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At least 397 records · Page 22Linked to original sources
PubMed2006

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

Burkholderia↗
PubMed1990

Circular single-stranded RNA replicon in Saccharomyces cerevisiae.

Circular RNA replicons have been reported in plants and, in one case, in animal cells. We describe such an element in yeast. In certain yeast strains, a 20S RNA species appears on transfer of cells to acetate medium. This phenotype shows cytoplasmic (non-Mendelian) inheritance and the 20S RNA is associated with 23-kDa protein subunits as a 32S particle. We demonstrate that yeast 20S RNA is an independent replicon with no homology to host genomic, mitochondrial, or 2-microns plasmid DNA or to the L-A, L-BC, or M1 double-stranded RNA viruses of yeast. The circularity of the 20S RNA is shown by the apparent absence of 3' and 5' ends, by two-dimensional gel electrophoresis, and by electron microscopy. Replication of yeast 20S RNA proceeds through an RNA-RNA pathway, and a 10,000-fold amplification occurs on shift to acetate medium. The copy number of 20S RNA is also reduced severalfold by the SKI gene products, a host antiviral system that also lowers the copy numbers of yeast double-stranded RNA viruses. Yeast 20S RNA and the hepatitis delta virus show some similarities.

Genotype↗
PubMed1999

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Closterovirus↗
PubMed2006

Inhibition of hcv replication in HCV replicon by shRNAs.

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.

Hepacivirus↗
PubMed2003

Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system.

The use of type I interferon (IFN), in combination with ribvirin, to treat chronic hepatitis C virus (HCV) infection has many drawbacks that prevent widespread application, ultimately leading to a significant unmet clinical need. Potential improvements in IFN therapy through targeted delivery, molecular alteration, and combination with other agents are ongoing in an attempt to decrease adverse effects and increase efficacy. In this report, the HCV replicon cell culture system was used to assess potential synergistic antiviral effects of multiple IFN species when administered in combination. Quantitative analysis of HCV replicon RNA by TaqMan (PE Applied Biosystems, Foster City, CA) and qualitative analysis of HCV protein expression were used to measure the antiviral efficacy of individual and combination IFN treatments, and synergistic responses of IFN combinations were determined through statistical analysis of the TaqMan results. We found that when administered simultaneously, type I/II IFN combinations (IFN-alpha2b + IFN-gamma or IFN-beta + IFN-gamma) resulted in dramatic antiviral synergy, whereas a type I/I combination (IFN-alpha2b + IFN-beta) demonstrated a slightly antagonistic profile. The synergistic effect is likely due to differential cell surface receptors and signaling pathways employed by types I and II IFNs. Conversely, all type I IFN species bind the same receptor and signal through similar pathways, possibly accounting for the nearly additive response observed. In support of this hypothesis, IFN treatment resulted in differential induction of Stat1 phosphorylation at Tyr 701. In conclusion, simultaneous type I/II IFN combination treatment may allow an overall decreased effective IFN dose, which may reduce the side effect profiles that hinder current therapy.

Antiviral Agents↗
PubMed1987

Mode of replicon fusion mediated by the duplicated insertion sequence IS21 in Escherichia coli.

The insertion sequence IS21 (2.1 kb) originating from the broad-host-range IncP plasmid R68 transposes infrequently; by contrast, the IS21 tandem repeat found on the derivative R68.45 is highly active in transpositional mobilization of other replicons in a variety of Gram-negative bacteria. The mobilized plasmids are joined to R68.45 by single IS21 copies in direct orientation. The formation of IS21 tandem duplications was observed in cointegrates between R68.45 and pBR325::IS21 and also in an RP1::IS21 plasmid derivative in which a segment located between two directly repeated copies of IS21 was deleted spontaneously. We speculate that IS21 tandem repeats can arise when the termini of two IS21 elements are specifically joined in a transposition or deletion event. A resistance gene flanked by two IS21 elements in direct orientation did not behave as a transposon. The omega fragment carrying transcription and translation stop signals was inserted into various sites of the IS21 tandem repeat; in this way it could be shown that the left IS21 element (which is next to the kanamycin resistance gene in R68.45) was 100 times more active in cointegrate formation than was the right-hand element. Cointegrates between the conjugative plasmid R751 and pBR325 derivatives carrying IS21 and IS21::omega in tandem contained a single IS21 at one replicon junction and a single IS21::omega at the other. In the IS21 duplications the inner IS21 ends were preferentially recognized (presumably by IS21 transposase), whereas the outer termini were not required for cointegrate formation. Based on these findings a conservative (simple) pathway of transposition is proposed for R68.45 and other plasmids with an IS21 tandem repeat.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Deletion↗
PubMed1986

Evolution of R plasmids by replicon fusion.

The occurrence of plasmid-plasmid fusions in nature and their possible influence on the spread of bacterial drug resistance is considered. Three ways in which replicon fusions can be formed are described; specifically fusions mediated by IS elements, "one-ended transposition" systems derived from Tn3 and like transposons, and pUB2380, a prototype for a new type of transposition. Non-transposition based systems which mediate replicon fusion are also briefly considered.

Biological Evolution↗
PubMed1991

Effect of UV light on DNA chain growth and replicon initiation in xeroderma pigmentosum variant cells.

The effects of UV light on DNA replication were examined in wild-type and xeroderma pigmentosum (XP) variant cells using DNA fiber autoradiography. Replication segments were significantly shorter in UV-exposed XP variant cells than they were in UV-exposed wild-type human cells immediately after exposure. This is consistent with data obtained by others using alkaline sucrose gradients suggesting that there is more blockage of DNA fork progression at UV-induced lesions in XP variant cells. With time, (2.5-5.0 h) the lengths of replication segments increased in both cell lines, suggesting that some type of bypass was occurring in XP variant cells or that excision repair was removing the blocking lesions. To determine if the post-replication defect in XP variant cells involved a failure to activate alternative sites of replicon initiation, high/low specific activity labeling was performed. The results obtained indicated that XP variant cells were able to activate alternative sites of replicon initiation. Therefore the unique phenotype of the XP variant cells is probably due to some other defect.

Animals↗
PubMed1996

Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.

Animals↗
PubMed2000

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Amino Acid Sequence↗
PubMed2000

The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 10(45)-fold amplification.

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85-95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura(+) colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

Bromovirus↗
PubMed1989

Characterization of the RepFVII replicon of the haemolytic plasmid pSU233: nucleotide sequence of an incFVII determinant.

We have isolated a replication region (designated RepFVII) from the IncFVII plasmid, pSU233. Hybridization experiments showed that RepFVII has high homology to the RepFIII replicon of the plasmid pSU316 and, therefore, that pSU233 is another member of the RepFIIA family. We have also located the incompatibility gene (incFVII) from RepFVII. The analysis of its sequence revealed the same organization described for several other replicons belonging to the same family.

Base Sequence↗
PubMed1998

Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes.

The authors previously identified large plasmids in Mycobacterium xenopi, M. branderi and M. celatum which appeared to have a linear topology. This study has confirmed the presence of such linear plasmids in mycobacteria, including M. avium, and demonstrated that the ends of these replicons are covalently bound with protein(s), suggesting an invertron-like structure. The termini of one 25 kb plasmid, designated pCLP, from M. celatum were cloned and the first 500 bp of each terminus were sequenced. The termini of this plasmid show the characteristic features of invertrons with terminal inverted repeats of 45 bp (with imperfect matches) and several palindromic sequences. Moreover, similarity existed in the structure and terminal nucleotide sequence of pCLP and the termini of linear replicons of Streptomyces and Rhodococcus species, indicating a conservation of these linear extrachromosomal elements within the Actinomycetales.

Base Sequence↗
PubMed2000

A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon.

A 9.2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 4.2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the Mycobacterium celatum plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of Mycobacterium avium pLR7, M. fortuitum pJAZ38 and Mycobacterium scrofulaceum pMSC262. A region containing a putative ori site was located upstream of the rep gene; this region displayed high homology at the nucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carrying the 4.2 kb HindIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen Mycobacterium tuberculosis, making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 2.1 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

Amino Acid Sequence↗
PubMed2003

Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences.

In this report, it is shown that the rolling circle replicon pG(+)host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria, it is shown that IS1223 and IS1201 transpose in L. bulgaricus.

Base Sequence↗
PubMed2004

Evolution of naturally occurring 5' non-translated region variants of hepatitis C virus genotype 1b in selectable replicons.

Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5' non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5' end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5' NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 5' NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5' NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system.

5' Untranslated Regions↗
PubMed2005

Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon.

Hepatitis C virus non-structural NS5A protein inhibits epidermal growth factor (EGF)-stimulated activation of the Ras-ERK mitogen-activated protein kinase pathway at a point upstream of Ras activation. To determine the mechanism of this inhibition, the events occurring between the EGF receptor and Ras in Huh-7 cells harbouring the HCV subgenomic replicon were investigated. It was shown that, following EGF stimulation, these cells exhibited decreased EGF receptor tyrosine phosphorylation, aberrant recruitment of the adaptor proteins ShcA and Grb2 to the EGF receptor, reduced phosphorylation of ShcA and reduced Ras activation in comparison with control cells. These data are consistent with effects of NS5A and/or other components of the replicon on multiple events occurring upstream of Ras.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC.

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.

Amino Acid Sequence↗