Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.     

体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kan^r)基因对成团肠杆菌固氮质粒pEA9进行活体遗传标记。将来自质粒pEA9的3.0kb片段(nif ENX)克隆到pBR322载体中,再将卡那霉素抗性(Kan^r)基因插入到3.0kb的片段中,构建成供体质粒pST5。将该质粒转化到含有待标记质粒pEA9的E.a.339菌株中,然后在AP培养基中消除供体质粒,筛选得到40个失去了pST5并保持卡那霉素抗性的克隆,分析表明它们不是质粒pEA9和pST5的共整合体,而是卡那霉素抗性基因通过两个质粒在nifENX区域内的DNA间的同源重组整合到了质粒pEA9上。     

Some characteristics of transgenic clones of mouse R1 line embryonic stem cells

Transgenic clones of mouse embryonic stem cells of the R1 line were received by transfection of plasmid linear vectors. The changes in the transgene structure during its integration into the genome of the target cells were investigated. Displacements were found on the flanks of the integrated transgene. It was found that multicopy tandem structures are formed in head–tail orientation at the transgene integration. It was noted that the number of copies of the integrated transgenes varies considerably, but the introduction of DNA fragments isolated from the nuclear envelopes into the flanks of the transgene normalizes the number of its copies.     

Cloning of a multicopy plasmid from the actinorhizad nitrogen-fixing bacterium Frankia sp. and determination of its restriction map

An 8.3-kb multicopy plasmid, pFQ31, from the nitrogen-fixing Frankia sp. strain ArI3, was cloned into Escherichia coli plasmid vectors and analysed physically. pFQ31 has no detectable sequence homology with another Frankia plasmid, pFQ32, which is present in the same host. Derivatives of pFQ31 with an antibotic resistance marker were introduced into Streptomyces lividans, which is taxonomically related to Frankia, but no stable replication could be achieved.     

Production of supercoiled multimeric plasmid DNA for biopharmaceutical application

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.     

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.     

Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62×10^-4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.Abbreviations DOPE dioleoylphosphatidylethanolamine - DOPC dioleoylphosphatidylcholine - Chol cholesterol - OA oleic acid - PEG polyethylene glycol 6000 - NAA -napthaleneacetic acid - BAP 6-benzylaminopurine     

Detection of displacement (“D”) loops with the properties of a replicating intermediate synthesized by a DNA/membrane complex derived from the low-copy-number plasmid RK2

A significant fraction of the plasmid DNA extracted from an RK2 miniplasmid DNA/membrane complex after incubation in vitro with appropriate substrates and cofactors contains "D" or displacement loops in one of at least three loci in the genome. These include the origin of replication and two transposon regions that code for kanamycin and tetracycline resistance. The relationship of these loops to early replicating intermediates for plasmid and transposon replication is discussed.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Transposition of the maize transposable element Ac in Solanum tuberosum

Summary The maize transposable element Ac has been introduced into potato via the T-DNA (transferred DNA) of Agrobacterium tumefaciens. Ac was inserted within the untranslated leader region of a neomycin phosphotransferase II (NPT-II) gene such that excision restored NPT-II activity. Two approaches to monitor Ac excision were used. (i) Using an Agrobacterium strain harbouring plasmid pGV3850::pKU3, leaf discs were selected on kanamycin (Km) after exposure to Agrobacterium. (ii) Using a strain containing plasmid pGV3850HPT::pKU3, the leaf discs were selected on hygromycin (Hm) and the resulting shoots were checked for NPT-II expression. Thirteen kanamycin resistant shoots transformed with pGV3850::pKU3 were isolated, suggesting that Ac had excised from the NPT-II gene. Out of 43 hygromycin resistant shoots transformed with pGV3850HPT::pKU3, 22 expressed the NPT-II gene, indicating that Ac had undergone excision in approximately 50% of the hygromycin resistant shoots. Southern analysis revealed that all kanamycin resistant plants contained the DNA restriction fragments expected when Ac excises from the NPT-II gene. The presence of Ac at new locations within the genomic DNA of several transformants was also detected.     

A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells.

We describe the ability of novel episomally maintained vectors to efficiently promote gene expression in embryonic stem (ES) cells as well as in established mouse cell lines. Extrachromosomal maintenance of our vectors is based on the presence of polyoma virus DNA sequences, including the origin of replication harboring a mutant enhancer (PyF101), and a modified version of the polyoma early region (LT20) encoding the large T antigen only. Reporter gene expression from such extrachromosomally replicating vectors was approximately 10-fold higher than expression from replication-incompetent control plasmids. After transfection of different ES cell lines, the polyoma virus-derived plasmid variant pMGD20neo (7.2 kb) was maintained episomally in 16% of the G418-resistant clones. No chromosomal integration of pMGD20neo vector DNA was detected in ES cells that contained episomal vector DNA even after long term passage. The vector's replication ability was not altered after insertion of up to 10 kb hprt gene fragments. Besides undifferentiated ES cells, the polyoma-based vectors were also maintained extrachromosomally in differentiating ES cells and embryoid bodies as well as in established mouse cell lines.     

Stability and expression of bacterial genes in replicating geminivirus vectors in plants.

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.     

Classification of plasmid vectors using replication origin, selection marker and promoter as criteria

Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a classification method for commonly used plasmid vectors. Plasmid vectors were classified into different classes based on their replication origin, selection marker and promoter information. The replication origins of plasmid vectors were classified as: prokaryotic replication origin, eukaryotic replication origin and viral replication origin. Selection markers of plasmid vectors were mainly classified as ampicillin, kanamycin, neomycin, chloramphenicol, gentamycin, tetracycline, erythromycin, streptomycin, vancomycin and spectinomycin resistance gene markers. Promoter sequences were also classified as prokaryotic, eukaryotic and viral promoters. Finally, the nomenclature of common plasmid vectors has three determinants. We believe that the classification of plasmid vectors can provide useful information for researchers employing molecular cloning procedures. A web service of the plasmid classification was established and it is available from http://www.computationalmedicalbiology.org/plasclas.aspx.     

Gene silencing from plant DNA carried by a Geminivirus

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.     

A vehicle for the introduction of transposons into plant-associated pseudomonads

A recombinant plasmid with wide-host-range transfer functions, narrow-host-range replication functions, and carrying a kanamycin-resistant transposon transferred kanamycin resistance to a number of plant-associated pseudomonads. Southern hybridization studies suggest that only a small portion of the plasmid, coinciding with the location of the transposon, is present in the kanamycin-resistant Pseudomonas derivatives. The plasmid sequences appear to be inserted at a number of different sites in the recipient genome. This plasmid can thus be used as a vehicle for the introduction of transposons into some plant-associated pseudomonads and should be useful in both genetic and ecological studies of these bacteria.