Phage lambda and plasmid expression vectors with multiple cloning sites and lacZ alpha-complementation

Two new lambda vectors were constructed which permit cloning of genes that are potentially lethal if cloned in analogous plasmid vectors. lambda DL10 and lambda DL11 contain the alpha-complementing fragment of lacZ and multiple cloning sites found in the polylinker region of M13mp10 and M13mp11, respectively. DNA cloned into the unique cloning sites of these vectors can be detected by inactivation of alpha-complementation. These lambda vectors provide a lac promoter for expression of foreign genes as well as the ability to make fusion proteins. Two plasmid expression vectors, pPR110 and pPR111, were constructed from lambda DL10 and lambda DL11 respectively, and pCQV2. These plasmids, which express lacZ alpha-complementing activity from the lambda PR promoter, contain multiple cloning sites immediately downstream of the PR promoter. They allow cloning of genes under the control of the PR promoter and the plasmid-encoded thermosensitive (cI857) repressor, and allow easy detection of inserted fragments by inactivation of alpha-complementation.     

Construction of linear plasmid vectors for cloning in Escherichia coli cells]

Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends. The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites. As a result a family of linear plasmid vectors was constructed. Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.     

Construction of new shuttle plasmid vectors for Escherichia coli-Bacteroides transgeneric cloning

Abstract An Escherichia coli-Bacteroides shuttle vehicle (pKBF367-1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis ; and [2] the 4.2 kb Eco RI-B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin-resistant transconjugants upon helper plasmid-mediated transfer to a recipient strain of Bacteroides distasonis . To improve the potential of pKBF367-1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn 501 (Hg^r) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.     

The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation

The versatility of insertional inactivation of β-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile Polylinker specifies 17 restriction sites in the β-galactosidase α-complementing gene fragment. One of the new polylinkers was inserted into M 13 DNA to produce a vector (M13mIC7) with nine cloning sites.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites

A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles. Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes. The plasmid has the additional advantages of very high copy number and altered incompatibility. The latter permits it to be stably maintained in the same host as pBR322.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis

Summary Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tc^r gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec⁺ recipients. Loss of sequences from hybrid plasmids was not prevented in a r ^- m ^- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.     

Versatile low-copy-number plasmid vectors for cloning in Escherichia coli

Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques. pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin. pLG338 (7.3 kb) has unique restriction endonuclease sites for BamHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene. pLG339 (6.2 kb) lacks the KpnI site, but has unique SphI and PvuII sites. These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids.     

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

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.     

New plasmid vectors for cloning and expression of genes

Plasmids of the pFPCP series have been constructed, containing the whole gene for the filamentous phage main coat protein or its regulatory elements along with unique restrictase sites.     

Construction of a plasmid containing the complete coding region of human elongation factor 2.

A plasmid pUChEF-2 containing the coding sequence as well as the complete 3'-untranslated region (3'UTR) of human EF-2 mRNA was constructed. The plasmid construct was assembled from a cDNA insert of pHGR81 (Rapp et al., (1988) Biol. Chem. Hoppe-Seyler 369, 247-250) comprising the C-terminal portion of the coding region and the 3'UTR, as well as a polymer chain reaction PCR fragment (Rapp et al., (1989) Biol. Chem. Hoppe-Seyler 370, 1071-1075) covering the missing part of the coding region from the amino-terminus.     

Construction of a family of universal expression plasmid vectors

A family of universal expression vectors based on the pUC8 and pUC9 plasmids has been constructed. These vectors cover all three possible reading frames in both directions, allowing any synthetic DNA, genomic DNA or cDNA to be expressed under control of the lac promoter. The four new vectors retain the useful features of the pUC plasmids, including the blue to white color change on X-gal plates indicating the presence of an insert. This family of expression vectors is expected to be quite useful in allowing direct immunological screening of cDNA or genomic DNA banks.     

A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors

We have constructed a plasmid cloning vector, pGB2, which is derived from the Escherichia coli plasmid pSC101. The plasmid, which specifies resistance to spectinomycin and streptomycin, contains unique restriction sites for the enzymes HindIII, PstI, SalI, BamHI, SmaI and EcoRI. pGB2 shows no sequence homology, as detected by DNA-DNA hybridization, to several widely used vectors such as pBR322, pUC8 and phage lambda L47.1. Amongst other applications, DNA fragments can be cloned into the plasmid and then radioactive plasmid DNA can be used as a probe to screen recombinant DNA libraries.     

Abundant conserved microRNA target sites in the 5′-untranslated region and coding sequence

Recent studies have shown that miRNAs can target the promoter and CDS region. Thus, we predicted miRNA target sites in the 5′-UTR, CDS and 3′-UTR of Homo sapiens, Mus musculus and Drosophila melanogaster using miRanda and TargetScan. Target-site densities normalized with the average region length were higher in the 5′-UTR than 3′-UTR in all three organisms but were lower in the negative data set. Interestingly, the putative target sites were more conserved than non-target regions in both the 5′-UTR and 3′-UTR, implying that target sites in the 5′-UTR are subject to high selective pressure and might be functional. In Drosophila, 48 of 78 (61.5%) miRNAs showed high similarities with predicted siRNAs. Based on the results of previous experimental studies and a large-scale statistical analysis, we conclude that miRNA-mediated regulation is not limited to the 3′-UTR. However, the functionality of target sites in the 5′-UTR and CDS requires thorough investigation.