Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12

ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector.     

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.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Cloning, characterization and expression in Escherichia coli of a leucine biosynthetic gene from Streptomyces rochei

Leucine and histidine biosynthetic genes from Streptomyces rochei HP1 that complemented auxotrophic mutations in S. lividans TK54 were cloned in pIJ61. DNA from one leucine recombinant plasmid was subcloned into pBR322. From the latter, a recombinant plasmid was obtained that complemented the leuA mutation in Escherichia coli CV512 but not other leucine markers in E. coli. Analysis of this and several subclones, including mutant plasmids constructed in vitro, established that the cloned S. rochei gene was expressed in E. coli from the tetracycline promoter of pBR322 to produce a polypeptide of 67 kDa; the corresponding coding region was shown to be within a 1.7 kbp DNA fragment. Blot hybridization revealed corresponding homologous genes in several other streptomycetes.     

Immunological screening of standard cDNA libraries in pBR322 vectors: detection of human fibrinogen and prothrombin cDNA clones

The in situ immunological detection of antigens encoded by cDNA inserted into the PstI site of pBR322 plasmids was optimized. It was found that sensitivity of the detection was dramatically increased by in situ amplification of the recombinant plasmids on chloramphenicol-containing medium followed by a brief incubation without chloramphenicol during which protein synthesis resumes. In addition, several modifications of the previously described methods which permit total suppression of background and false positives are described. These techniques allowed easy detection of cDNA clones for human B beta- and gamma-fibrinogen and -prothrombin using a human liver double-stranded cDNA recombinant plasmid library in pBR322 vectors.     

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Expression of the replication region of phage lambda DNA cloned into pBR322 in E. coli minicells

Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII. The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations. Recombinant plasmids were transferred to the minicell-producing strain of E. coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis. All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322. The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.     

A bacterial cell that synthesizes a protein containing the antigenic determinants of rat prolactin.

Bacterial minicells containing three different recombinant plasmids with rat prolactin cDNA sequences inserted at the Pst I site of pBR322 via the poly(dG):poly(dC) joining technique were examined for the expression of rat prolactin antigenic determinants. The three prolactin coding sequences were in the same orientation as the coding sequence of the ampicillin-resistance gene of pBR322. The presence of each of the three recombinant plasmids induced some prolactin synthesis by the bacteria as measured by immunoprecipitation with anti-prolactin antisera. About 10% of the protein synthesized from one of the plasmids, prl 3, precipitated with the antisera. These prolactin antigenic determinants were part of a larger fused protein.     

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control. II. DNA fragment insertion at the vicinity of the lac UV5 promoter.

Summary Recombinant plasmids containing the entire 16S RNA gene from the rrn B cistron of E. coli inserted in Col E1 and pBR322 plasmid vectors have been constructed. These plasmids have been mapped using several restriction endonucleases as well as by DNA-RNA hybridization. These maps reveal previously undetected restriction sites in the rrn B cistron and in Col E1 plasmid DNA.     

Sequence rearrangements may alter the in vivo superhelicity of recombinant plasmids

Electrophoretic resolution of topoisomers was used to compare the in vivo superhelicity of recombinant plasmids containing a fragment of cDNA for an immunoglobulin light chain, cloned in the two possible orientations into the BamHI site of pBR313 or pBR322. Previously, frequent transpositions of IS1 or IS5 were observed into the sequence upstream to the cloned fragment in recombinants in one orientation [(+) plasmids] but not in recombinants in the opposite, (-) orientation [(1982) Nucleic Acids Res. 10, 4525-4542]. The results of the present analyses show that, on average, (-) plasmids are less negatively supercoiled than (+) plasmids, or pBR322. These results suggest that primary sequence rearrangements in plasmids could affect their in vivo topological state, and consequently, perhaps, their effectiveness as recipients of transposable elements.     

Increased stability of recombinant plasmids by Tn1000 insertion in chemostat cultures of recombinantEscherichia coli GT123

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.     

B-Z DNA junctions contain few, if any, nonpaired bases at physiological superhelical densities

The reactions of bromoacetaldehyde (BAA) with recombinant plasmids that contain sequences which can adopt left-handed Z structures or, at other locations, cruciforms were studied as a function of supercoil density. The sequence in pRW756 that undergoes a supercoil induced transition from a right to left-handed helix was (dC-dG)16 and regions near the replication origin of the pBR322 vector were converted from linearforms to cruciforms. The locations of the most nonpaired structural features were mapped by S1 nuclease cleavage of the "wedged open" duplexes after linearization of the DNAs. Three cruciforms in the pBR322 portions of the plasmids were specifically detected by BAA reaction at physiological supercoil densities (sigma = -0.067). However, the B-Z junctions did not react with BAA under these conditions although the junctions were present since the (dC-dG)16 was shown to be left-handed. Thus, the B-Z junctions have less single-stranded character than the pBR322 cruciforms (3-6 nonpaired bases) and may be fully paired. At much higher superhelical densities (sigma = -0.11-0.12), the B-Z junctions as well as the cruciforms react with BAA indicating a change in the nature of the junctions. Studies were also performed with pRW777 which harbors the mouse kappa immunoglobin sequence (dT-dG)32 . (dC-dA)32 that adopts a left-handed helix under appropriate conditions; the results were similar to those found with pRW756.     

C1 and cro repressors of lambda phages. I. Construction of vectors for expression of cro repressor of bacteriophage lambda imm434

Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised. These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434. The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped. The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

A dominant role for DNA secondary structure in forming hypersensitive structures in chromatin

Previous work has suggested that potential information in DNA secondary structure might be used by cells to define DNAase I- and S1-sensitive chromatin structures associated with promoter and terminator regions. To test this hypothesis, supercoiled pBR322 was cotransfected into L cells. For the majority of transfected clones supercoil-induced S1-sensitive sites in pure pBR322 DNA are also S1-sensitive in L-cell nuclei. These results suggest that the potential of certain DNA sequences to form specific secondary structures in chromatin can be a dominant characteristic. A recombinant chicken β^A-lobin supercoiled plasmid was reconstituted in vitro with histones. The reconstituted chromatin also retained the ability to form S1-sensitive sites. Evidence suggests that DNA sequences capable of forming S1-sensitive sites in supercoiled plasmids may bind nucleosomes poorly after reconstitution with histones.