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PubMed1999

Plasmid-encoded anthranilate synthase (TrpEG) in Buchnera aphidicola from aphids of the family pemphigidae.

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819-3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC1, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC2, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.

Amino Acid Sequence↗
PubMed1972

Positive and negative control of R-factor replication in Proteus mirabilis.

Replication of the 50 and 58 moles per cent guanine plus cytosine (%GC) components of R factor 222 in Proteus mirabilis during growth in the presence and absence of chloramphenicol and after shifting exponential- and stationary-phase cells to conditions which inhibit host protein or deoxyribonucleic acid (DNA) synthesis was examined. Chloramphenicol reduced the growth rate but increased the amount of both R-factor components; the 58% GC component showed a larger proportionate increase. This was inferred to indicate reduced synthesis of an inhibitor that acts on both R-factor components and an initiator for replication of the 50% GC component. Replicative patterns observed after shifting exponential- and stationary-phase cells grown with or without chloramphenicol to minimal medium or chloramphenicol for one generation, or puromycin for 3 hr, corroborated this interpretation. After shifts of exponential cells from either medium, replication of the 50% GC components paralleled host replication, thus indicating a requirement for protein synthesis; replication of the 58% GC component increased due to reduced inhibitor synthesis. R-factor DNA remained constant after shifting stationary cells from drug-free medium, thus indicating that the cells contained effective concentrations of the regulatory inhibitor, whereas increased replication of the 58% GC component occurred after identical shifts of chloramphenicol-grown cells of the same chronological age. Similar responses were observed after shifts to 5 C or to medium containing streptomycin or tetracycline. Absence of replication of the 50% GC component after shifting to medium containing nalidixic acid or phenethanol and its hereditary persistence during growth indicated that the 50% GC replicon was attached to the membrane. Thus, in P. mirabilis the three replicons of R factor 222 are regulated as follows: The composite and transfer portion (RTF) replicons represented by the 50% GC component require protein synthesis and membrane attachment and are negatively regulated by an inhibitor; the 58% GC or resistance-determinants replicon exists cytoplasmically and is subject only to negative control.

Alcohols↗
PubMed1989

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.

Cloning, Molecular↗
PubMed1989

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage.

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.

Bacteriophages↗
PubMed2006

Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging.

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Animals↗
PubMed1996

Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer.

Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.

Animals↗
PubMed2001

Phenotypic characterization of three phylogenetically conserved stem-loop motifs in the mengovirus 3' untranslated region.

An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3' untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (DeltaII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37 degrees C). In a few of the DeltaII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 microg per 3 x 10(6) cells). Viruses from two DeltaII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the DeltaII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D(pol)), and the other was localized to the 3' UTR, upstream of stem I.

3' Untranslated Regions↗
PubMed2003

Transcription strategy in a Closterovirus: a novel 5'-proximal controller element of Citrus Tristeza Virus produces 5'- and 3'-terminal subgenomic RNAs and differs from 3' open reading frame controller elements.

Citrus tristeza virus (CTV) produces more than thirty 3'- or 5'-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5'-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3' genes deleted produce only the larger LMT1 RNAs. These 5'-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5'TR) surrounding the putative termination sites (nt approximately 550 to 1000) was duplicated in the 3' end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparently by termination. Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR. A large 3'-terminal sgRNA resulting from the putative promoter activity of the native 5'TR was not observed, possibly because of the down-regulation of a promoter approximately 19 kb from the 3' terminus. However, we were able to observe a sgRNA produced from the native 5'TR of a small defective RNA, which placed the native 5'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5'TR (in the 3' position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3'- and 5'-terminal sgRNAs. These mutations, when introduced in frame in the native 5'TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5'TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3' open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3'-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

Base Sequence↗
PubMed2004

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.

Antiviral Agents↗
PubMed2005

Dissecting the interferon-induced inhibition of hepatitis C virus replication by using a novel host cell line.

The Hepatitis C virus (HCV), a member of the family Flaviviridae, is a major cause of chronic liver disease. Patients are currently treated with alpha interferon (IFN-alpha) that is given alone or in combination with ribavirin. Unfortunately, this treatment is ineffective in eliminating the virus in a large proportion of individuals. IFN-induced antiviral activities have been intensively studied in the HCV replicon system. It was found that both IFN-alpha and IFN-gamma inhibit HCV replicons, but the underlying mechanisms have not yet been identified. Of note is that nearly all of these studies were performed with the human hepatoma cell line Huh-7. Here, we report that genotypes 1b and 2a replicons also replicate in the human hepatoblastoma cell line HuH6. Similar to what has been described for Huh-7 cells, we observed that efficient HCV replication in HuH6 cells depends on the presence of cell culture-adaptive mutations and the permissiveness of the host cell. However, three major differences exist: in HuH6 cells, viral replication is (i) independent from ongoing cell proliferation, (ii) less sensitive to certain antiviral compounds, and (iii) highly resistant to IFN-gamma. The latter is not due to a general defect in IFN signaling, as IFN-gamma induces the nuclear translocation of signal transducer and activator of transcription 1 (STAT1), the enhanced transcription of several IFN-regulated genes, and the inhibition of unrelated viruses such as influenza A virus and Semliki Forest virus. Taken together, the results establish HuH6 replicon cells as a valuable tool for IFN studies and for the evaluation of antiviral compounds.

Antiviral Agents↗
PubMed2003

Simple sequence repeats and compositional bias in the bipartite Ralstonia solanacearum GMI1000 genome.

BACKGROUND: Ralstonia solanacearum is an important plant pathogen. The genome of R. solananearum GMI1000 is organised into two replicons (a 3.7-Mb chromosome and a 2.1-Mb megaplasmid) and this bipartite genome structure is characteristic for most R. solanacearum strains. To determine whether the megaplasmid was acquired via recent horizontal gene transfer or is part of an ancestral single chromosome, we compared the abundance, distribution and composition of simple sequence repeats (SSRs) between both replicons and also compared the respective compositional biases. RESULTS: Our data show that both replicons are very similar in respect to distribution and composition of SSRs and presence of compositional biases. Minor variations in SSR and compositional biases observed may be attributable to minor differences in gene expression and regulation of gene expression or can be attributed to the small sample numbers observed. CONCLUSIONS: The observed similarities indicate that both replicons have shared a similar evolutionary history and thus suggest that the megaplasmid was not recently acquired from other organisms by lateral gene transfer but is a part of an ancestral R. solanacearum chromosome.

Base Composition↗
PubMed2004

Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex.

Positive-strand RNA viruses replicate their RNA genome within a ribonucleoprotein (RNP) complex that is associated with cellular membranes. We used a two-step method of purification to isolate hepatitis C virus (HCV) RNP complexes from human hepatoma cell line Huh7, which stably expresses HCV subgenomic replicons. The procedure involved hybridization of replicon-expressing cellular lysates with oligonucleotides tagged with biotin and digoxigenin at their respective termini complementary to subgenomic replicon RNA followed by avidin-agarose enrichment of the mixture and subsequent immunoprecipitation of biotin-eluted material with anti-digoxigenin antibody. The immunoprecipitates were immunoblotted with antisera against HCV nonstructural (NS) proteins. The analysis revealed the association of all the HCV NS proteins (NS3, NS4a, NS4b, NS5a, and NS5b) that are encoded by the subgenomic replicon RNA. The HCV RNP complex migrated in a native polyacrylamide gel with an approximate molecular mass of 450 kD. The association of these viral proteins in the RNP complex reinforces the widely acknowledged notion that RNA viruses accomplish replication within a membranous RNP complex.

Chromatography, Affinity↗
PubMed1998

Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints.

Three DNA damage-responsive cell cycle checkpoints can be shown to operate in diploid human fibroblasts. One checkpoint arrests growth in G1, another inhibits replicon initiation in S phase cells, and the third delays progression from G2 into mitosis. Progression from G2 into M is controlled in part by a cyclin-dependent kinase (cyclin B/Cdk1) that is regulated by tyrosine phosphorylation. Phosphorylation of Tyr15 on Cdk1 is inhibitory for kinase activity. Activation of cyclin B/Cdk1 at the onset of mitosis is accomplished by a phosphatase, Cdc25C, that interacts with cyclin B/Cdk1 in an autocatalytic feedback loop to remove the inhibitory phosphate at Tyr15 and activate kinase activity. DNA damage triggers G2 delay by inhibiting formation of the autocatalytic feedback loop so that dephosphorylation of Tyr15 does not occur. This suppression of activation of cyclin B/Cdk1 appears to account for the failure of damaged G2 cells to progress into mitosis. Once the damage to DNA is repaired, cells resume progression into mitosis as the cycle is re-engaged. The isoflavone genistein inhibits tyrosine kinases, including one that phosphorylates Cdk1 on Tyr15. This kinase, p56/p53lyn is rapidly induced by treatments that trigger cell cycle checkpoints (ionizing radiation, cytosine arabinoside), suggesting that this kinase may actively delay the onset of mitosis by phosphorylating Tyr15 on Cdk1. Genistein also inhibits type II DNA topoisomerase to produce a form of DNA damage that triggers all of the DNA damage-responsive cell cycle checkpoints. A brief 10 min incubation with the topoisomerase poison amsacrine was sufficient to trigger the S phase checkpoint response and inhibit replicon initiation. Inhibition of replicon initiation by 1 microM amsacrine was maximal 20-30 min after drug treatment and by 120 min, the checkpoint response had decayed to allow near control rates of replicon initiation. Topoisomerase II poisons also are powerful clastogens inducing lethal and carcinogenic chromosomal aberrations. Type II topoisomerase can break DNA in a region of chromosome 11q23 that contains the ataxia telangiectasia gene (ATM). The ATM gene controls all of the DNA damage-responsive cell cycle checkpoints. Chromosomal aberrations in 11q23 are frequently seen in acute myeloid leukemia that develops as a consequence of etoposide chemotherapy. Thus, topoisomerase poisons such as genistein may trigger chromatid breakage to inactivate AT gene function, disable cell cycle control, and induce genetic instability.

DNA Damage↗
PubMed1986

Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity.

tsFT20 cells derived from a mouse mammary carcinoma cell line FM3A have temperature-sensitive DNA polymerase alpha activity (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1761-1765). DNA replication in tsFT20 cells at the restrictive temperature (39 degrees C) has been characterized in detail. DNA-synthesizing ability of these cells was measured by [3H] thymidine incorporation and autoradiography. The incorporation of [3H]thymidine decreased rapidly after temperature shift-up, and the incorporation was less than 20% of the initial level after 4 h at 39 degrees C. The rapid decrease correlated well with the decrease in the grain number in the individual nucleus but not with the number of cells with labeled nuclei. Alkaline sucrose gradient sedimentation analysis and DNA fiber autoradiography revealed that DNA chain elongation proceeded normally within a replicon in the temperature-sensitive cells incubated at the restrictive temperature and the DNA elongation rate did not change during the incubation at the restrictive temperature up to at least 6 h. On the other hand, the maturation of replicon-sized DNA to higher molecular weight DNA was retarded or inhibited in the temperature-sensitive cells at the restrictive temperature. The analysis of the center to center distance between replicons by DNA fiber autoradiography revealed that the frequency of replicon initiation decreased in tsFT20 cells at 39 degrees C.

Animals↗
PubMedDate not supplied

Alphavirus vectors and vaccination.

Alphaviruses are positive-stranded RNA viruses that have a broad host range and therefore are capable of replicating in many vertebrate and invertebrate cells. The single-stranded alphavirus genome is divided into two ORFs. The first ORF encodes the nonstructural proteins that are translated upon entry of the virus into the cytoplasm and are responsible for transcription and replication of viral RNA. The second ORF is under the control of a subgenomic promoter and normally encodes the structural proteins, which are responsible for encapsidation of viral RNA and final assembly into enveloped particles. Expression vectors have been engineered from at least three alphaviruses in which the structural protein gene region has been replaced by heterologous genes and have been shown to express high levels of the heterologous protein in cultured cells. These RNA vectors, known as replicons, are capable of replicating on their own but are not packaged into virus-like particles unless the structural proteins are provided in trans. Thus, replicons are single cycle vectors incapable of spreading from infected to noninfected cells. Because of these features, alphavirus replicon vectors are being developed as a platform vaccine technology for numerous viral, bacterial, protozoan and tumour antigens where they have been shown to be efficient inducers of both humoral and T cell responses. In addition, as the alphavirus structural proteins are not expressed in vaccine recipients, antivector immune responses are generally minimal, allowing for multiple effective immunisations of the same individual.

Alphavirus↗
PubMed2000

Stable transduction of actively dividing cells via a novel adenoviral/episomal vector.

Many gene therapy indications would benefit from vectors capable of achieving efficient in vivo delivery and long-term transgene expression in either dividing or nondividing cells. Such vector systems are not yet available. To achieve both goals, we have used noncytotoxic E1- and E4-deleted adenoviral vectors as vehicles for delivering an Epstein-Barr virus-based self-replicating episome (replicon) via Cre/loxP site-specific recombination. Co-infection of human cells with a proreplicon-encoded and a Cre-expressing adenovirus resulted in efficient delivery and excision of a functional replicon in the absence of vector-induced cytotoxicity. In addition, replication and nuclear retention of the replicon in the cell progeny translated into a prolonged transgene expression in actively dividing cells, both in vitro and in vivo. Combining desired features from different viruses within a single hybrid vector system should expand the range of clinical indications currently amenable to gene transfer.

Adenoviruses, Human↗
PubMed1997

Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain.

The species type strain of Pseudomonas alcaligenes contains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively. Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related. pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C. This suggested that pECB3 is especially rich in these bases. Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1. A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kb Pseudomonas-specific plasmid isolated from Pseudomonas syringae pv. savastonoi. The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced. Introduction of the putative replicon into the Escherichia coli plasmid pUC19 created a recombinant vector able to replicate in both E. coli and Pseudomonas spp. Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1--a finding supported by sequencing studies. Attempts at plasmid curing were unsuccessful. A phenotypic comparison with a non-plasmid-harboring strain of P. alcaligenes, based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth. Transformation of the non-harboring strain with pECB1-3, followed by selection on a minimal medium containing benzoate, gave no colonies. The advantages gained by possession of pECB1-3, if any, are at present unknown.

Amino Acid Sequence↗
PubMed1995

An unusual mutation in RepA increases the copy number of a stringently controlled plasmid (Rts1 derivative) by over one hundred fold.

A copy number mutant of the Rts1 replicon (copy number 1-2 copies/cell) was obtained. A one-base substitution in the repA region results in a single amino acid change from histidine to asparagine at position 159. This mutation increased the plasmid copy number by up to 120-fold depending upon the growth conditions. At 42.5 degrees C the plasmid with the wild type replicon was unstable while the mutated replicon was relatively stable.

Bacterial Proteins↗