Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.     

Mobilization of the pACYC184 plasmid by hybrid pAS8-121 delta plasmids in Escherichia coli cells

The plasmid pACYC184 is shown to be mobilized for conjugal transfer in Escherichia coli cells by the deleted (Tn7-TcR) derivatives of the hybrid conjugative plasmid pAS8-121 (RP4-Co1E1). Both the mobilized and mobilizing plasmids are autonomously inherited by the recipient cells when the mobilizing plasmid carries single copy of IS8 (the plasmid pAS8-121 delta 16). Cointegrates pAS8-121 delta 16D:: ::pACYC184 are found in the recipient cells with pACYC184 being inserted between two repeats of IS8 if the derivate plasmid pAS8-121 delta 16D having the duplication of IS8 is used to mobilize pACYC184 for conjugal transfer. The insertion of pACYC184 between IS8 repeats in the plasmid pAS8-121 delta 16D eliminates the plasmid ability to be inserted with high frequency into the chromosome of the phototrophic bacterium R. sphaeroides 2R. The cointegrate pAS8-121 delta 16D:: pACYC184 is stable but can be resolved during the transformation deriving the plasmid pACYC184:: IS8. The latter may be used as a probe for isolation and analysis of IS8 DNA sequences and for constructing the vectors on the basis of pACYC184.     

Cloning and expression of Thiobacillus versutus cryptic plasmid pTAV-1 DNA in Escherichia coli

pTAV-1 is an approximately 100 kb Thiobacillus versutus cryptic plasmid. pTAV-1 DNA was cloned in Escherichia coli. Nine recombinant plasmids containing pTAV-1 DNA inserted into the EcoRI restriction site of pACYC184 were constructed. The origin of DNA inserts was confirmed by Southern blot hybridization. The expression of mixotrophic T. versutus plasmid genes was demonstrated in E. coli.     

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

Abstract A Tn 5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn 5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn 5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.     

Molecular cloning and expression of the Streptococcus lactis tagatose 1,6-bisphosphate aldolase gene in Escherichia coli

Abstract A 4.4 kb Eco RI DNA fragment of the Streptococcus lactis H₁ plasmid pDI1 was cloned into the Escherichia coli plasmid pACYC 184. The recombinant plasmid expressed d -tagatose 1,6-bisphosphate aldolase activity in E. coli . Enzyme activity was at the same level as in the original S. lactis host but was not repressed by glucose.     

Plasmid pACYC184 contains an ssi signal for initiation of single-strand phage DNA replication

Using the plaque assay system for screening the single-strand (ss) initiation determinant (ssi) sequences, we have found that 119-bp region in pACYC184, a derivative of the plasmid P15A of Escherichia coli, can direct such ss DNA initiation. This region is located downstream from the P15A origin of replication and conserves consensus sequences of the ssi signals found in the other plasmids. Signals for ss DNA initiation are defined as nucleotide sequences present on ss DNA templates and required for priming DNA synthesis. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. In this region, we found a potential stem-and-loop structure. The 119-bp DNA segment of plasmid pACYC184 cloned in f1R199 filamentous phage could direct rifampicin-resistant conversion of the ss DNA to the double-stranded replicative form.     

Molecular cloning of herpes simplex virus type 2 DNA

Restriction enzyme HindIII digestion of the whole genome of herpes simplex virus type 2 strain 186 yielded 10 DNA fragments with molecular weights ranging from approximately 22 X 10(6) to 1.2 X 10(6), which were cloned into the HindIII site of bacterial plasmid pACYC 184. The cloned fragments were identified by hybridization to HSV-2 virus DNA and by double digestion with restriction endonucleases. The recombinant plasmids, even if they carried DNA sequences with molecular weights of more than 10(7), were efficiently replicated in E. coli HB101.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase.

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.     

Plasmid R1drd-19-mediated superoligomerization of the plasmid pACYC184in the recB mutant of Escherichia coli K12

It was found that monomers of the pACYC184 plasmid undergo superoligomerization in a recB mutant of Escherichia coli K12 which is deficient in ATP-dependent RecBC nuclease and carries the drug resistance plasmid R1drd-19. The observed effect is specifically related to the ability of R1drd-19 to determine an ATP-dependent exonucleolytic activity which is functionally similar but not identical to the RecBC nuclease. The oligomerization of pACYC184 is accompanied by the formation of high-order circular structures, and this leads to elimination of the plasmid from cells growing under non-selective conditions.     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

The atp operon: nucleotide sequence of the region encoding the alpha-subunit of Escherichia coli ATP-synthase

Part of the atp (or unc) operon encoding the alpha, beta, gamma, delta, and epsilon subunits of Escherichia coli ATP-synthase has been cloned into the plasmid pACYC 184. The DNA coding for the largest of these proteins, the alphas subunit, has been sequenced by cloning into the bacteriophage M13 and sequencing with dideoxy nucleotide chain terminators. It comprises 1539 nucleotides corresponding to a protein of 513 amino acids.     

Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments

Restriction enzyme XbaI DNA fragments that represent 99% of the sequences from the long and short unique as well as the repeat sequences of the human cytomegalovirus (CMV) genome have been cloned into bacterial plasmid pACYC184. The viral DNA sequences associated with the recombinant plasmids were analyzed by restriction mapping and by hybridization to fragments of authentic viral DNA. The relationship of the cloned viral DNA fragments to the XbaI physical map of the viral genome is demonstrated. Even though large recombinant plasmids ranging from approx. 39 to 1.8 kb were isolated, most if not all of the viral DNA fragments were stable during propagation in Escherichia coli HB101.     

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Natural plasmid transformation in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli.     

pMS76, a plasmid capable of amplification by treatment with chloramphenicol

pMS76 is a nonconjugative, 5.54-megadalton plasmid. This plasmid is present in Escherichia coli K12 cells at about 20 copies per chromosome. In addition, the pMS76 plasmid can be mobilized by conjugative plasmids and it shares a number of other properties with the amplifiable ColE1 plasmid, including the ability to amplify copy number in the presence of chloramphenicol. However, pMS76 is compatible with ColE1-like replicons, pBR313, and with other multicopy plasmids, RSF1030 and pACYC184. Also the replication of pMS76 is rifampicin sensitive and requires DNA polymerase 1.     

Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili

A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.