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At least 1,441 records · Page 80Linked to original sources
PubMed1978

DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromo-deoxyuridine substituted DNA.

Combinations of 5-bromodeoxyuridine (BrdUrd) and 3H-deoxyadenosine (3H-DAdo) short pulses were given in the synchronous DNA-replication period of Physarum polycephalum. After a chase period, UV-photolysis products were analyzed on alkaline sucrose gradients. This strategy has allowed the following conclusions. a) at the time of master-initiation of DNA replication, points separated by 1.1-2.2x10(7) daltons of single strand DNA may initiate DNA synthesis. b) among these, only selected groups of replicons actually proceed in DNA replication at this time, while others appear to hold (later temporal sets of replicons). The origins of the ones that proceed in replication are separated from each other by a distance corresponding to 1.1-2.x10(7) daltons. c) regions in actual replication are separated from each other by increasing distances (up to 1.5x10(8) daltons single strand DNA) at later times in S.

Bromodeoxyuridine↗
PubMed1979

Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine.

Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [(3)H]thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rates of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. Our observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair.

Caffeine↗
PubMed2004

RNAi expression mediated inhibition of HCV replication.

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Cell Line, Tumor↗
PubMed2000

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata.

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

Amino Acid Sequence↗
PubMed1992

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

DNA Repair↗
PubMed2000

Gene transfer to Clostridium cellulolyticum ATCC 35319.

Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CCE:I, is an isoschizomer of MSP:I (HPA:II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMss1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BSU:FI methylase in vivo or MSP:I methylase in vitro. Plasmids based on the pAMss1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10(2) transformants per microg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10(5) and 10(7) transformants per microg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

Cellulose↗
PubMed2026

Genomic epidemiology and antimicrobial resistance profile of Shigella isolated from diarrhoea diseases in under-five children in Blantyre, Malawi.

Antimicrobial resistance (AMR) in Shigella is rising globally, complicating shigellosis management. Whole-genome sequence analysis (WGSA) has advanced our understanding of AMR and transmission dynamics, yet contemporary whole-genome sequencing data from Shigella in sub-Saharan Africa remain scarce. In this study, we applied WGSA to 27 Shigella isolates collected from children presenting with diarrhoea at Ndirande Health Centre in Malawi (2022-2023), as part of the Enterics for Global Health Shigella surveillance study. Serotyping, AMR profiling and phylogenetic analysis revealed Shigella sonnei as the dominant serogroup, with distinct genetic clustering relative to global reference strains among S. sonnei, Shigella flexneri and Shigella boydii. We identified 16 AMR genes linked to ten antimicrobial classes with qnrS1 and qnrB19 genes conferring resistance to fluoroquinolone, alongside IncFIB(K) and IncFII plasmid replicon markers. Importantly, no azithromycin resistance determinants were detected both genotypically and phenotypically, providing baseline evidence that warrants continued surveillance of current first-line treatment. However, the detection of fluoroquinolone resistance genes with plasmid replicon markers in the absence of phenotypic resistance might indicate a silent reservoir with epidemic potential. This is the first contemporary Shigella data from a large-scale diarrhoea disease surveillance study in Malawi, providing essential baseline information for guiding antibiotic treatment and future vaccine development efforts, contributing to the efforts to combat shigellosis in Malawi and other similar regions.

Humans↗
PubMed2004

FixJ-regulated genes evolved through promoter duplication in Sinorhizobium meliloti.

The FixLJ two-component system of Sinorhizobium meliloti is a global regulator, turning on nitrogen-fixation genes in microaerobiosis. Up to now, nifA and fixK were the only genes known to be directly regulated by FixJ. We used a genomic SELEX approach in order to isolate new FixJ targets in the genome. This led to the identification of 22 FixJ binding sites, including the known sites in the fixK1 and fixK2 promoters. FixJ binding sites are unevenly distributed among the three replicons constituting the S. meliloti genome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries the fixJ gene. Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes, smc03253 and proB2. This FixJ-dependent regulation appears to be mediated by a duplication of the whole fixK promoter region, including the beginning of the fixK gene. Similar duplications were previously reported for the nifH promoter. By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways in S. meliloti.

Amino Acid Sequence↗
PubMed2006

Characterization of the theta-type plasmid pCD3.4 from Carnobacterium divergens, and modulation of its host range by RepA mutation.

The complete nucleotide sequence of the 3475 bp plasmid pCD3.4 from Carnobacterium divergens LV13, which encodes the bacteriocin divergicin A, was determined. Nucleotide sequence, deletion and complementation analyses revealed the presence of a trans-acting replication protein, RepA, and DNA sequences involved in plasmid replication and copy-number control. The DNA region preceding the repA gene probably contains the origin of replication. This sequence includes four and a half direct repeats (iterons) of 22 bp, to which RepA is thought to bind, and an AT-rich region containing a 12 bp repeat, at which initiation of DNA might occur. Further upstream of this sequence resides a fifth iteron required for optimal plasmid replication. The RepA protein shows homology to replication proteins of the pUCL287 subfamily of theta-type replicons. Two ORFs were found downstream of the repA gene that could be deleted without affecting replication and stability of the plasmid. pCD3.4 has a narrow host range, and could only be maintained in Carnobacterium spp.; however, a mutant of the plasmid was obtained that enabled the pCD3.4 replicon to replicate in Enterococcus faecium, but not in Carnobacterium spp. The mutation was located in the C-terminal region of the RepA protein, changing a proline into a serine. This is believed to be the first example of such plasmid-host-range modulation in Gram-positive bacteria.

Bacterial Proteins↗
PubMed2005

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria.

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Bacterial Proteins↗
PubMed2004

The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity.

The hepatitis C virus (HCV) non-structural NS5A protein has been shown to associate with a variety of cellular signalling proteins. Of particular interest is the observation that a highly conserved C-terminal polyproline motif in NS5A was able to interact with the Src-homology 3 (SH3) domains of the adaptor protein Grb2. As it has previously been shown that specific polyproline motifs can interact with a range of SH3 domains, we investigated whether NS5A was capable of interacting with other SH3 domain-containing proteins. We show here that NS5A interacts with the SH3 domains of members of the Src family of tyrosine kinases: a combination of in vitro binding assays and co-immunoprecipitation experiments revealed an interaction between NS5A and Hck, Lck, Lyn and Fyn, but interestingly not Src itself. Mutational analysis confirmed that the polyproline motif responsible for binding to Grb2 also bound to the SH3 domains of Hck, Lck, Lyn and Fyn. Furthermore, a previously unidentified polyproline motif, adjacent to the first motif, was also able to mediate binding to the SH3 domain of Lyn. Using transient transfections and Huh-7 cells harbouring a persistently replicating subgenomic HCV replicon we demonstrate that NS5A bound to native Src-family kinases in vivo and differentially modulated kinase activity, inhibiting Hck, Lck and Lyn but activating Fyn. Lastly, we show that signalling pathways controlled by Src-family kinases are modulated in replicon cells. We conclude that the interactions between NS5A and Src-family kinases are physiologically relevant and may play a role in either virus replication or pathogenesis.

Animals↗
PubMed2004

Complementarity, sequence and structural elements within the 3' and 5' non-coding regions of the Bunyamwera orthobunyavirus S segment determine promoter strength.

The genome of Bunyamwera virus (BUN; family Bunyaviridae) consists of three segments of negative-sense, single-stranded RNA that are called L (large), M (medium) and S (small), according to their size. The genomic RNAs are encapsidated by the viral nucleocapsid protein to form ribonucleoprotein complexes (RNPs). The terminal 3' and 5' non-coding sequences are complementary and interact to give a panhandle-like structure to the RNP. Located within these non-coding sequences are elements that control replication and transcription. The sequences of the terminal 11 nt are conserved among the genome segments and are followed by shorter, complementary nucleotide motifs that are conserved on a segment-specific basis. Here, a detailed analysis of the 3' and 5' non-coding regions of the BUN S segment is presented. By using a mini-replicon system, it was shown that a functional BUN S promoter requires complementarity, as well as defined sequences, within the terminal 15 nt of either end. It was also shown that the minimal requirement for transcription is localized within the terminal 32 nt of the S segment. A comparison of known strong BUN promoters led to the prediction of a structural element outside the terminal 15 nt; introduction of this motif into the BUN S sequence resulted in increased antigenome and mRNA levels and increased expression of S segment proteins, as shown by mini-replicon assays, as well as recovery of a recombinant virus.

3' Untranslated Regions↗
PubMed2006

Insertion and deletion analyses identify regions of non-structural protein 5A of Hepatitis C virus that are dispensable for viral genome replication.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays an essential role in viral genome replication. A series of transposon-mediated insertion mutants and deletion mutants of NS5A was used to examine the colony-forming ability of HCV subgenomic replicons encoding the mutant proteins. The results reveal that two regions of NS5A can tolerate insertions: one spanning residues 240-314, which contain the interferon sensitivity-determining region (ISDR), and the other spanning residues 349-417 at the carboxy terminus. The majority of these sites also tolerated insertion of enhanced green fluorescent protein. Furthermore, replicons encoding NS5A with deletions in ISDR or in the carboxy-terminal regions were replication-competent, indicating that these regions of NS5A are not necessary for replication. Taken together, the results suggest that the central region spanning the ISDR and the carboxy-terminal region of the molecule are dispensable for the functions of NS5A in viral genome replication.

Gene Deletion↗
PubMed1993

Improved cloning vectors and transformation procedure for Lactococcus lactis.

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.

Base Sequence↗
PubMed2005

Development of food-grade cloning and expression vectors for Lactococcus lactis.

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

Base Sequence↗
PubMed1993

Linear plasmids and chromosomes in bacteria.

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.

Bacteria↗
PubMed1996

Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome.

We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density-shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow-cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.

Chromosomes, Bacterial↗
PubMed2006

Polymerization of SopA partition ATPase: regulation by DNA binding and SopB.

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for most ATPases (the Walker-box type) the mechanism is unknown. Also unknown is how the host cell contributes to partition. We have examined the effects of non-sequence-specific DNA on in vitro self-assembly of the SopA partition ATPase of plasmid F. SopA underwent polymerization provided ATP was present. DNA inhibited this polymerization and caused breakdown of pre-formed polymers. Centromere-binding protein SopB counteracted DNA-mediated inhibition by itself binding to and masking the DNA, as well as by stimulating polymerization directly. The results suggest that in vivo, SopB smothers DNA by spreading from sopC, allowing SopA-ATP polymerization which initiates plasmid displacement. We propose that SopB and nucleoid DNA regulate SopA polymerization and hence partition.

Adenosine Triphosphate↗