional control in cis exerted by the antisense promoter and to a translational control in trans mediated by the antisense RNA. Inactivation of one mechanism of control cannot be compensated by the remaining one.

Cloning, Molecular↗
PubMed1981

The effect of added H1 histone and polylysine on DNA synthesis and cell division of cultured mammalian cells.

Addition of H1 histone or polylysine (10 microgram/ml) to cultured Friend erythroleukemia cells or to two mouse lymphoma cell lines (el-4 and S-49) increased levels of cell division in these cultures. There is a stimulation of incorporation of labeled thymidine into DNA in cultures containing H1 histone and polylysine. DNA fiber autoradiographic experiments revealed that replicon size is decreased in the cells cultured with H1 histone and polylysine at later periods of culture.

Animals↗
PubMed1984

Histone protein and DNA synthesis in HeLa cells after thermal shock.

Exposure of suspension-cultured HeLa cells to a 45 degrees thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (Do = 7 min) and a less sensitive (Do = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (Do in excess of 200 min). When HeLa cells were heated at 45 degrees for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.

DNA↗
PubMed1989

Altered formation of DNA replication intermediates in cells growing in different culture conditions.

In human melanoma cells one can detect two discrete DNA replication intermediates, 10-kb DNA intermediates and Okakzaki fragments. Both intermediates are seen when cells are rapidly growing in medium supplemented with fetal calf serum. When the medium is supplemented with newborn calf serum, one can detect Okakzaki fragments but not 10-kb DNA intermediates. In contrast we do not detect changes in the replicon sizes in the two media.

Cells, Cultured↗
PubMed1996

The ori of filamentous phage phi Lf is located within the gene encoding the replication initiation protein.

phi Lf is a filamentous phage of Xanthomonas campestris pv. campestris. In this study, the origin for phi Lf replication was located in a 121-bp TaqI fragment within gII, the gene encoding the replication initiation protein. This fragment, ligating with a GmR catridge, was able to be maintained as a plasmid (pT2) in strain Xc17 with the gII being provided in trans. ssDNA of pT2 was detected in the cells, indicating that pT2 may replicate by a rolling circle replication mechanism. Upon superinfection of Xc17 containing pT2 with phi Lf, transducing particles containing ssDNA of pT2 were released, suggesting the presence of packaging signal in the 121-bp TaqI fragment. This fragment contains a sequence homologous to the nicking sites for superfamily I Rep proteins of the rolling circle-replicating replicons, in concert with the presence of conserved amino acid sequence motifs of the superfamily I Rep proteins in the phi Lf gIIP.

Bacteriophages↗
PubMed1995

Differential aldehyde sensitivity of newly replicated chromatin from Drosophila melanogaster embryos.

By using a series of formalin concentrations we have found that high aldehyde levels in the fixation buffer of Miller spreads are correlated with the appearance of nonnucleosomal stretches in newly replicated chromatin of embryos from Drosophila melanogaster. These nucleosome-free gaps are found 0-500 nm behind the replication fork and do not correspond to naked DNA. The analysis of the distribution of nucleosome-free gaps on newly replicated DNA has revealed some structural details about the maturation of nucleosomes and provides direct evidence that parental nucleosomes have an altered structure at the replication fork. Finally, these stretches of nonnucleosomal chromatin are located in a trans disposition inside the active replicon, although there exists a considerable variability.

Animals↗
PubMed2000

Dispersive initiation of replication in the Chinese hamster rhodopsin locus.

Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells.

Animals↗
PubMed1993

Precise deletions in large bacterial genomes by vector-mediated excision (VEX). The trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts.

We have developed a simple and efficient method of vector-mediated excision (VEX) for in vivo generation of precisely defined deletions in large bacterial genomes. The system uses homologous recombination with small cloned fragments on specialized pVEX plasmids to insert directly repeated bacteriophage P1 loxP sites at positions flanking the region to be deleted. In the presence of Cre recombinase, the loxP sites are efficiently recombined to produce the deletion. Deletion endpoints can be directed to specific nucleotides because they are defined by the termini of small homology-bearing fragments cloned into the pVEX plasmids. We have used VEX to delete trfA, the only known replication initiator gene of the 56.4 kb broad host-range plasmid RK2. The RK2 delta trfA mutant was found to be conjugation proficient, but unable to replicate in the RK2 hosts Acinetobacter calcoaceticus, Caulobacter crescentus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Rhizobium meliloti, or Rhodobacter sphaeroides. These results show that trfA is essential for replication in these hosts and indicate that the broad host-range of RK2 does not involve multiple replicons.

Conjugation, Genetic↗
PubMed1996

The RecG branch migration protein of Escherichia coli dissociates R-loops.

The RuvAB and RecG proteins of Escherichia coli promote branch migration of Holliday junction intermediates in genetic recombination. Both are structure-specific helicases that unwind and rewind DNA at the junction point. The helicase activities of these proteins were investigated using RNA:DNA hybrid molecules. RuvAB catalyses the unwinding of RNA:DNA partial duplexes of at least 218 bp in a reaction that requires both RuvA and RuvB, ATP and Mg2+. RecG failed to unwind these substrates even when the duplex region was reduced to 35 bp. In contrast, RecG rapidly removes a 218 nt RNA from an R-loop substrate, whereas RuvAB does not. RecG's ability to dissociate R-loops is correlated with an ability to reduce the copy number of pUC plasmids and other constructs based on the ColE1 replicon. Copy number is reduced severely when the plasmid carries recG+. RecG is assumed to reduce copy number by interfering with RNA II's ability to form an R-loop at the plasmid origin of replication and prime DNA synthesis. The dissociation of R-loops by RecG is discussed in terms of the functions needed to promote recombination and to prime DNA replication at D-loops formed during the early stages of RecA-mediated recombination.

Bacterial Proteins↗
PubMed1998

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Algorithms↗
PubMed1999

Inversion/dimerization of plasmids mediated by inverted repeats.

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8 %) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

Base Sequence↗
PubMed2000

Molecular clocks reduce plasmid loss rates: the R1 case.

Plasmids control their replication so that the replication frequency per plasmid copy responds to the number of plasmid copies per cell. High sensitivity amplification in replication response to copy number deviations generally reduces variation in copy numbers between different single cells, thereby reducing the plasmid loss rate in a cell population. However, experiments show that plasmid R1 has a gradual, insensitive replication control predicting considerable copy number variation between single cells. The critical step in R1 copy number control is regulation of synthesis of a rate-limiting cis-acting replication protein, RepA. De novo synthesis of a large number of RepA molecules is required for replication, suggesting that copy number control is exercised at multiple steps. In this theoretical kinetic study we analyse R1 multistep copy number control and show that it results in the insensitive replication response found experimentally but that it at the same time effectively prohibits the existence of only one plasmid copy in a dividing cell. In combination with the partition system of R1, this can lead to very high segregational stability. The R1 control mechanism is compared to the different multistep copy number control of plasmid ColE1 that is based on conventional sensitivity amplification. This implies that while copy number control for ColE1 efficiently corrects for fluctuations that have already occurred, R1 copy number control prevents their emergence in cells that by chance start their cycle with only one plasmid copy. We also discuss how regular, clock-like, behaviour of single plasmid copies becomes hidden in experiments probing collective properties of a population of plasmid copies because the individual copies are out of phase. The model is formulated using master equations, taking a stochastic approach to regulation, but the mathematical formalism is kept to a minimum and the model is simplified to its bare essence. This simplicity makes it possible to extend the analysis to other replicons with similar design principles.

Bacteriocin Plasmids↗
PubMed2001

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form.

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.

Base Sequence↗
PubMed1999

Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.

Animals↗
PubMed1993

Identification and characterization of a mobilization gene in the streptococcal plasmid, pVA380-1.

The 4.2-kb cryptic streptococcal plasmid, pVA380-1, has a broad host range and replicates via a rolling circle mode. The nucleotide base sequence of the 2.5-kb basic replicon of this plasmid, containing a plus origin, a minus origin, and a rep gene, was reported previously. The completed nucleotide base sequence of pVA380-1 revealed the presence of two additional open reading frames, ORF2 and ORF3. The protein predicted by ORF2 shared 74% amino acid identity with the Mob protein of the Group B streptococcal plasmid, pMV158. The 170 bases immediately 5' to ORF2 and to mob of pMV158 shared 87% homology and included a putative -10 region and a RSa site. A derivative of pVA380-1 into which a kanamycin resistance gene was inserted outside of the above 170 base region and ORF2 was mobilized by the conjugative streptococcal plasmid, pAM beta 1, into Enterococcus faecalis and Streptococcus sobrinus. Interruption of ORF2 by the same kanamycin resistance gene abolished the mobilizability of pVA380-1.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.

Base Sequence↗
PubMed1996

Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.

Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.

Animals↗