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At least 1,387 records · Page 77Linked to original sources
PubMed2002

Genome sequence of the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

Bacterial Proteins↗
PubMed2001

A high-throughput alphavirus-based expression cloning system for mammalian cells.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

Animals↗
PubMed2004

ATR and ATM regulate the timing of DNA replication origin firing.

Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Here, we establish that modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promoted rapid and synchronous origin firing; conversely, inhibition of Cdc25A slowed DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. We propose that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. Our observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage.

Animals↗
PubMed2006

Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.

Antiviral Agents↗
PubMed2005

Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids.

The use of Salmonella for the delivery of plasmid-encoded heterologous antigens to eukaryotic host cells has proven successful in experimental systems, but its general applicability is still hampered by a severe instability of transformants carrying these expression plasmids. To overcome the problem of plasmid instability, new low copy number expression plasmids were constructed using different replicons. Comparative studies between transformants of the high copy number plasmid pCMVbeta and the different low copy number plasmids that contain the pMB1, p15A or pSC101 replicons on the pCMVbeta backbone, revealed a dramatic increase in plasmid stability both in vitro and in vivo. Analysis of the resulting immune responses against antigens encoded by these vectors indicated that the increased stability resulted in a strong and reproducible induction of both antigen-specific CD4(+) and CD8(+) T-cell and antibody responses even after a single application. In addition, protective immunity was induced against Listeria monocytogenes using listeriolysin as antigen, regardless of the copy number of the delivery plasmid employed. Finally, Salmonella expressing two independent antigens on compatible low copy number plasmids elicited robust responses to either antigen that is as effective as Salmonella transformed with each plasmid singly adding further versatility to this delivery system.

Administration, Oral↗
PubMed2005

Cellular factors influencing Semliki Forest Virus vector biology.

Viral vectors are currently the best tools for gene delivery in a therapeutic setting, especially for in vivo use. Alphaviruses, a family of positive singlestranded RNA viruses, have been engineered to allow the formation of a highly efficient replicon. Using these replicons, it is possible to generate recombinant particles. Parental viruses and recombinant vectors share certain pathways while interacting with their target cells. In this review, we describe the consecutive events leading to transduction, and transgene expression, in view of the cellular factors that affect each individual step. Classical virology will benefit from the knowledge accumulated studying vectors, and such work will shed light on crosstalk between intruding viruses and their hosts. Ultimately, these data should help the design of vectors adapted to specific target cells.

Cells↗
PubMed2005

A vaccination strategy consisting of Semliki-Forest-virus (SFV) DNA prime and fowlpox-virus boost significantly protects mice from a recombinant (HIV-1) vaccinia-virus infection.

The use of DNA vectors based on the SFV (Semliki Forest virus) replicon have not been reported in the modality of DNA prime virus boost. In the present study, SFV DNA vectors (DNA vectors based on the SFV replicon) bearing the HIV-1 TAB9 multiepitopic polypeptide minigene were evaluated as priming DNA immunogens followed by a recombinant fowlpox expressing the TAB9 mutiepitope (FPTAB9LZ) boost. The results indicated that mice primed with pSFV(k)tab9 and boosted with FPTAB9LZ significantly decreased the HIV-1 recombinant (VVTAB13, a recombinant vaccinia virus expressing the TAB13 multiepitope) vaccinia virus replication, compared with groups given pSFV(k)tab9 vector and FPTAB9LZ virus alone. Additionally, the viral titre in ovary correlated with the number of specific gamma-interferon-secreting T-cells in spleen. These results support the possible use of SFV DNA vectors in prime-boost approaches implemented in therapeutic/prophylactic treatments for infectious diseases such as HIV-1.

Animals↗
PubMed2002

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection.

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Animals↗
PubMed1996

Streptomyces linear plasmids that contain a phage-like, centrally located, replication origin.

Unlike previously studied linear replicons containing 5' DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA-binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single-stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.

Adenosine Triphosphatases↗
PubMed1996

The centromere-like parC locus of plasmid R1.

The parA partitioning system of plasmid R1 consists of three components: the cis-acting centromere-like parC locus, and two proteins, ParM and ParR. The parC locus contains two sets of five direct repeats (iterons) to which the ParR protein binds. The parA promoter is located in the core region between the two sets of iterons. Mini-R1 replicons carrying parC are stabilized by the simultaneous presence of ParM and ParR. The parC locus present on a co-resident plasmid leads to instability of the mini-R1 replicon (incompatibility). Here we present a genetic analysis of the stability and incompatibility phenotypes associated with parC. We show that all 10 iterons are required for maximum stabilization and incompatibility. Replacement of the core promoter region between the repeats by a foreign promoter region did not reduce stabilization. Thus, the only structural components in parC seem to be the two sets of iterons. The parA promoter, P parA, is repressed by ParR. We show that all 10 iterons are required for full repression of the promoter. The activity of the promoter was influenced by sequences located outside the core region. An A-rich region located upstream of the -35 element of PparA was found to increase promoter activity. The region encoding the parA mRNA leader region also strongly influenced the expression level of PparA- lacZ fusions. We show that this high expression (hex) element is a transcriptional antiterminator that prevents Rho-dependent termination.

Bacterial Proteins↗
PubMed1997

Plasmid replication and partition in Escherichia coli: is the cell membrane the key?

The DNA-membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329-348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.

Cell Membrane↗
PubMed1997

Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii.

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination-deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.

Archaeal Proteins↗
PubMed1997

Dynamic bacterial genome organization.

Recently completed projects of sequencing chromosomal fragments and entire chromosomes, as well as physical mapping of genomes, have opened novel inroads to the understanding of the biology of bacterial genomes. From these studies one may draw some conclusions. (i) The organization of orthologous genes on the bacterial chromosome is not conserved during evolution. (ii) The bacterial genome is more complex and also more flexible than hitherto thought. Genetic elements are sometimes part of the chromosome, while at other times they are independent elements or parts of alternative replicons (e.g. large plasmids). Such replicons, carrying essential genes, now seem to deserve the designation 'secondary chromosomes'. A study of the regulation of replication and segregation of these essential genetic elements will be of great interest.

Bacteria↗
PubMed1999

Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles.

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30 )2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site-specific dimerization that leads to (IS30 )2 establishment. The other process is the dissolution of (IS30 )2 dimer, when the element is excised from an IR-IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle-generating processes. Our focus was to the dissolution of IR-IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied minicircle formation. The results present the possibility of host genome reorganization by IS30-like transposition.

DNA Transposable Elements↗
PubMed2001

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi.

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Bacteriophages↗
PubMed2002

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida.

The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.

Bacterial Proteins↗
PubMed1998

Improved cloning vectors for Bifidobacterium spp.

The recombinant plasmids pDLI41, pDGA7 and pDCO7 were constructed by cloning in pDG7, a vector based on Bifidobacterium longum replicon pMB1, the following heterologous genes: Pseudomonas fluorescens lipase, Bacillus licheniformis alpha-amylase and Streptomyces sp. cholesterol oxidase. The hybrid plasmids efficiently transformed Bifidobacterium belonging to five different species. A novel Escherichia coli-Bifidobacterium set of shuttle vectors based on the replicon pMB1 (pLF5, pCLJ15, pSPEC1) featuring chloramphenicol, erythromycin and spectinomycin resistance genetic determinants as selection marker for bifidobacteria, was developed. The plasmid pTRE3, a derivative of pLF5, was the smallest (2.8 kb) Bifidobacterium vector, possessed a convenient multicloning site and presented high structural and segregational stability.

Bifidobacterium↗
PubMed2005

Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins.

BACKGROUND & AIMS: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. METHODS: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. RESULTS: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. CONCLUSIONS: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.

Base Sequence↗