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PubMed2003

The human rhinovirus internal cis-acting replication element (cre) exhibits disparate properties among serotypes.

It has been reported previously that the Human rhinovirus 14 (HRV-14) RNA genome contains a cis-acting replication element (cre) that maps to the capsid coding (P1) sequence [19]. Further characterization of the HRV-14 cre in the present study established that by moving the cre stem-loop structure downstream, adjacent to the 3'NCR, that its position is not critical for function. When the P1 sequences of two closely related serotypes of HRV-14 were analyzed for the presence of a cre, both HRV-3 and HRV-72 were found to contain similar sequence at the same positions as HRV-14. Moreover, sequence at these positions produced structures from MFOLD analysis that closely resembled the HRV-14 cre. It was also discovered that neither HRV serotypes 1a or 16 harbor replication elements that map to the P1 segments of their genomes. Computer and mutational analyses suggest that the cre in these latter HRV serotypes map instead to the 2A gene, as has been reported for HRV-2. The putative HRV-3 cre was determined to be unable to support replication when placed in an HRV-14 replicon background. Similarly, the previously identified HRV-2 cre was unable to support replication of the HRV-14 genome. This finding is in contrast to the cardiovirus cre, which has been shown to be functionally active between two members of its family, and further suggests that there is a close link between the evolution of the human rhinoviruses and the mechanisms of RNA replication.

3' Untranslated Regions↗
PubMed2001

Investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants.

There has been a great deal of interest in the possibility that geminiviruses might be used as infectious gene vectors for expression of foreign proteins in plants. However, generic mastreviruses such as Maize streak virus (MSV) have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants with it and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vector could be complemented in trans by co-transfected wild type virus, true systemic movement of either the vector, or of co-complementing constructs, did not occur. However, recombination between the two complementing viral constructs frequently occurred to generate wild-type virus genomes. The results therefore suggest that formation of ssDNA and size of the viral replicon are not the sole determinants of whether the MSV movement proteins can mobilise viral sequences and move them systemically in plants.

Blotting, Southern↗
PubMed2001

A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature.

Genetic manipulation of Antarctic bacteria has been very limited so far. This article reports the isolation and molecular characterization of a novel plasmid, pMtBL, from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAC 125. This genetic element, 4,081 bp long, appeared to be a multicopy cryptic replicon with no detectable transcriptional activity. By an in vivo assay, the pMtBL autonomous replication sequence was functionally limited to an AluI plasmid fragment of about 850 bp. This novel cold-adapted replication element showed quite a broad host range profile: it was cloned into a mesophilic genetic construction, obtaining a cold-adapted expression vector that was able to promote the production of P. haloplanktis A23 alpha-amylase in a psychrophilic bacterium. This study represents the first report of successful recombinant production of a cold-adapted protein in an Antarctic host.

Antarctic Regions↗
PubMed1980

Mode of action of 9-beta-D-arabinosyladenine and 1-beta-D-arabinosylcytosine on DNA synthesis in human lymphoblasts.

The effects of 9-beta-D-arabinosyladenine (AraAde), 1-beta-D-arabinosylcytosine (AraCyt) and 2'-deoxyadenosine on DNA replication in cultured human lymphoblasts (CCRF-CEM line) were studied by pulse-labeling cells with [3H]thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients. At doses that reduced the overall rate of DNA synthesis to 50--70% of control values, both AraAde and AraCyt profoundly inhibited the formation of new replicons, with secondary effects on chain elongation contributing to the total inhibition of DNA synthesis. In contrast, the suppression of DNA synthesis by 2'-deoxyadenosine stemmed mainly from an inhibition of chain elongation. These studies also disclosed that about 100 times more AraAde than AraCyt was required to produce a similar inhibition of DNA replication in CCRF-CEM cells. Determination of intracellular concentrations of the nucleoside triphosphates (AraCTP and AraATP) indicated to 90% inhibition of DNA synthesis was achieved at 1.6 and 25 pmol/1 . 10(6) cells, respectively. Studies with cell lysates revealed that the replicative machinery in CCRF-CEM cells is more sensitive to AraCTP than to AraATP. This finding contrasts with earlier research, in which the inhibtion of purified DNA polymerase by either AraATP of AraCTP yielded essentially the same Ki value. The difference in sensitivity of the cell lysate to these arabinonucleotides may reflect either a target enzyme other than DNA polymerase or, more plausibly, some subtle interaction of the polymerase with other components of the replicative process.

Cells, Cultured↗
PubMed1986

DNA synthesis studies in pre-meiotic mouse oogenesis.

DNA fiber autoradiography was used in mouse oogenesis to test Callan's hypothesis that a longer S phase results from a reduction in the number of active initiation sites. The data indicated that the premeiotic increase in the duration of S phase in mouse oogenesis was characterized by a rapid initial rate of chain growth and a larger replicon size when compared with replicating DNA of somatic mouse cells. These findings were at variance with those in mouse spermatogenesis and also did not support the Callan hypothesis of activation site repression.

Animals↗
PubMed1986

Interaction between gene II protein and the DNA replication origin of bacteriophage f1.

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

Bacteriophages↗
PubMed1982

Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3.

The tnpR gene product, resolvase, of the transposable element gamma delta mediates site-specific recombination between two copies of gamma delta directly repeated on the same replicon, and it negatively regulates transcription of the tnpA (transposase) gene and its own gene. The recombinational site, res, and the regulatory region both are located in the tnpA-tnpR intercistronic region. In studying the interaction of purified resolvase with DNA fragments derived from gamma delta and the related transposon, Tn3, that span this region, we have demonstrated that three sites specifically bind resolvase. Site I overlaps the recombinational crossover point and both transcriptional promoters. Sites II and III cover most of the DNA between the crossover point and the translational start of the tnpR gene. These are the only binding sites we have detected in a region of about 400 base pairs centered on the crossover point. Studies of cointegrates that contain only part of the region that binds resolvase indicate that site I is not sufficient for efficient site-specific recombination and suggest that all three sites are probably required.

Binding Sites↗
PubMed1985

tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution.

We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein. The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo. A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences. Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments. Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene. We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.

Bacterial Proteins↗
PubMed1986

Insertion of an R1 plasmid into the origin of replication of the E. coli chromosome: random timing of replication of the hybrid chromosome.

A 16 bp BgI II fragment was deleted in vitro from the minimal origin of replication of the Escherichia coli chromosome, oriC, and was replaced by a 10 kb R1 miniplasmid, pKN1562, containing the basic R1 replicon and a kanamycin resistance gene. The deletion-insertion was transferred by homologous recombination into the chromosome of a dnaA(ts) strain. P1 transduction separated the origin "mutation" from the dnaA46 allele. Integration of mini-R1 into oriC was verified by Southern blotting and by analysis of the R1 incompatibility phenotype. It was possible to isolate normal R1 miniplasmids from the integrated R1. Chromosome replication was initiated at random times after a short delay. The constructed strains grew 20%-30% slower than the wild type and showed more heterogeneous cell sizes.

Chromosomes, Bacterial↗
PubMed1990

Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes.

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria. As indicated by nucleic acid hybridisation, the genes in pIP811 conferring resistance to chloramphenicol, erythromycin, and streptomycin were closely related to plasmid-borne determinants that are common in enterococci-streptococci. Plasmid pIP811 shared extensive sequence homology with pAM beta 1, the prototype broad host range resistance plasmid in these two groups of gram-positive cocci. These results suggest that emergence of multiple antibiotic resistance in Listeria spp is due to acquisition of a replicon originating in enterococci-streptococci. The dissemination of resistance to other strains of L monocytogenes is likely.

Chloramphenicol Resistance↗
PubMed1980

Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region.

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol., 118, 1123-1131).

Cell Division↗
PubMed1983

Escherichia coli minichromosomes containing the pSC101 partitioning locus are stably inherited.

The par region of pSC101, required in cis to promote its stable inheritance, was joined, in combination with the tetr determinant of pBR325, to large and small minichromosomes. These hybrid minichromosomes were examined for stability and found to be no more stable than their parent minichromosomes. Indeed, one recombinant plasmid, pEH21, showed reduced stability, which was not attributable to a reduced copy number. Neither pEH21 nor pEH22, a plasmid composed of the same DNA arranged differently, was stabilized by the presence of a Par+ pSC101 derived replicon in the same cell. We conclude that the par region of pSC101 does not stabilize minichromosomes.

Cell Division↗
PubMed1983

Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.

A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA*. The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms. These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria.

DNA Replication↗
PubMed1984

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans.

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.

Base Sequence↗
PubMed1984

Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2.

The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol. 141, 213-222). They include the origin of replication (oriRK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions oriRK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.

Bacterial Proteins↗
PubMed1985

Specific-purpose broad-host-range vectors.

Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression.

Chromosome Mapping↗
PubMed1986

Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis.

A range of Tn1723 transposon mutants of the oriV region of broad-host-range plasmid RK2 have been isolated, and the internal EcoRI fragment of the transposon has been deleted from each to reduce the insertion size from 9.6 kb (Tn1723) to 35 bp (delta Tn1723). Sequencing from the delta Tn1723-derived EcoRI site has allowed the precise mapping of these insertions to various points dispersed through the origin region. Using these mutants we have determined which regions of oriV RK2 are of functional importance to plasmid establishment following transformation of the host species Escherichia coli, Pseudomonas putida, and P. aeruginosa. Insertions into an A/T-rich region, and a region containing five direct repeat sequences prevented successful transformation of each host species tested, but the continuity of sequences adjacent to the five repeats were essential only in E. coli and P. putida. The establishment and maintenance in E. coli of a mini-RK2 replicon was found to be inhibited by transcription from an inducible promoter positioned to read into oriV RK2 against the direction of replication. Assays of transcription emerging from Tn1723 demonstrated significant levels from one end of the transposon only. Four mutants with insertions downstream of oriV RK2 were unable to become established in E. coli, and contained Tn1723 in the orientation which would supply transcription toward the oriV RK2 region. These results demonstrate both that the sequence requirements for oriV RK2 function differ between host bacterial species, and that origin function may be further influenced by the genetic environment in which it lies.

Base Sequence↗
PubMed1987

A new IncQ plasmid R89S: properties and genetic organization.

The new small (8.18 kb) streptomycin-resistant multicopy plasmid R89S of the Q group incompatibility is described. In contrast to other IncQ plasmids, replication of R89S is dependent on DNA polymerase 1 and proceeds in the absence of de novo protein synthesis. According to our data up to now, the host spectrum of the plasmid R89S is limited to Enterobacteriaceae. A genetic map of the plasmid R89S has been prepared through the construction of deletion and insertion derivatives. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to streptomycin, and the region essential for mobilization of R89S. The origin of vegetative replication has been located within a 0.7-kb fragment. Another region highly homologous to oriV of the plasmid RSF1010, but not functioning as an origin of replication, was localized. Two regions involved in the expression of incompatibility have also been identified. The data from the restriction analyses, DNA-DNA hybridization, and genetic experiments enable us to assume that the plasmid R89S is a naturally occurring recombinant between part of an IncQ plasmid and another narrow host range replicon of unknown incompatibility group.

Chromosome Mapping↗