Tetracycline Resistance Gene Of pBR322 Research Articles

Analysis of the Proteins in the Leaves of Transgenic Tobacco Plants Expressing Double-Stranded RNA Upon Viral Infection

Generation of transgenic tobacco plants, producing double-stranded RNA with no homology to tobacco genome sequences is reported. The RNA synthesis is mediated by a construct containing an inverted repeat of the pBR322 tetracycline-resistance gene fragment under control of the 35S CaMV promoter. Analysis of the resistance of transgenic plants to the tobacco mosaic virus revealed the changes in the protein spectra of the infected plants. The 25- and 30-kDa proteins found were not detected in the extracts of normal plants. Amino acid sequencing of the 30-kDa peptide with subsequent computer database search revealed the homology of this protein to the hydrolases belonging to the group of plant β-glucanases. The role of the novel polypeptides in an increase of the resistance of transgenic plants to TMV, and also the possibility of the regulation of their expression by nonhomologous dsRNA are discussed.

Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

Adjacent Upstream Superhelical Writhe Influences an Escherichia coliPromoter as Measured by in VivoStrength and in VitroOpen Complex Formation

This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the −35 consensus sequence of Escherichia colipromoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately upstream of the β-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. colicells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve showed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivofor the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity.

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

Mutation spectra of the two guanine adducts of the carcinogen 4-nitroquinoline 1-oxide in Escherichia coli. Influence of neighbouring base sequence on mutagenesis.

When the chemical carcinogen 4-nitroquinoline 1-oxide binds to DNA in vitro, two major adducts are formed, both at guanine residues, but at different positions, i.e. the C8 or the N2 position. Well-defined adducts (either C8 or N2 guanine adducts) can be formed in vitro by reacting DNA with 4-actoxyaminoquinoline 1-oxide (Ac-4HAQO) under different reaction conditions. Forward mutations induced by each of both main 4NQO adducts in the tetracycline resistance gene of pBR322 were determined. In total, 30 independent 4NQO-induced mutations were characterized, showing mainly base-pair substitution mutations and some frameshift mutations. We have observed that the 5' neighbouring base influences the specificity of dGuo-N2-AQO induced base-pair substitutions mutagenesis; a similar effect does not occur with dGuo-C8-AQO. This study reveals the importance of the N2 guanine adduct in the mutagenesis induced by 4NQO in vivo.

Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase.

The nrdB gene, which encodes the B2 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1), was cloned into multicopy plasmid pSPS2. This vector, which contains the pL promoter of bacteriophage lambda and the tetracycline resistance gene of pBR322, was transformed into a lysogenic host with a thermolabile repressor. In the newly constructed strain, subunit B2 constituted approximately 25% of the soluble protein after heat induction, an overproduction of several hundredfold relative to the wild-type strain. Purification to homogeneity of the overproduced protein was accomplished by using DEAE and quaternary aminoethyl ion-exchange resins.

Rec-dependent and Rec-independent recombination of plasmid-borne duplications in Escherichia coli K12.

Plasmidic recombination in E. coli K12 has been previously demonstrated to be dependent on the host rec genotype. The construction of plasmids that carry a duplication within an antibiotic-resistance gene is described. Recombination between the direct repeats recreates an active antibiotic-resistance gene, allowing quantitative analysis of recombination frequencies in a closely related set of E. coli K12 strains carrying various rec mutations. Using this system, intraplasmidic recombination of a duplication within the pBR322 tetracycline-resistance gene is shown to be rec-dependent while recombination of a similar duplication within the kanamycin-resistance gene of Tn903 is shown to be independent of recA, recB, recC, recE, recF and sbcB.

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.

Cloning of phosphoenolpyruvate carboxylase gene from a cyanobacterium, Anacystis nidulans, in Escherichia coli.

The phosphoenolpyruvate carboxylase gene (ppc) from Anacystis nidulans, a cyanobacterium (blue-green alga), was cloned in Escherichia coli. Chromosomal DNA of A. nidulans was partially digested with Sau3AI, and the obtained DNA fragments were ligated in the BamHI site of pBR322. The hybrid plasmids were first transformed into E. coli K802 (hsdR-, hsdM+) to obtain the gene bank of A. nidulans. The bank consisted of about 12,000 clones. These hybrid plasmids were then transformed into E. coli PCR1 (ppc2-, recA1-, hsdR+, hsdM+), and the transformants were selected by complementation of the ppc mutation (phenotype of glutamate requirement). In the cell-free extracts of E. coli strains having the cloned ppc gene, PEPCase activities were detected, but their properties were different from those of the E. coli enzyme. Analysis by subcloning showed that the ppc gene was included in a DNA fragment 3,500 base pairs long and the maxicell method revealed that the molecular weight of the gene product was about 108,000. It is suggested that the ppc gene is expressed in E. coli mainly by read-through transcription, being initiated by the promoter of tetracycline-resistance gene of pBR322, but the significant expression in reversed orientation of the cloned ppc gene indicates that the gene includes a promoter capable of functioning in E. coli cells.

A 140 base-pair DNA segment from the kanamycin resistance region of plasmid R1 acts as an origin of replication and promotes site-specific recombination

A 140 base-pair DNA segment situated just upstream of the kanamycin resistance gene of transposon Tn2350, a transposon carried by the plasmid R1, was found to act as an origin of replication and allow autonomous replication of a plasmid composed only of the segment and of the tetracycline resistance gene of pBR322. This segment also promotes site-specific recombination: when cloned in pBR322 it promotes multimer formation in a recA − strain. If two copies are cloned on the same plasmid they promote either deletion or inversion of the intervening region, depending on their orientation relative to each other. DNA gyrase seems to be involved in this process since the inversion rate, in a plasmid carrying sequences in opposite orientations, varies in different nalidixic acid-resistant strains ( gyrA mutants) independently isolated.

Mutagenic SOS repair of site-specific psoralen damage in plasmid pBR322

The mutagenic repair of psoralen damage was examined by transforming Escherichia coli with psoralen-treated pBR322. Plasmid DNA randomly reacted with psoralen was repaired only when the E. coli was uvrA + and recA +, and only when the cells were pre-irradiated with far-ultraviolet light. The rec A dependence and requirement for pre-irradiation are characteristics of SOS repair. Psoralens were placed specifically near the BamHI site, in the tetracycline-resistance gene of pBR322, using a sulfhydryl-containing psoralen derivative. Repair of this damage also required pre-irradiation of the host cells. This repair was accompanied by a 4% frequency of mutagenesis to a tetraeycline-sensitive phenotype. Sequence analysis of these mutant plasmids revealed that 75% had mutations within the targeted region, while 25% had no sequence changes within 100 bases of the BamHI site. In up to five independent isolates only one kind of mutation was observed at each site, suggesting that mutagenic SOS repair is influenced by DNA structure at the site of the psoralen. Most mutations were transitions, primarily G-C to A-T changes. Some transitions occurred at sites where psoralen crosslinks could not have formed, and these may have arisen from the repair of psoralen monoadducts.

Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene

The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a model for the ultimate metabolite of the rat liver carcinogen 2-acetylaminofluorene, AAF). In vivo studies have shown the mutagenicity of AAF and its derivatives in both bacterial and eukaryotic systems. N-AcO-AAF reacts in vitro with DNA leading mainly to the formation of a guanine adduct, N-2-(deoxyguanosin-8-yl)-acetylaminofluorene (80%) and to at least three minor adducts. Studies by our group showed that binding of N-AcO-AAF to DNA resulted in a local distortion of the DNA helix around the C-8 adduct (the insertion-denaturation model). We describe here the analysis of forward mutations induced in the tetracycline-resistance gene of pBR322 by directing the chemical reaction of the carcinogen to a small restriction fragment (BamHI-SalI) inside the antibiotic-resistance gene. Mutants are selected for ampicillin (Ap) resistance and tetracycline (Tc) sensitivity. The plasmid DNA of such mutants was analyzed for sequence changes in the fragment where the AAF binding had been directed. We show here that the mutations are mainly frameshifts involving GC base pairs and that certain base pairs (hotspots) are affected at high frequencies.

Local mutagenesis within deletion loops of DNA heteroduplexes.

An efficient method has been developed to generate base substitution mutations within deletion loops of DNA heteroduplexes. This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which two restriction sites had been removed from the vector. The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation. To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence. It was found that the proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20% up to 70%. Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.

BvuI: a site-specific endonuclease from Bacillus vulgatis

A site-specific endodeoxyribonuclease was partially purified from an extract of osmotically shocked spheroplasts of a Bacillus vulgatis strain; the enzyme has been designated BvuI and its activity was characterized. The locations of BvuI-generated cleavages on bacteriophage lambda and M13 derivatives mp7, mp8 and mp9, SV40 and pBR322 DNA molecules were determined. BvuI was shown to recognize the DNA sequence 5'-G-Pu-G-C-Py↓-C-3' 3'↑-C-Py-C-G-Pu-G-5' and cleaves it at the positions indicated by arrows. Two identical BvuI recognition sites separated by fourteen nucleotide pairs were shown to occur within the tetracycline resistance gene of pBR322; Bvul should be useful for molecular cloning in that plasmid vector.

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 pHVT1, 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 pHCVS contains the tetracycline-resistance gene of pBR322, and pHVTl contains the analogous region from the transposon Tn 10.