Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication

Improved expression vectors have been constructed which are derived from runaway-replication mutants of plasmid R1 and carry the strong leftward promoter (pL) of bacteriophage lambda. The activity of this promoter is controlled by a temperature-sensitive repressor, product of the phage gene cI cloned on a compatible plasmid. Heat induction leads to amplification of the plasmid copy number and at the same time turns on the promoter. At a short distance downstream from the promoter, unique EcoRI, BamHI, XbaI and HindIII sites are present. This system was used for high level expression of the T4 DNA-ligase gene; 3 h after induction the ligase amounted to about 20% of total cellular protein.     

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.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

Functional organization of the plasmid pT181 replication origin

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.     

Localization of the replication origin of plasmid pE194.

The pE194 replication origin was localized to a 265-base-pair interval by analyzing the ability of purified pE194 restriction fragments to direct replication of heterologous plasmids. Replication was dependent upon RepF protein supplied in trans. The origin region contained a GC-rich dyad symmetry which may serve as the RepF target.     

A family of cosmid vectors with the multi-copy R6K replication origin

A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.     

Isolation of the origin of replication of the IncW-group plasmid pSa

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

HU protein binding to the replication origin of the rolling-circle plasmid pKYM enhances DNA replication

The RepK protein, which is encoded by the rolling-circle plasmid pKYM, binds to the PR I site in the pKYM DNA replication origin. We have identified HU as a protein that binds to the PR II and PR III sites in the replication-enhancing region which is downstream of PR I. DNA footprinting assays show that HU binds to these two sites only when RepK is bound to PR I, and that HU also enhances the binding of RepK to PR I. In vivo, pKYM was unable to transform an HU null strain. Two mutant RepK proteins, RepKW179Y, which contains a Trp-to-Tyr exchange at position 179, and RepKD277L, which contains an Asp-to-Leu mutation at residue 277, initiate DNA replication in vivo in the absence of HU. In vitro, these mutant RepK proteins form more stable complexes with the pKYM origin region than does the wild-type RepK protein. These results indicate that HU plays a role in the formation of a stable RepK-origin complex, which is required for the initiation of pKYM DNA replication. Received: 24 July 1996 / Accepted: 30 December 1996     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication

All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.     

Characterization of the replication origin of the myxobacterial self-replicative plasmid pMF1

Thus far, pMF1 is the only endogenous myxobacterial plasmid whose replication mechanism is unclear. In this study, we determined that the plasmid replicates via the theta-mode. The pMF1.14 gene, located in the pMF1.13-pMF1.15 operon (repABC), encodes an essential replication initiation protein that was predicted to have no typical DNA/protein binding motifs but contains rich disordered regions. The pMF1 replication-related essential cis-acting DNA region, approximate 370bp, was located within pMF1.14, and was found to contain several directly and inverted atypical repeats. The unique characteristics of the pMF1 replicon are suggested to be the reason for its strict narrow host range in Myxococcus cells.     

Broad-host-range cloning vectors derived from the W-plasmid Sa

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (M_r 5.6?7.2 × 10⁶), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,     

The nucleotide sequence of the replication origin of plasmid NTP1.

The sequence of the DNA of the origin region of NTP1 has been obtained. Analysis of the sequence indicates that: (1) there is great sequence homology in the DNA upstream from the origin in NTP1, ColE1, CLODF13, PBR345 AND PBR322; (2) only seven base pairs of NTP1 are identical with the sequence downstream from the origin in ColE1, although some homology exists for 140 bases downstream; (3) two ten base pair direct repeats are present in NTP1 which are also conserved in all four plasmids named above; (4) probably no polypeptide greater than fifteen amino acids in length is encoded by the NTP1 origin region, since no single open reading frame is conserved in all five plasmids.     

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.     

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.