Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.     

Functional importance and local environments of the cysteines in the tetracycline resistance protein encoded by plasmid pBR322

The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined. Cysteines are important but not essential for tetracycline transport activity. None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.     

Construction of frameshift mutation hot spots within the tetracycline resistance gene of pBR322

The chemical carcinogen, N-2-acetylaminofluorene (AAF) when bound covalently to DNA induces a majority (greater than 90%) of frameshift mutations. The mutations occur with high frequencies at defined sequences (i.e. mutation hot spots). Two classes of mutation hot spots were found: at repetitive sequences and at specific non-repetitive sequences. Mutations at the repetitive sequences depend upon a functional umuC gene whereas mutations at specific non-repetitive sequences are umuC-independent. The first discovered sequence of this class is the NarI restriction enzyme recognition sequence (5'GGCGCC3'). In an attempt to define a family of such sequences we constructed a related sequence 5'GCGCGC3' within the tetracycline resistance gene of pBR322. This sequence was also found to be an--AAF induced--2 frameshift mutation hot spot in both wild type and umuC strains.     

High-copy-number derivatives of the plasmid cloning vector pBR322

A stable copy-number mutant of a pBR322-derived plasmid was isolated. The mutation was found to be a single G → T transversion located near the 3' end of a DNA segment coding for the regulatory RNA I. The resulting copy number for this plasmid is approx. 1000 per cell or 65 % of total cellular DNA. Several cloning vectors have been constructed from this copy-number mutant and their practical application is discussed.     

Stable maintenance in chemostat-grownEscherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene

Plasmid stability was studied in antibiotic-free chemo-stat cultures . Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.Issued as NRCC publication No. 23992.     

A new revision of the sequence of plasmid pBR322

A revised sequence in the region immediately upstream from the rop gene of pBR322 is reported. Two base pairs in the accepted sequence do not exist in the plasmid DNA. Specifically, a TA base pair is missing at sequence coordinate 1893 [Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77–90] and an AT base pair is missing at position 1915, giving a total size for pBR322 of 4361 bp. These changes are in a potential translation initiation sequence and probably reflect errors in the original sequence rather than recent evolution of the plasmid.     

Genetic analysis of the tetA(C) gene on plasmid pBR322.

The TetA(C) protein, encoded by the tetA(C) gene of plasmid pBR322, is a member of a family of membrane-bound proteins that mediate energy-dependent efflux of tetracycline from the bacterial cell. The tetA(C) gene was mutagenized with hydroxylamine, and missense mutations causing the loss of tetracycline resistance were identified at 30 distinct codons. Mutations that encoded substitutions within putative membrane-spanning alpha-helical regions were scattered throughout the gene. In contrast, mutations outside the alpha-helical regions were clustered in two cytoplasmic loops, between helices 2 and 3 and helices 10 and 11, suggesting that these regions play a critical role in the recognition of tetracycline and/or energy transduction. All of the missense mutations encoded a protein that retained the ability to rescue an Escherichia coli strain defective in potassium uptake, suggesting that the loss of tetracycline resistance was not due to an unstable TetA(C) protein or to the failure of the protein to be inserted in the membrane. We postulate that the mutations encode residues that are critical for the active efflux of tetracycline, except for mutations that result in the introduction of charged residues within hydrophobic regions of the TetA(C) protein.     

In vitro sodium bisulfite mutagenesis of restriction endonuclease recognition sites

Sodium bisulfite treatment of single-stranded DNA deaminates exposed cytosine residues to form uracil, resulting in cytosine-to-thymidine transition mutations following DNA replication. We have used this reaction in vitro to destroy the recognition sequences for the restriction endonucleases HindIII and XmaI in the aminoglycoside 3'-phosphotransferase I coding region of plasmid pUC4K. This procedure should be applicable to the mutation of any recognition sequence of restriction endonucleases which generate cytosine-containing single-stranded ends. The possibility of mutagenesis of restriction sites to generate stop codons in coding regions is discussed.     

The function of the tet gene in the plasmid pBR322 is not inhibited by expression of an anti-tet gene

The possibility of gene suppression by the expression of anti-sense sequences has been tested for tet gene of pBR322 plasmid. Anti-tet gene has been inserted into lac-promoter regulated site of M13mp 10 single-stranded high copy phage vector. To achieve that, HihdIII-BamHI fragment of pBR322 carrying part of the tet gene was inserted into poly-linker of mp 10. The influence of the anti-tet gene expression on growth parameters of cells with or without tetracycline in the growth media was monitored for JM103 cells. The results indicate that in this system the detectable suppression of the tet gene by anti-tet expression was not manifested.     

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.