Recovery of activity of the gene coding for tetracycline resistance in the plasmin pBRS188

The spontaneous recovery of activity of tet gene deleted of the promoter region was studied. Plasmid pBRS188 was used as a model for studying this problem. The plasmid has the fragment of tet gene of pBR322, from which it originates, between the sites of restriction endonucleases EcoRI and HindIII cleavage resulting in inactivation of tet promoter. E. coli cells harbouring the plasmid were shown to revert the TcR phenotype with the frequency 10(-9). The gene activation coincided with intraplasmid recombination revealed by restriction analysis. In some cases the recovery of tet gene activity coincided with the formation of multimeric plasmids.     

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.     

Genetic analysis of Saccharomyces cerevisiae transformed by plasmid containing a supressor transfer ribonucleic acid gene.

The behavior in Saccharomyces cerevisiae of plasmid pYTE1, which contains yeast tyrosine-inserting ochre suppressor SUP4.o, a 4-kilobase EcoRI fragment of yeast 2muDNA, and the bacterial plasmid pBR322, has been studied. Selection of yeast transformants was by suppression of multiple ochre mutations. About 10(3) to 10(4) transformants per microgram of pYTE1 dfeoxyribonucleic acid were obtained. The majority of transformants contained both an integrated copy of the SUP4.o gene plus pBR322 deoxyribonucleic acid sequences and autonomously replicating forms of the plasmid. The integrated copy was extremely stable mitotically and meiotically, but the associated nonintegrated copies were lost at meiosis. The chromosomally integrated pBR322 sequences were linked to the SUP4.o gene. The integration site was at the SUP4+ locus. In transformants with only nonintegrated copies of pYTE1, the expression of suppression was reduced, and the plasmid was unstable in mitosis. Plasmid deoxyribonucleic acid preparations from both types of transformant could be used to retransform yeast cells. Plasmid pYTE1 has restriction enzyme sites useful for the high frequency and stable transformation of other genes into yeasts. The potential uses of this plasmid for transformation of other organisms is discussed.     

Cloning of the aspartase gene (aspA) of Escherichia coli

The aspartase gene (aspA) of Escherichia coli has been isolated in two plasmids, pGS73 and pGS94, which contain segments of bacterial DNA (12.5 and 2.8 kb, respectively) inserted into the tet gene of the vector pBR322. The plasmids were constructed by sequential sub-cloning from a larger ColE1-frd+ hybrid plasmid. The location of the aspA gene confirmed predictions based on a correlation between the genetic and restriction maps of the corresponding region. The aspartase activities of plasmid-containing aspA mutants were amplified four- to sixfold relative to aspA+ parental strains. The aspA gene product was tentatively identified as a polypeptide of Mr 55 000, which is somewhat larger than previous estimates (Mr 45000 to 48000) for aspartase.     

Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and Tn10

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.     

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.     

DNA rearrangements generating artificial promoters

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.     

Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.     

Expression of the gene for hybrid beta-lactamase pBR322 with C-terminal fragment containing tuftsin (Thr-Lys-Pro-Arg)

A hybrid beta-lactamase gene with a synthetic tuftsin-coding DNA fragment inserted at the Pst I-site of pBR322 plasmid has been obtained and its expression has been studied. Radioactive amino acids have been used to show that in E. coli chi 925 minicells up to 30% of newly synthesized chimeric protein is secreted into periplasm providing the tuftsin transport. After hybrid protein cleavage with CNBr, tuftsin has been isolated using ion-exchange and thin-layer chromatography.     

The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.     

Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12

The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold. The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184. In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid. The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells.