Plasmid cloning vectors that can be nicked at a unique site

Summary We describe ColE1-type plasmids, with relaxed DNA replication, based on pMB9, and carrying the Cm^R determinant of R1, in addition to the Tc^R determinant of pMB9. One of the plasmids, pPH207, has unique sites for EcoRI, HindIII, BamI, SalI and Hpal. Insertion of foreign DNA into all but the last of these inactivates cither the Cm^R or the Tc^R determinant.The original Cm^R Tc^R plasmid (pCM2) contains a copy of IS1 which produces deletions to left and to right. Most of these inactivate either the Cm^R or the Tc^R determinant. An internal 280 bp deletion of IS1 DNA in pPH207 greatly reduces the frequency at which deletions are observed.The main feature of these plasmids is a site that is cleaved by some preparations of EcoRI in only one strand of the DNA duplex (the EcoRIⁿ site). This site facilitates strand separation of sequences inserted at the HindIII, BamI and SalI sites of the Tc^R gene, and also of any inserted at the true EcoRI site by a method that destroys that site. Since the oricntation of the EcoRIⁿ site is known, the orientation of sequences inserted at the neighbouring sites can be easily determined.Plasmid pPH207 is not mobilised by a Hfr, but its mobilisation is promoted by ColE1. It is therefore Mob ^- bom ⁺. Experiments with minicells show that it directs the copious synthesis of chloramphenicol transacetylase.     

Improved cloning vectors for Bifidobacterium spp.

The recombinant plasmids pDLI41, pDGA7 and pDCO7 were constructed by cloning in pDG7, a vector based on Bifidobacterium longum replicon pMB1, the following heterologous genes: Pseudomonas fluorescens lipase, Bacillus licheniformis α-amylase and Streptomyces sp. cholesterol oxidase. The hybrid plasmids efficiently transformed Bifidobacterium belonging to five different species. A novel Escherichia coli-Bifidobacterium set of shuttle vectors based on the replicon pMB1 (pLF5, pCLJ15, pSPEC1) featuring chloramphenicol, erythromycin and spectinomycin resistance genetic determinants as selection marker for bifidobacteria, was developed. The plasmid pTRE3, a derivative of pLF5, was the smallest (2·8 kb) Bifidobacterium vector, possessed a convenient multicloning site and presented high structural and segregational stability.     

Transformation and mobilization of cloning vectors in Acinetobacter spp.

R300B-, RSF1010-, and RK2-derived plasmids were introduced into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413 by transformation and conjugal mobilization. The transformation frequencies of BD413 were 4.2 X 10(6) to 6.3 X 10(6) transformants per micrograms of DNA per 10(9) recipient cells. Conjugal mobilization frequencies were 1.1 X 10(-1) to 8.5 X 10(-1) per recipient. An improved method for the transformation of A. calcoaceticus BD413 is reported.     

Plasmid vectors with multiple cloning sites and cat-reporter gene for promoter cloning and analysis in animal cells

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581.

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.     

ColD-derived cloning vectors that autoamplify in the stationary phase of bacterial growth

The construction of cloning vectors based on the replicon of plasmid ColD-CA23 is reported. These vectors, like ColD itself, autoamplify when cultures of host bacteria enter the stationary phase of growth, thereby resulting in a substantial increase in the expression of cloned genes as a consequence of the increase in gene dosage. The principal advantage of these vectors is that, unlike the situation pertaining to other expression vectors, the increase in expression of genes cloned in ColD vectors does not require any experimental intervention (i.e., occurs naturally), and takes place at high cell densities. The vectors show high stability in Escherichia coli strains and are compatible with ColE1-type cloning vectors.     

Plasmid deoxyribonucleic acid replication in Streptomyces griseus

A series of electron micrographs showing the presence of different molecular forms representing various replication stages of plasmid deoxyribonucleic acid from Streptomyces griseus was obtained. Based upon an analysis of these electron micrographs, a tentative model for plasmid deoxyribonucleic acid replication in S. griseus is proposed.     

Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.     

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24. 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.     

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

Plasmid cloning vectors resistant to ampicillin and tetracycline which can replicate in both E. coli and Haemophilus cells

We have constructed two plasmid vectors, pHCV5 and pHVTl, which will replicate both in Haemophilus and in Escherichia coli. Both contain the ampicillin-resistance gene and the replication origin from a Haemophilus plasmid, pRSF0885. Both also contain the pBR322 origin and therefore can be amplified in E. coli by chloramphenicol treatment. The plasmid pHCV5 contains the tetracycline-resistance gene of pBR322, and pHVT1 contains the analogous region from the transposon Tn10.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

Plasmid pSE21 of Streptomyces erythraeus strains

Crossing of S. erythraeus 4, a laboratory strain (NRRL 2338) containing a family of plasmids with S. erythraeus 1, a plasmid-free strain resulted in formation of strain 6. A multi-copy plasmid pSE21 11.5 kb in length was isolated from S. erythraeus 6. A detailed restriction map of plasmid pSE21 was constructed. Its cloning to E. coli YM83 on vector pUC19 showed that plasmid pSE21 was not stable in E. coli. It was found that the status of plasmid pSE21 changed in relation to the physiological state of S. erythraeus 6. Southern hybridization of the plasmid pSE21 DNA with the total DNA of the cultures of S. erythraeus 1 maintained for various periods at 4 degrees C demonstrated that plasmid pSE21 was present in S. erythraeus 1 in an autonomous state in 0.1 to 0.2 copies per genome. The number of the plasmid pSE21 copies could be decreased. The chromosomal DNA of S. erythraeus 1 contained the DNA sequences highly homologous to those of plasmid pSE21. It was assumed that during the crossing of S. erythraeus 4 with S. erythraeus 1 the genetic element from the donor strain was transferred to the recipient strain which in some way changed the plasmid pSE21 status and imparted the multicopy pattern to it. Further investigation of the plasmid pSE21 properties and construction of a vector for S. erythraeus on the plasmid basis are under way.     

Replication bypass and mutagenic effect of alpha-deoxyadenosine site-specifically incorporated into single-stranded vectors.

alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that alpha constituted a moderate block to DNA replication. The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of alpha introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.     

Plasmid vectors used in various strains of Erwinia species

Summary Two multiple-copy plasmid vectors, pBEH3-5 and pBEH8-2 were constructed from a Erwinia plasmid, pEC3 or pEC8, and the Escherichia coli plasmid pBR328. Part of sequence homology between pEC3 and pEC8 was found by Southern hybridization. The two vectors were efficiently transferred into members of the species E. amylovora, E. carotovora, E. carotovora subsp. carotovora, and E. herbicola using a binary plasmid system with RP4. The transformation system examined in strains of these Erwinia species yields about 2 to 4x10² transformants per g of pBEH3-5 DNA. These host-vector systems make potentially useful tools for the study of genes involved in the plant pathogenesis of Erwinia species.