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.     

Use of transposon-promoted deletions in DNA sequence analysis

The usefulness of the dideoxy method for DNA sequencing can be greatly extended by the use of transposon-generated deletions. These deletions have the unique property of extending from a fixed nucleotide at the transposon terminus to various sites outside it. A plasmid (pAA3.7) carrying Tn9, which allows positive selection of such deletions as galactose-resistant colonies of Escherichia coli, is described. A cloned gene can thus be subdivided into a series of overlapping sequences, all of which are fused to a common sequence at the transposon terminus. Restriction fragments carrying the segments fused by deletions are cloned in M13, and sequenced using a primer complementary to the Tn9 terminus. Complete nucleotide sequence of the gene is assembled from sequence overlaps found in deletions with end-points approximately 350 base-pairs apart. The method is rapid, requires minimal in vitro manipulation, and is free from redundant information normally produced in shotgun sequencing.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.     

Isolation of a new plasmid pIRL19: its use in construction of small vectors and detection of specific sequence in a foreign DNA

A new plasmid, pIRL19, was constructed by ligating a 1875-bp HaeII fragment carrying the ampicillin-resistance (Apr) gene to a 370-bp HaeII fragment containing the replication origin of the plasmid pBR322. The plasmid essentially contains only the basic replicator and the Apr gene. This basic replicator provides a valuable initial building block for in vitro construction of other very small vectors with antibiotic-resistance determinants. To illustrate this potential, we have transferred the chloramphenicol-resistance (Cmr) gene and a part of the Apr gene from the plasmid pBR329 into pIRL19 such that the new plasmid pIRL20 acquired the Cmr gene and maintained the integrity of its Apr structural gene.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.     

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.     

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

Lethality of palindromic DNA and its use in selection of recombinant plasmids

A plasmid derived from ColE1 is constructed so that the removal of one restriction endonuclease HindIII fragment allows the ends of the remaining single fragment (the replicator) to be joined, generating a palindromic sequence 2394 bp in length. The circular species thus produced gives rise to transformants of E. coli at very low frequency. Since the palindromic sequence is effectively lethal to a plasmid containing it, the replicator will give rise to more transformants when the restriction fragment originally removed from it is replaced by another. This principle can be exploited to allow the efficient molecular cloning of unselected restriction fragments.     

A modified pBR322 vector with improved properties for the cloning, recovery, and sequencing of blunt-ended DNA fragments

The construction of a plasmid vector which facilitates the cloning and recovery of blunt-ended DNA fragments is described. This plasmid, called pHP34, differs from pBR322 by a 10-bp insertion which introduces a unique SmaI site immediately flanked by two EcoRI sites. Blunt-ended DNA fragments cloned in the SmaI site can be recovered by digestion with EcoRI. Small cloned fragments can be chemically sequenced using a strategy which does not require their purification. The use of a plasmid related to pHP34 for in vitro mutagenesis by the insertion of a DNA linker fragment conferring an antibiotic resistance is also discussed.     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.     

Site-specific recA-independent recombination (fusion) of pMB8-related replicons in Escherichia coli in the region of the replication origins

Escherichia coli recA+ and recA- cells were co-transformed with a mixture of pMB9 (Tcr) and pST8-26 (Apr, pBR325 derivative) plasmid DNAs followed by selection on plates containing both tetracycline and ampicillin. A set of stable Tcr Apr derivatives was isolated from these transformants. Many of the stable Tcr Apr segregants contained fused pMB9::pST8-26 plasmids with lengths that were about 0.8 kb longer than the sum of the lengths of the parental plasmids; one plasmid (pTF8) was about 1.5 kb shorter. The fusion was not stimulated by UV irradiation of co-transformants and occurred both in recA+ and recA- genetic backgrounds. Restriction analysis of the fused plasmids showed the two replicons were in the same relative orientation, and also indicated unique points of fusion in most cases (in 9 out of 10) which are localised within the 1.6-kb regions around the replication origins (RO). Because the fusion of plasmids of the type used in this study was not described before we have tentatively named it RO-fusion (Replication-Origin-fusion).     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.