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At least 1,711 records · Page 95Linked to original sources
PubMed2003

Identification and nucleotide sequence analysis of a cryptic plasmid, pRM21, from Rhodothermus marinus.

Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus. pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli. A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified. Of 40 additional R. marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.

AT Rich Sequence↗
PubMed2003

Sequence analysis and characterization of plasmids from Streptococcus thermophilus.

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.

Bacillus subtilis↗
PubMed2000

Use of a plasmid of a yersinia enterocolitica biogroup 1A strain for the construction of cloning vectors.

A plasmid with a size of 2,682 base pairs isolated from the Yersinia enterocolitica biogroup 1A strain # 29807 was characterized in respect to its suitability as a basic replicon for cloning vectors. The copy number of the plasmid was determined to be approximately 14 copies per cell and it was shown to be compatible with vectors with an origin of replication derived from ColE1 and p115A. The replication region of the plasmid encodes a primer RNAI and countertranscript RNAII. Two vectors, pIV1 and pIV2, containing a kanamycin resistance gene and the lacZalpha fragment with the multiple cloning site of pBluescriptSK + were constructed. A mobilizable derivative was successfully introduced into different bacteria belonging to the family Enterobacteriacea. To prove the applicability of the novel vectors for cloning purposes, a 13 kb hemolysin operon of Escherichia coli was inserted into pIV1, and the resulting recombinant plasmid was stably maintained and expressed in E. coli and Y. enterocolitica.

Base Sequence↗
PubMed2003

Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms.

Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes. Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase. Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression. Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks. Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures. We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.

Cell Cycle Proteins↗
PubMed2003

Immune responsiveness, tolerance and dsRNA: implications for traditional paradigms.

Double-stranded (ds)RNA motifs are central to immune regulation and block tolerance to tumor-associated, self- and foreign antigens. In addition, they could explain why DNA vectors encoding self-replicating mRNA (replicons) are more immunogenic than conventional DNA vaccines. Accumulating evidence on the immunomodulatory roles of dsRNAs warrants a reconsideration of various models of immune homeostasis.

Animals↗
PubMed2004

Gastrointestinal basic science 2002-2003: the year in review.

The Board of Editors of Gastroenterology identified 6 topics in basic gastroenterology research in which exciting advancements have been made during the past year. These topics are (1) NOD2 variants in Crohn's disease, (2) Heliobacter pylori and CagA and VacA pathogenesis, (3) beta-catenin function in normal colonic epithelia and colon cancer, (4) DNA methylation in colonic cancer, (5) the HCV replicon, and (6) the use of small interfering RNA in somatic cell genetics. Basic research in gastroenterology has been characterized during the past year by the rapid translation from fundamental research into pathophysiological systems. New technological advances are being applied to difficult areas of gastrointestinal research. Well-defined patient populations are critical to extend the knowledge derived from the human genome to gastrointestinal diseases.

Animals↗
PubMed2001

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli.

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.

Base Sequence↗
PubMed2006

Benzimidazole derivatives bearing substituted biphenyls as hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors: structure-activity relationship studies and identification of a potent and highly selective inhibitor JTK-109.

Following the discovery of a new series of benzimidazole derivatives bearing a diarylmethyl group as inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase (HCV NS5B RdRp),1,2 we extended the structure-activity relationship (SAR) study to analogues bearing a substituted biphenyl group and succeeded in a significant advancement of activity. Starting from compound 1, optimization of the A, B, and C rings afforded potent inhibitors with low nanomolar potency against genotype 1b NS5B. The compounds, which have a substituent with a carbonyl function at the 4-position of the B-ring, efficiently blocked subgenomic viral RNA replication in the replicon cell assay at low submicromolar concentrations. Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.

Animals↗
PubMed2006

Discovery of conformationally constrained tetracyclic compounds as potent hepatitis C virus NS5B RNA polymerase inhibitors.

We report a new series of hepatitis C virus NS5B RNA polymerase inhibitors containing a conformationally constrained tetracyclic scaffold. SAR studies led to the identification of 6,7-dihydro-5H-benzo[5,6][1,4]diazepino[7,1-a]indoles (19 and 20) bearing a basic pendent group with high biochemical and cellular potencies. These compounds displayed a very small shift in cellular potency when the replicon assay was performed in the presence of human serum albumin.

Antiviral Agents↗
PubMed1980

Effects of methylated xanthines on mammalian cells treated with bifunctional alkylating agents.

Caffeine has been previously reported to enhance the lethal potential of many DNA-damaging agents in rodent cells1-5. This effect has most commonly been ascribed to the binding of caffeine to single-stranded DNA6, and the resulting inhibition of post-replication repair7-10, which is associated with the synthesis of abnormally small nascent DNA fragments7, 11-13. However, certain aspects of this theory remain unclear:(1) why does the addition of caffeine to damaged cells elevate the level of DNA synthesis when it supposedly blocks post-replication repair10,14, and (2) as pointed out by Cleaver15, why does caffeine continue to exert its synergistic lethal effects until completion of the S phase16, 17, even though the size of newly synthesized DNA seems normal much earlier18-20? The present studies with nitrogen mustard (HN2) fail to demonstrate any effect of non-lethal concentrations of methylated xanthines (MXs) on removal of DNA damage or post-replication repair in conditions producing synergistic lethal effects. We demonstrate an influence by MXs on initiation of DNA synthesis in damaged replicons, and propose that this effect is primarily responsible for the synergistic lethal properties of these drugs.

Animals↗
PubMed1993

Replication structure of the human beta-globin gene domain.

The animal cell genome is organized into a series of replicons with an average size of 50-300 kilobases; each of these units is characterized by its own origin of replication which serves as the point of initiation for DNA synthesis. In animal viruses, origin usage can be regulated by cis-acting elements, and in some cases, replication may be cell-type specific. Little is known, however, about the organization and control of endogenous tissue-specific gene replication. To understand this process, we have used a replication direction assay to examine DNA fragments covering more than 200 kilobases of the human beta-like globin domain, and have identified a single bidirectional origin located upstream of the beta-globin itself. This locus is used to initiate DNA synthesis in expressing cells, where the globin domain replicates early, and in non-expressing cells, which are characterized by late replication of the same region. Deletion of this origin sequence, as occurs in the haemoglobin Lepore syndrome, cancels bidirectional DNA synthesis at this site and leads to a striking reversal of replication direction upstream to the locus. This represents the first genetic proof of the existence of specific, discrete origins of replication in animal cells.

Cell Line↗
PubMed2004

Interferon type I gene expression in chronic hepatitis C.

Hepatitis C virus (HCV) frequently causes chronic liver disease. The cause of viral persistence might be an inappropriate type I interferon (IFN) induction. To analyze the host's IFN response in chronic hepatitis C, we measured the transcription level of type I IFN genes as well as type I IFN-regulated genes in liver tissue and corresponding blood samples from patients with chronic hepatitis C, nonviral liver diseases, and a suspected but later excluded liver disease. Competitive and real-time RT-PCR assays were used to quantify the messenger RNA (mRNA) levels of all known IFN-alpha, IFN-beta, and IFN-lambda genes and those of some IFN-regulated genes. We failed to detect any hepatic type I IFN mRNA induction, although liver tissue of chronic hepatitis C patients contained high numbers of some type I IFN-inducible effector mRNA molecules. Analysis of peripheral blood samples, however, showed a clear type I IFN induction. Parallel experiments employing HCV replicon cell lines revealed that replication of HCV RNA is not sufficient to induce any type I IFN nor to induce directly type I IFN-regulated genes such as MxA. In conclusion, our data provide evidence for the absence of an induction of type I IFN genes by HCV in the human liver and argue for a further development of type I IFN-based therapies.

Adolescent↗
PubMed1998

Enhancing immune responses using suicidal DNA vaccines.

We describe a DNA vaccine strategy that allows antigens to be produced in vivo in the context of an alphaviral replicon. Mice immunized with such vectors developed humoral and cellular immune responses at higher levels than mice that received a conventional DNA vaccine vector. Immunized animals acquired protective immunity to lethal influenza challenge. Compared with traditional DNA vaccine strategies in which vectors are persistent and the expression constitutive, the expression mediated by the alphaviral vector was transient and lytic. As a result, biosafety risks such as chromosomal integration, and the induction of immunological tolerance, could be circumvented.

Adjuvants, Immunologic↗
PubMed2004

In search of the holy replicator.

After 40 years of searching for the eukaryotic replicator sequence, it is time to abandon the concept of 'the' replicator as a single genetic entity. Here I propose a 'relaxed replicon model' in which a positive initiator-replicator interaction is facilitated by a combination of several complex features of chromatin. An important question for the future is whether the positions of replication origins are simply a passive result of local chromatin structure or are actively localized to coordinate replication with other chromosomal activities.

Animals↗
PubMed1998

The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family.

Toxin-antitoxin systems are defined as a group of plasmid- and chromosome-encoded loci that specify a cell toxin and a protein antitoxin. Plasmid-encoded toxin-antitoxin systems stabilize their replicons by killing plasmid-free cells. Here, we show that the relBE genes of Escherichia coli K-12 have all the basic features previously connected with toxin-antitoxin systems: (i) relE encodes a cytotoxin lethal or inhibitory to host cells; (ii) relB encodes an antitoxin that prevents the lethal action of the relE-encoded toxin; (iii) the relBE genes stabilize a mini-R1 test plasmid; and (iv) the RelB antitoxin autoregulates the relBEF operon at the level of transcription. Using database searching, we found relBE homologues on the chromosomes of E. coli K-12, Haemophilus influenzae and Vibrio cholerae. A fifth relBE homologue was identified on the enterotoxin encoding E. coli plasmid P307. Indirect evidence suggests that the toxicity of RelE may be related to the inhibition of protein synthesis. Based on these observations, we propose a model that explains the delayed relaxed phenotype associated with mutations in relB.

Amino Acid Sequence↗
PubMed1999

The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus.

A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co-transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading.

Amino Acid Sequence↗
PubMed2001

Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS.

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5'-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.

Amino Acid Sequence↗
PubMed2003

Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system.

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Centrifugation, Density Gradient↗