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PubMed1990

Topological DNA target size model.

This study presents a model that explains the difference in radiosensitivity between dividing and resting mammalian non-lymphoid tissue cells (liver, kidney, respiratory tract, muscle cells, neurons), based on the topological organization of DNA. In dividing cells, the target for radiation might be identified in replicon clusters or domains (7 X 10(8)-5.8 X 10(9) Da of DNA), in contrast with resting cells, in which the target could be limited to the size of chromatin loops or replicons (10(7)-10(8) Da). Hence, the target theory, D37(cGy) = 0.58 X 10(12)/weight of DNA in Da, indicates that the D37 dose (low-LET radiation) needed to inactivate 63% of the replicon clusters contained by the genome is around 100-850 cGy, and the D37 doses that could damage 63% of chromatin loops increase to 5800-58,000 cGy, with a value of 10,000 cGy for medium size replicons (5.8 X 10(7) Da). Accordingly, most dividing cells have D37 doses of 35 to 650 cGy, and the D37 values for the interphase death of non-lymphoid resting cells increase to several tens of Gy or more. These data are consistent with the idea that killing of dividing cells is correlated with the inactivation of most replicon clusters (about 720-6000 domains per genome), induced mainly by DNA single-strand breaks (SSBs), associated with double-strand breaks (DSBs); while the death of resting cells occurs when the majority of replicons comprised by the cell nucleus (about 72,000 chromatin loops) are damaged by radiation (SSBs, DSBs), which might prevent the process of transcription.

Cell Division↗
PubMed2005

Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation.

The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5' untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5'UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5'UTR was necessary for translation; interestingly, when plasmids were used the 5'UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SLL) that has a structure and stability similar to SL's found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SLL had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SLL or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5'UTR of the SG RNA is required. Further investigation of the differing effects of the SLL on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SLLs could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SLL only functions in a "cytopathic environment" in which cell translation has been altered.

Animals↗
PubMed2002

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector.

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus-Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.

Bacterial Proteins↗
PubMed2003

Replication functions of new broad host range plasmids isolated from polluted soils.

The nucleotide sequencing of replicons isolated from three new broad host range plasmids, pMOL98, pEMT8, and pEMT3, originating from polluted soils, showed a typical organization of iteron replicons replicating by the theta mode. In the pMOL98 replicon, the origin region and the rep gene were identified in complementation experiments. Sequence comparisons showed that the regions bearing these features are highly identical to regions in pIP02T and pSB102 and that the Rep proteins (but not the origin regions) of these three plasmids show some identity to the Rep proteins of the IncW group of plasmids. This suggests that pMOL98, pIPO2T, and pSB102 constitute a new Inc/Rep family, distantly related to the IncW group, but having an incompatibility phenotype different from the IncW phenotype. The pEMT8 replicon displayed an orf whose conceptually translated product is related to the Rep proteins of four plasmids, pSD20, pSW500, pMLb, and pALC1, not yet classified into any known incompatibility group. The vegetative origins of these plasmids were not similar, suggesting that the five plasmids could belong to a new family with similar Rep proteins but different incompatibility phenotypes. The pEMT3 replicon is clearly related to IncP replicons (sequence similarities and incompatibility phenotype), although sequence comparisons revealed some divergence with respect to the two well-documented subgroups IncPalpha and IncPbeta. This suggests that in these plasmids, despite the existence of a powerful system of centralized control over replication, maintenance, and transfer functions, plasticity and evolution of these functions are at work. Our analysis confirms the extreme genetic flexibility of plasmids and the absolute necessity of using multiple techniques (PCR, DNA sequencing, DNA chips, and databases) to analyze the role of broad host range plasmids in the capture, recombination and spread of genetic traits among bacteria.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1999

Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species.

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed-field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two-replicon structure is common throughout Vibrio species.

Chromosome Mapping↗
PubMed2004

Expression of gastrointestinal glutathione peroxidase is inversely correlated to the presence of hepatitis C virus subgenomic RNA in human liver cells.

There is great medical need to develop novel therapies for treatment of human hepatitis C virus (HCV). By gene expression analysis of three HCV-subgenomic RNA replicon cell lines, we identified cellular proteins whose expression is affected by the presence of HCV and therefore may serve as drug targets. Data from cDNA array filter hybridization, as well as from Northern and Western blotting, revealed that the gastrointestinal-glutathione peroxidase (GI-GPx) was drastically down-regulated (up to 20-fold) in all replicon cell lines tested. Concomitantly, total cellular glutathione peroxidase activity was drastically reduced, which rendered these human liver cells more susceptible toward oxidative stress. Interferon alpha caused down-regulation of the HCV-replicon followed by recovery of GI-GPx expression to nearly normal levels. Furthermore, expression of GI-GPx in replicon cells by gene transduction caused down-regulation of HCV RNA in a dose-dependent manner. Moreover, activating the endogenous gene coding for GI-GPx by all-trans-retinoic acid (RA) was sufficient to cause down-regulation of the HCV replicon. A small interfering RNA duplex abrogated GI-GPx up-regulation by RA and concomitantly suppression of HCV. The RA effect was dependent on the presence of sodium selenite, was reversible, and was independent of RNA-activated protein kinase. Taken together, these results show that HCV inhibits the expression of GI-GPx in replicon cells to promote its intracellular propagation. Modulation of GI-GPx activity may open new avenues of treatment for HCV patients.

Base Sequence↗
PubMed2001

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen.

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.

Animals↗
PubMed1988

Recovery from effects of heat on DNA synthesis in Chinese hamster ovary cells.

The hyperthermic inhibition of cellular DNA synthesis, i.e., reduction in replicon initiation and delay in DNA chain elongation, was previously postulated to be involved in the induction of chromosomal aberrations believed to be largely responsible for killing S-phase cells. Utilizing asynchronous Chinese hamster ovary cells heated for 15 min at 45.5 degrees C, an increase in single-stranded regions in replicating DNA (as measured by BND-cellulose chromatography) persisted in heated cells for as long as replicon initiation was affected. Alkaline sucrose gradient analyses of cells pulse-labeled immediately after heating with [3H]thymidine and subsequently chased at 37 degrees C revealed that these S-phase cells can eventually complete elongation of the replicons in operation at the time of heating, but required about six times as long relative to control cells which completed replicon elongation within 4 h. DNA chain elongation into multicluster-sized molecules was prevented for up to 18 h in these heated cells, resulting in a buildup of cluster-sized molecules (approximately 120-160 S) mainly because of the long-term heat damage to the replicon initiation process. Utilizing bromodeoxyuridine (BrdU)-propidium iodide bivariate analysis on a flow cytometer to measure cell progression, control cells pulsed with BrdU and chased in unlabeled medium progressed through S and G2M with cell division starting after 2 h of chase time. In contrast, the majority of the heated S-phase cells progressed slowly and remained blocked in S phase for about 18 h before cell division was observed after 24 h postheat. Our findings suggest that possible sites for where the chromosomal aberrations may be occurring in heated S-phase cells are either (1) at the persistent single-stranded DNA regions or (2) at the regions between clusters of replicons, because this long-term heat damage to the DNA replication process might lead to many opportunities for abnormal DNA and/or protein exchanges to occur at these two sites.

Animals↗
PubMed2000

The RNA encompassing the internal ribosome entry site in the poliovirus 5' nontranslated region enhances the encapsidation of genomic RNA.

Poliovirus replicons were constructed which contain the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) substituted for the poliovirus IRES. To monitor gene expression and encapsidation, the gene encoding firefly luciferase was substituted for the P1 gene. Replicons can be encapsidated following serial passage in the presence of a recombinant vaccinia virus, VV-P1, which expresses the poliovirus P1 protein following infection. Encapsidation of the wild-type replicon (PV-Luc) was accomplished at either 33 or 37 degrees C; the lower temperature actually resulted in greater amounts of encapsidated replicon. In contrast, the replicon with the EMCV IRES element (EMCV-Luc) was not efficiently encapsidated at 37 degrees C and, following serial passage with VV-P1 at 37 degrees C, was not amplified. EMCV-Luc was efficiently encapsidated, however, following serial passage with VV-P1 at 33 degrees C. Using the encapsidated EMCV-Luc obtained at 33 degrees C, we found that cells infected with EMCV-Luc at 33 or 37 degrees C produced similar amounts of luciferase. Encapsidated EMCV-Luc and PV-Luc had similar thermal stability at 33 and 37 degrees C. A single-round encapsidation analysis revealed that less EMCV-Luc was encapsidated at 37 than at 33 degrees C; less EMCV-Luc was encapsidated at 33 degrees C compared to PV-Luc at either 37 or 33 degrees C. The results of our studies suggest that in addition to influencing translation/replication, the IRES region of poliovirus can function to enhance encapsidation.

5' Untranslated Regions↗
PubMed1981

Chromosomal replication of Drosophila virilis. II. Organization of active origins in diploid brain cells.

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 micron/min at 25 degrees C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. - Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (less than 10 micron) which correlate with replicating satellite sequences. - The majority of replicons fall into size classes less than 100 micron. The frequency distribution is skewed towards larger replicon sizes; it spans 2-238 micron, has a mean of x = 25.6 micron and a median of approximately 21.0 micron. If the distribution is corrected for supposed satellite replicons, the median increases of approximately 31.0 micron. - In experiments using warm-hot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length.

Autoradiography↗
PubMed1999

Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata.

The basic replicon of the narrow-host-range plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized. The replicon consists of a 1.7-kb DNA fragment, which contains the genetic information required for autonomous replication and stable maintenance. Analysis of its sequence revealed the presence of two ORFs which seem to form one transcription unit. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology to the theta-replicase (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to ColE2. The product encoded by ORF2 showed a certain similarity to the RepB proteins of the same family of replicons and perhaps represents the pHE1 RepB function. Deletion analysis suggests that the pHE1 origin of replication (ori) is located in an 800-bp region upstream of repA. A third putative gene, incA, was found on the complementary strand to the leader region of the repA mRNA. This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structures, suggests that the incA gene encodes a small antisense RNA. A possible control mechanism for pHE1 replication is proposed, involving an RNA molecule which sequesters the translational initiation region of the replication protein RepA. The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning and expression vectors for moderate halophiles.

Amino Acid Sequence↗
PubMed1983

Effects of Hoechst 33258 on different cell cycle events. I. Inhibition of synthetic activities in bone-marrow cells of the mole rat Bandicota bengalensis.

A study of the effects of the A-T base-specific ligand Hoechst 33258 on the cell cycle events of the bone-marrow cells of the mole rat Bandicota bengalensis reveals that (1) the G2 phase is prolonged by 2 h; (2) the chromosomes are undercondensed to varying degrees in about 30% metaphases; (3) DNA as well as RNA synthesis is partially inhibited. Study on DNA fibre autoradiograms shows that despite the inhibition of DNA synthesis the average replicon size and rate of fork migration remain unchanged, but there is a marked asynchrony of initiation of replicons in replicon clusters in H-treated cells in comparison with control. It is suggested that these inhibitory effects result from the stabilization of the double-stranded organization of DNA (dsDNA) by the drug so that the unwinding of DNA at the replication or transcription origin sites is greatly impaired. It is likely that this introduces asynchrony in the initiation of replicons within replicon clusters which may retard the overall rate of replication. In addition, it appears that the reduction in transcription is at least one cause for the prolongation of G2 phase.

Animals↗
PubMed2005

Enhancement of antiviral activity against hepatitis C virus in vitro by interferon combination therapy.

Alpha, beta, and gamma interferons (IFN-alpha, IFN-beta, IFN-gamma) have been shown to be effective inhibitors of HCV replication in human cell lines carrying HCV replicons. To help define the divergent cellular processes involved in the control of intracellular HCV replication by these agents, we have characterized the activity of monotherapies and combination therapies with the major types of human interferons against HCV replication in the HCV replicon-containing cell line, AVA5. IFN-alpha, IFN-beta, and omega interferon (IFN-omega) were equally effective at inhibiting HCV replication, while IFN-gamma was approximately 10-fold more potent. In kinetic experiments, IFN-beta and IFN-gamma inhibited HCV replication more rapidly, and for a more prolonged period following the removal of treatment, than IFN-alpha. Combination interferon therapies produced enhanced anti-HCV activity in most cases, and displayed a diverse range of interactions. Mixtures of IFN-alpha and IFN-beta exhibited generally additive to slightly antagonistic interactions, IFN-alpha or IFN-beta combined with IFN-omega were strongly antagonistic, while IFN-alpha/IFN-gamma and IFN-beta/IFN-gamma combinations displayed the most enhanced and strongly synergistic antiviral effects. Simultaneous administration of interferons in the combination treatments was found to be superior to sequential administration. Ribavirin did not exhibit any selective anti-HCV activity in cell culture, consistent with in vivo monotherapies, and did not influence the effectiveness of IFN-alpha in combination treatments. A panel of human cytokines and immune response modifiers induced by interferon and ribavirin therapies in vivo did not demonstrate anti-HCV activity in HCV replicon-containing cultures. Combination therapy can be effectively modeled using HCV replicon technology yielding potentially more effective treatment regimens. HCV replicon technology has potential utility in designing combination therapies to significantly enhance the anti-HCV activity of IFN-alpha.

Antiviral Agents↗
PubMed2006

Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034.

HCV NS3 protease variants resistant to the protease inhibitor SCH 503034 were selected. Three mutations, T54A, V170A and A156S mutations conferred low to moderate levels of resistance (<20-fold). Longer exposure (>10 passages) or selection with higher levels of compound led to the selection of a more resistant variant, A156T (>100-fold). [Lin, C., Lin, K., Luong, Y.P., Rao, B.G., Wei, Y.Y., Brennan, D.L., Fulghum, J.R., Hsiao, H.M., Ma, S., Maxwell, J.P., Cottrell, K.M., Perni, R.B., Gates, C.A., Kwong, A.D., 2004. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J. Biol. Chem. 279(17), 17508-17514; Lu, L., Pilot-Matias, T.J., Stewart, K.D., Randolph, J.T., Pithawalla, R., He, W., Huang, P.P., Klein, L.L., Mo, H., Molla, A., 2004. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 48(6), 2260-2266.] Combination with IFN-alpha drastically reduced the number of emergent colonies. Resistant colonies showed no change in sensitivity to IFN-alpha. Although the A156T mutation conferred the highest level of resistance to SCH 503034, it significantly reduced the colony formation efficiency (CFE) of the mutant replicon RNA, and rendered replicon cells less fit than those bearing wild-type replicons. Replicon cells bearing mutation A156S were less fit than wild-type in co-culture growth competition assays but showed no impact on CFE. The V170A mutation, on the other hand, did not affect replicon fitness in either assay, which was consistent with its emergence as the dominant mutant after 12 months of continuous selection. The reduced fitness of the most resistant variant suggests that it may be rare in naïve patients and that development of high-level resistance may be slow. Combination therapy with IFN-alpha should also greatly reduce the potential emergence of resistance.

Blotting, Western↗
PubMed2026

Comparative genomic epidemiology of food- and patient-derived diarrheagenic Escherichia coli from sentinel surveillance in Southeast China.

Diarrheagenic Escherichia coli (DEC) remains an important foodborne pathogen, yet long-term comparative genomic surveillance data jointly characterizing food-derived and patient-derived isolates remain limited. This surveillance-based comparative study integrated antimicrobial susceptibility testing and whole-genome sequencing to characterize diarrheagenic Escherichia coli isolates recovered from food and patient sources in Lishui, Southeast China, during 2018-2025, with emphasis on occurrence, resistance profiles, genomic backgrounds, and plasmid replicon-associated features. Antimicrobial susceptibility testing was performed for 258 selected isolates, and whole-genome sequencing was conducted for a curated analytical subset of 204 isolates. The sequenced subset was used for diversity-oriented comparative genomic analysis rather than for unbiased prevalence estimation of the entire DEC collection. EAEC predominated in both sources, although food-associated occurrence was heterogeneous across categories, with the highest recovery rate observed in raw meat. Patient-derived isolates showed a broader overall resistance burden, whereas food-derived isolates retained substantial resistance to tetracycline, chloramphenicol, and florfenicol. Phylogenetic analysis showed partial overlap in genomic backgrounds between food-derived and patient-derived isolates, while representative resistance determinants displayed both broadly distributed and lineage-enriched patterns. Replicon-based plasmid profiling identified 42 plasmid types, including 12 detected in both sources, with IncF-related replicons predominating among these shared profiles. Several food-derived isolates carried multiple plasmid replicon types that were also observed in patient-derived isolates. Overall, food-derived and patient-derived DEC showed partial overlap in genomic backgrounds, resistance determinants, and replicon-defined plasmid profiles within this surveillance setting, while retaining source-associated heterogeneity. These findings should be interpreted as surveillance-based comparative evidence rather than as evidence of direct source attribution or transmission.

Humans↗
PubMed2005

Nonstructural protein 5A does not contribute to the resistance of hepatitis C virus replication to interferon alpha in cell culture.

The hepatitis C virus (HCV) subgenomic replicon system was used to study a possible involvement of nonstructural protein 5A (NS5A) in the mechanisms of HCV resistance to interferon alpha (IFN-alpha). A series of chimeric HCV replicons was constructed. In these replicons, the NS5A gene in the backbone of the Con1 replicon was swapped by corresponding fragments obtained from four IFN-alpha responder and four IFN-alpha nonresponder patients that had been infected with the same HCV AD78 strain. Experiments with transfected Huh7 cells did not reveal significant differences in sensitivity of HCV RNA replication to IFN-alpha in cell clones, bearing chimeric Con1/AD78 replicons with NS5A sequences from IFN responders and nonresponders. Thus, these data provide no evidence that the NS5A protein contributes to the resistance of HCV replication to IFN-alpha.

Amino Acid Sequence↗
PubMed2000

Multiple chromosomes in Burkholderia cepacia and B. gladioli and their distribution in clinical and environmental strains of B. cepacia.

Burkholderia cepacia is found in soils and waters, it can be used in biocontrol and bioremediation but is also a human pathogen. It is not yet clear what differentiates pathogenic from non-pathogenic strains of the organism. In this study the multiple replicon structure was investigated in 28 strains of B. cepacia by pulsed field gel electrophoresis. All strains examined, whether of clinical, environmental or plant pathogenic origin, were found to have two, three or four large (> 500 kbp) replicons. Many strains also contained small replicons. Clinical strains were more likely to have three or four large replicons than non-clinical strains. Multiple replicon structure was also demonstrated in B. gladioli and Alcaligenes eutrophus.

Burkholderia↗
PubMed1980

Initiation of DNA replication in a ColE1-type plasmid: isolation of mutations in the ori region.

We have constructed a plaque-forming hybrid phage, lambda SN4, which behaves as a composite replicon of the lambda phage and a mini-ColE1 plasmid. From the hybrid phage, plaque-type mutants altered in the ability to replicate as a ColE1 replicon were isolated. These mutations were designated as cer, signifying ColE1 replication defective. One of such mutants, lambda SN4cer6, was studied further. The mutant DNA was unable to replicate in vivo if expression and function of its lambda replicon were inhibited. The defect could not be complemented in trans. DNA sequence determination of the mutant phage revealed a single base pair (bp) alteration, C-G to T-A, at 160 bp upstream from the ori site of its ColE1 replicon. From lambda SN4cer6, revertants were obtained that had regained function of the ColE1 replicon, and they could be classified into two groups that showed a full and a partial recovery in the rates of ColE1-driven DNA synthesis. DNA sequence determination of revertant DNA indicated that the former group contained true revertants, T-A to C-G, at the cer6 site, whereas one of the partial revertants was found to sustain a secondary-site mutation, G-C to A-T at 187 bp upstream of the ori site. It was possible to construct a hairpin structure that starts by hydrogen bonding of bases at the site -160 and -187.

Bacteriocin Plasmids↗