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At least 739 records · Page 41Linked to original sources
PubMed2003

An episomal mammalian replicon: sequence-independent binding of the origin recognition complex.

An extrachromosomally replicating plasmid was used to investigate the specificity by which the origin recognition complex (ORC) interacts with DNA sequences in mammalian cells in vivo. We first showed that the plasmid pEPI-1 replicates semiconservatively in a once-per-cell-cycle manner and is stably transmitted over many cell generations in culture without selection. Chromatin immunoprecipitations and quantitative polymerase chain reaction analysis revealed that, in G1-phase cells, Orc1 and Orc2, as well as Mcm3, another component of the prereplication complex, are bound to multiple sites on the plasmid. These binding sites are functional because they show the S-phase-dependent dissociation of Orc1 and Mcm3 known to be characteristic for prereplication complexes in mammalian cells. In addition, we identified replicative nascent strands and showed that they correspond to many plasmid DNA regions. This work has implications for current models of replication origins in mammalian systems. It indicates that specific DNA sequences are not required for the chromatin binding of ORC in vivo. The conclusion is that epigenetic mechanisms determine the sites where mammalian DNA replication is initiated.

Animals↗
PubMed2004

A topological view of the replicon.

The replication of circular DNA faces topological obstacles that need to be overcome to allow the complete duplication and separation of newly replicated molecules. Small bacterial plasmids provide a perfect model system to study the interplay between DNA helicases, polymerases, topoisomerases and the overall architecture of partially replicated molecules. Recent studies have shown that partially replicated circular molecules have an amazing ability to form various types of structures (supercoils, precatenanes, knots and catenanes) that help to accommodate the dynamic interplay between duplex unwinding at the replication fork and DNA unlinking by topoisomerases.

DNA Replication↗
PubMed2002

The origin of a developmentally regulated Igh replicon is located near the border of regulatory domains for Igh replication and expression.

The 3' Ig heavy chain locus (Igh) regulatory region is the most downstream known element of the murine Igh gene cluster. We report here that the nearest non-Igh genes-Crip, Crp2, and Mta1-are located approximately 70 kb further downstream and are beyond the end of the domain of Igh transcriptional regulation. We have localized an origin of replication in MEL cells to a 3-kb segment located between the 3' Igh regulatory region and Crip. Sequences downstream of this origin are replicated by forks that move in both directions. Sequences upstream of this origin (Igh-C, -D, and -J) are replicated in a single direction through a 500-kb segment in which no active bidirectional origins can be detected. We propose that this origin may lie at or near the end of the Igh regulation domain.

Animals↗
PubMed1999

Mechanism of DNA segregation in prokaryotes: ParM partitioning protein of plasmid R1 co-localizes with its replicon during the cell cycle.

The parA locus of plasmid R1 encodes a prokaryotic centromere-like system that mediates genetic stabilization of plasmids by an unknown mechanism. The locus codes for two proteins, ParM and ParR, and a centromere-like DNA region (parC) to which the ParR protein binds. We showed recently that ParR mediates specific pairing of parC-containing DNA molecules in vitro. To obtain further insight into the mechanism of plasmid stabilization, we examined the intracellular localization of the components of the parA system. We found that ParM forms discrete foci that localize to specific cellular regions in a simple, yet dynamic pattern. In newborn cells, ParM foci were present close to both cell poles. Concomitant with cell growth, new foci formed at mid-cell. A point mutation that abolished the ATPase activity of ParM simultaneously prevented cellular localization and plasmid partitioning. A parA-containing plasmid localized to similar sites, i.e. close to the poles and at mid-cell, thus indicating that the plasmid co-localizes with ParM. Double labelling of single cells showed that plasmid DNA and ParM indeed co-localize. Thus, our data indicate that parA is a true partitioning system that mediates pairing of plasmids at mid-cell and subsequently moves them to the cell poles before cell division.

Adenosine Triphosphatases↗
PubMed2001

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

Amino Acid Sequence↗
PubMed1990

Ambiguities in results obtained with 2D gel replicon mapping techniques.

Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11). The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment. We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.

DNA, Fungal↗
PubMed2000

Enhanced flexibility and aphidicolin-induced DNA breaks near mammalian replication origins: implications for replicon mapping and chromosome fragility.

Common fragile sites are chromosomal loci prone to breakage and rearrangement that can be induced by aphidicolin, an inhibitor of DNA polymerases. Within these loci, sites of preferential DNA breaks were proposed to correlate with peaks of enhanced DNA flexibility, the function of which remains elusive. Here we show that mammalian DNA replication origins are enriched in peaks of enhanced flexibility. This finding suggests that the search for these features may help in the mapping of replication origins, and we present evidence supporting this hypothesis. The association of peaks of flexibility with replication origins also suggests that some origins may associate with minor levels of fragility. As shown here, an increased sensitivity to aphidicolin was found near two mammalian DNA replication origins.

Animals↗
PubMed2000

Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus.

Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).

Defective Viruses↗
PubMed1999

Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons.

Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.

Amino Acid Sequence↗
PubMed2003

Only one catalase, katG, is detectable in Rhizobium etli, and is encoded along with the regulator OxyR on a plasmid replicon.

The plasmid-borne Rhizobium etli katG gene encodes a dual-function catalase-peroxidase (KatG) (EC 1.11.1.7) that is inducible and heat-labile. In contrast to other rhizobia, katG was shown to be solely responsible for catalase and peroxidase activity in R. etli. An R. etli mutant that did not express catalase activity exhibited increased sensitivity to hydrogen peroxide (H(2)O(2)). Pre-exposure to a sublethal concentration of H(2)O(2) allowed R. etli to adapt and survive subsequent exposure to higher concentrations of H(2)O(2). Based on a multiple sequence alignment with other catalase-peroxidases, it was found that the catalytic domains of the R. etli KatG protein had three large insertions, two of which were typical of KatG proteins. Like the katG gene of Escherichia coli, the R. etli katG gene was induced by H(2)O(2) and was important in sustaining the exponential growth rate. In R. etli, KatG catalase-peroxidase activity is induced eightfold in minimal medium during stationary phase. It was shown that KatG catalase-peroxidase is not essential for nodulation and nitrogen fixation in symbiosis with Phaseolus vulgaris, although bacteroid proteome analysis indicated an alternative compensatory mechanism for the oxidative protection of R. etli in symbiosis. Next to, and divergently transcribed from the catalase promoter, an ORF encoding the regulator OxyR was found; this is the first plasmid-encoded oxyR gene described so far. Additionally, the katG promoter region contained sequence motifs characteristic of OxyR binding sites, suggesting a possible regulatory mechanism for katG expression.

Amino Acid Sequence↗
PubMed2006

Inhibition of type I allergic responses with nanogram doses of replicon-based DNA vaccines.

BACKGROUND: Allergic diseases have become a major public health problem in developed countries; yet, no reliable, safe and consistently effective treatment is available. DNA immunization has been shown to prevent and balance established allergic responses, however, the high dose of conventional DNA vaccines necessary for the induction of anti-allergic reactions and their poor immunogenicity in primates require the development of new allergy DNA vaccines. We evaluated protective and therapeutic effects of a Semliki-Forest Virus replicase-based vs a conventional DNA vaccine in BALB/c mice using the model allergen beta-galactosidase. METHODS: Immunoglobulin (Ig)E suppression was determined by a basophil release assay as an in vitro correlate for allergen-specific crosslinking capacity of IgE reflecting the in vivo situation in an allergic individual. Th1 memory responses were measured by cytokine detection via enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). RESULTS: Nanogram amounts of a replicase-based vector triggered a Th1 response comparable with that achieved with the injection of 20,000-times more copies of a conventional DNA plasmid, and induced IgE suppression in both a protective and a therapeutic setting. CONCLUSIONS: Replicase-based DNA vaccines fulfill the stringent criteria for an allergy DNA vaccine, i.e. low dose, strong Th1 immunogenicity and memory, lack of 'therapy-induced' IgE production and anaphylactic side effects. Moreover, by triggering apoptosis in transfected cells, their unique 'immunize and disappear' feature minimizes the hypothetical risks of genomic integration or induction of autoimmunity.

Allergens↗
PubMed2001

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon.

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.

DNA Probes↗