Conditional gene vectors regulated in cis

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.     

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.     

Isolation and characterization of a rolling-circle-type plasmid from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant-protein expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a theta-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Replication and Control of Circular Bacterial Plasmids

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.     

Primary sequence differences between Chido and Rodgers variants of tryptic C4d of the human complement system

The initiation sites of the Galleria mellonella L. nuclear polyhedrosis virus (G.m. NPV) DNA replication were revealed. For this purpose SCLd 135 cells permitting the G.m. NPV productive reproduction were transformed by the recombinant plasmids containing the viral genome individual fragments in pRSF 2124 and pBR 322 vectors. It was revealed that 2 of the 32 recombinant plasmids can autonomously replicate in the eucaryotic cells. According to the Maxam-Gilbert method the DNA G.m. NPV fragment (1300 bp) primary structure of pHBR plasmid was determined. The structure analysis revealed the typical regulator signals as in the replicons. The possible regulation mechanism of the DNA G.m. NPV synthesis initiation was supposed.     

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a θ-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110

Gateway^® cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method.     

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.     

Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics

Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin-spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum. The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families.     

Genetic analysis of bacterial plasmid promiscuity

The genetic basis of the promiscuous behaviour of bacterial plasmids has been investigated by study of the incompatibility P-1 group of conjugative plasmids of gram-negative bacteria. Both transposon mutagenesis and the construction of minireplicons linking varying combinations of the plasmid genome have shown that specific genomic regions control the conjugational transfer and vegetative replication of the plasmid in specific bacterial hosts. These include the plasmid DNA primase gene, the origin of plasmid transfer, a region near the origin of transfer, the origin of plasmid vegetative replication, thetrans- acting gene essential for the initiation of plasmid replication and a region involved in its regulation. DNA sequence analysis has identified the requirement of specific direct repeats within the origin of replication for plasmid replication in some but not in other hosts. The cloning of some of the trans-acting genes onto multicopy cloning vectors and complementation tests have shown that the requirements of these gene products vary in different hosts and that the plasmid has evolved genetic strategies for their optimal expression.     

A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid.

The major DNA-binding protein, ICP8, encoded by herpes simplex virus is localized to the infected cell nucleus where it plays a role in viral DNA replication and control of viral gene expression. To identify the parts of the ICP8 protein that are important for its localization and functions, we have developed a system to test the ability of recombinant plasmids to express functional ICP8. A recombinant plasmid containing the wild-type ICP8 gene was transfected into cells. The cells were later infected with a temperature-sensitive ICP8 mutant virus at the nonpermissive temperature. Sufficient wild-type ICP8 was expressed from the transfected plasmid to complement the replication of the mutant virus. This provides a genetic system to test the properties of ICP8 expressed from mutagenized plasmids without the establishment of a stable cell line or the reintroduction of the ICP8 gene into the herpes simplex virus genome.     

Effects of plasmids on chromosome metabolism in bacteria

In addition to many other functions of the cell, many bacterial plasmids are involved in repair, mutagenesis, replication, and recombination of the host chromosome. Numerous studies performed with wild-type strains and various mutants suggest that plasmids participate in these processes through three basic routes: (i) contribution to cell's regulatory systems; (ii) introduction of new pathways operating either independently of the existing ones or affecting the efficiency of the latter; these new pathways may or may not be subject to cellular regulation; (iii) replacement of defective proteins by functionally similar plasmid products or compensation for missing proteins by either activating existing pathways or introducing plasmid-born bypass pathways. The differences among individual plasmids in their effects on DNA metabolism are governed by intimate mechanisms of the metabolic process, the genetic background of the host, and the genetic constitution of the plasmid. The corresponding plasmid genetic determinants and the products thereof remain, for the most part, unidentified. However, the available evidence indicates that plasmids can confer on the cell additional resources which extend its DNA metabolism potential, thereby promoting evolutionary transformations.     

Transposase-mediated construction of an integrated adeno-associated virus type 5 helper plasmid

Adeno-associated viruses (AAVs) are replication-defective parvoviruses that require helper virusfunctionsfor efficient productive replication. The AAVs are currently premier candidates as vectors for human gene therapy applications. In particular; much recent interest has been expressed concerning recombinant AAV serotype 5 (rAAV-5) vectors, as they appear to utilize cellular receptors distinctfrom those of the prototypical AAV serotype (AAV-2) and have been reported to have transduction properties in vivo that differ significantly from those of the prototype. One of the most popular current methodsfor the production of rAAVs involves co-transfection of human 293 cells with three plasmids: (i) an adenovirus (Ad)-derived helper plasmid containing Ad genes required for AAV replication, (ii) an AAV-derived plasmid encoding complementing AAV genes (ie., the viral rep and cap genes), and (iii) a target plasmid containing a transgene of interestflanked by AAV inverted terminal repeats (ITRs) that confer packaging and replication capabilities upon the ITR-flanked heterologous DNA. Here we describe novel plasmid reagents designed for convenient and efficient production of rAAV-S. An integrated helper plasmid containing all Ad genes requiredfor the efficient production of recombinant AAV as well as the complementing AAV genes on the same plasmid backbone, was constructed via transposase-mediated insertion into an Ad helper plasmid of a transposable element containing the AAV-5 rep and cap genes linked to a selectable marker This simple strategy can be used in the rapid and efficient construction of integrated helper plasmids derived from any reported AAV serotype for which a molecular clone exists.     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation

Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells.     

Plasmids of corynebacteria

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.     

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.