Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Construction and characterization of new cloning vehicles. VII. Construction of plasmid pBR327par, a completely sequenced, stable derivative of pBR327 containing the par locus of pSC101

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.     

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.     

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.     

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.     

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.     

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.     

Transposition of a DNA fragment flanked by two inverted Tn1 sequences

The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Km^r gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids. We designated this new transposon Tn1755. In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F′ColVBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition. Tn1755 can integrate into various sites of the recipient plasmids. We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tc^r gene. Presumably, to form a new transposable structure, other features must also be of importance.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.     

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli

The thyA gene of Escherichia coli, which directs the synthesis of the enzyme thymidylate synthetase, has been subcloned from a recombinant λ phage (Hickson et al., 1982) into the multicopy plasmid pBR325 to give the plasmid pPE245. To identify the thyA gene product, the transposon Tn1000 was inserted into pPE245 and derivative plasmids isolated that were no longer able to complement thyA mutations. When proteins synthesised by these plasmids and by pPE245 were labelled and analysed on SDS-polyacrylamide gels a protein of 33000 M_r, presumably the thyA⁺ gene product was absent whenever the thyA gene was inactivated. On assaying cell extracts prepared from cells harbouring pPE245 for thymidylate synthetase, the level of this enzyme was found to be elevated by a factor of at least 25.     

Heteroduplex analysis of tRNA3bVal genes from the 90BC and 84D sites of Drosophila melanogaster

Two recombinant DNA plasmids coding for Drosophila tRNA_3b^Val hybridize to two different loci on the Drosophila genome. Plasmid pDt41R hybridizes at the 90BC site, and pDt78R hybridizes at the 84D site. Heteroduplexes of the two plasmids were examined by electron microscopy. The study shows that the Drosophila segments have significant homology only over the length of the tRNA_3b^Val genes.     

A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression

The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described. In this vector, the replicon and Ap^R gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment. However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the Tc^R phenotypes. Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site. The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.