Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Stabilization by the Mini-F Fragment of a pBR322 Derivative Bearing the Tryptophan Operon in Escherichia coli

In an Escherichia coli K-12 strain (trpA trpE tnd) cultured in LB broth without selective pressure, a pBR322 derivative bearing the E. coli tryptophan Operon (pBR322-trp) was rapidly lost: after 27 cell-number doublings, only 7% cells retained both tryptophan prototrophy (Trp⁺) and ampicillin resistance (Ap^r), and 17% were Ap^r but Trp^?. Insertion of the mini-F DNA from F factor into this plasmid effectively suppressed both the plasmid loss and the discoordinate loss of Trp⁺: the percentage of Trp^? cells per cell-number doubling was decreased more than 100-fold. Partial derepression of the trp operon due to 3-indole acrylic acid further decreased the stability of the pBR322-trp but not that of the mini-F-inserted pBR322-trp.     

Cloning and restriction map of the E. coli apt gene

The apt gene coding for Escherichia coli adenine phosphoribosyl transferase has been cloned in pBR322. The restriction map of a 1.6-kb fragment containing the apt gene is presented.     

Physical mapping of mutations in the structural gene encoding for the Escherichia coli aldohexuronate transport system

Summary Synthesis of both chromosomal and plasmid (pBR322) DNA was measured in E. coli strains differing in their relA allele (relA ⁺: CP78; relA: CP79). It was found that upon limitation of a required amino acid or after valine treatment to trigger a stringent response synthesis of pBR322 DNA was stimulated only in the relaxed strain and was inhibited in its stringent counterpart. The results suggest that replication of plasmid DNA is negatively controlled by the relA ⁺ allele.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA ^–] bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom ^– in both relA ^– and relA ⁺ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom ^–, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Transformation of pBR322-Derived Plasmids in Phytopathogenic Pseudomonas avenae and Enhanced Transformation in Its Proline-Auxotrophic Mutant

Efficient transformation of pBR322 and its derived plasmids, which have been widely used as cloning vectors in Escherichia coli, was observed in Pseudomonas avenae (K1), the pathogen of leaf blight disease in cereals. Moreover, there was a 10- to 50-fold transformation efficiency (1.3–3.0 × 10⁶/μg DNA) in the proline-auxotrophic mutant (Pr47), whose virulence to rice seedlings decreased. Similar enhancement of the frequency of transfer by mobilization of RSF1010, a broad host range plasmid, was observed in the recipient Pr47 strain in mating with donor Pseudomonas syringae. The plasmids harbored in these strains were maintained very stably after subcultures. Thus, a highly efficient transformation system with pBR322-derived plasmids used as a vector and Pseudomonas as a host bacterium was developed. Received: 13 July 1996 / Accepted: 26 August 1996     

A stable derivative of pBR322 conferring increased tetracycline resistance and increased sensitivity to fusaric acid

A hybrid trp-tet promoter was formed on pBR322 by insertion of a segment containing part of the trp promoter at the ClaI site. The product plasmid, pDR42, conferred resistance to higher concentrations of tetracycline than pBR322. Cells bearing pDR42 were sensitive to lower concentrations of fusaric acid than were those bearing pBR322. Since the difference in growth on fusaric acid between the E. coli RR1 alone and the strain with pDR42 is greater than is the case with pBR322, an improved selection of tetracycline-sensitive (Tc^s) colonies out of a background of pDR42 specified tetracycline-resistant (Tc^r) colonies was observed.     

Inhibition of mutation induction and unchanged mutational specificity inEscherichia coli K12 overproducing the RecA protein

Induced mutagenesis was studied inEscherichia coli K12 cells in relation to the level of KecA-protein (P-RecA). In experiments strains AB2497, AB2497(pBR322) and AB2497(pX02) were used. The multicopy plasmid pX02 is a recombinant of pBR322 and recA⁺ gene ofE. coli K12. Cells carrying this plasmid overproduce the P-RecA constitutively. Mutagenesis was induced by the decay of incorporated 6-³H-thymidine. Mutations of theargE3 (ochre) to Arg⁺ prototrophy were followed. Besides the frequency of mutations, mutagenic specificity was determined. In cells AB2497(pX02) which overproduce the P-RecA the yield of Arg⁺ revertants was markedly reduced compared with that in strains AB2497 or AB2497(pBR322), whereas the mutagenic specificity was not changed. In all the strains studied the predominant type of mutation produced was the base substitution in the A: T base pair.     

The effect ofrecA mutation on the expression ofEcoKI andEcoR124Ihsd genes cloned in a multicopy plasmid

Type I restriction-modification (R-M) endonucleases are composed of three subunits—HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of clonedEcoKI andEcoR124Ihsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wthsd genes in a multicopy plasmid inEscherichia coli recA ⁺ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the clonedhsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation inE. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.     

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives

Summary We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli

We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (O^c) mutations on the pBR322-derived plasmid pPY97. The rate of O^c mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the O^c phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.     

Establishment of Escherichia coli cells with an integrated high copy number plasmid

Summary The dnaA46 cells can grow at high temperature when a high copy number plasmid pKY31, a derivative of pBR322 carrying a segment of the E. coli chromosome, integrates into the bacterial chromosome. In contrast, the dnaA46 polA ^- cells with the integrated plasmid can not grow at high temperature. Therefore, integration of the plasmid can suppress the dnaA mutation and this suppression requires DNA polymerase I which has been known to be required for plasmid replication. Full reversion of polA or lysogenization of polA ⁺ is lethal for the dnaA46polA ^- bacteria that carry the plasmid only in integrated state. Partial reversion of polA allows these cells to grow at both low and high temperatures. Introduction of the plasmid pBR322 into cytoplasm of these bacteria suppresses the lethal effect caused by full reversion of polA or lysogenization of polA ⁺. This lethal effect expresses independent of the presence or absence of the dnaA mutation. In partial revertants of polA which have only integrated plasmid, the number of copies of a region near the replication origin of integrated plasmid increases. The number is reduced by the presence of extrachromosomal pBR322. It is suggested that the lethal effect of normal levels of DNA polymerase I in strains that carry only the integrated plasmid is due to excessive initiation of replication of the bacterial chromosome from the plasmid origin and high potential of initiation can be absorbed in many copies of cytoplasmic plasmid, probably, in their replication origins.Abbreviations Amp^r ampicillin resistant (resistance) - Tet^s tetracycline sensitive - Tet^r tetracycline resistant - MMS^r methyl methane sulfonate resistant (resistance) - ts temperature sensitive - Kb kilobase pairs     

Syntheses and Plant Growth Retardant Activities of Hydrazones Containing a Terpenoid Moiety Derived from Girard’s Reagent T

The genetic background of the protein-leaky mutant of Escherichia coli (PE4LA) was examined. This mutant preferentially excretes periplasmic proteins and outer membrane components. The results obtained from various crosses indicated that the mutation responsible for protein leakage was located near the purE gene. The amount of protein excreted decreased to about half whenever PE4LA was converted from Pur^? to Pur⁺ either by transduction or spontaneous reversion. When purine or pyrimidine auxotrophs were obtained from purE⁺ revertant of PE4LA, only auxotrophs whose blockages were located at a step identical or close to that governed by purE gene excreted proteins to the same extent as PE4LA. Excess adenine in the culture medium repressed exprotein accumulation by PE4LA. These results suggest that purE is involved in some way with protein excretion, and perhaps the accumulating metabolic intermediate(s) causes protein excretion due to a defect in purE enzyme.     

Isolation of physarum DNA segments that support autonomous replication in yeast

Summary Hybrid plasmids containing the bacterial resistance-transfer factor pBR322 and the yeast leu2 ⁺gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containing Physarum sequences transform leu2 ^-yeast to Leu⁺ at high frequency. The resulting Leu⁺ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu⁺ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu⁺ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.     

Stability of plasmid pBR 322 in escherichia coli nf 161 (rela+) and nf 162 (rel a) grown in a methionine limited chemostat

The stability of the plasmid pBR 322 was measured in E. coli NF 161 (rel A⁺) and NF 162 (rel A) grown in a methionine limited chemostat. A significant plasmid loss occurred only in E. coli NF 162 at a low dilution rate and if the fermenter had been inoculated with cells from the stationary phase. Obviously, this behaviour resulted from the high expression of β-lactamase due to amplification of the pBR 322 DNA in the inoculum cells of E. coli NF162.