Cloning and cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper strain).

Sequences representative of most of the bovine herpesvirus 1 (Cooper strain) DNa were cloned in the plasmid vector pBR322 at the HindIII site. EcoRI, HpaI, and BamHI restriction endonuclease sites were mapped in each of the cloned fragments, and this information was used to construct a restriction endonuclease cleavage site map of the entire viral genome for the four enzymes.     

Cloning and expression of the gene(s) for cephalosporinase production of Citrobacter freundii

The chromosomal gene(s) for cephalosporinase production of Citrobacter freundii GN346 has been cloned into vector plasmid pMK1, initially as a 7.3 kb EcoRI fragment. From the substrate profile and the response to anti-GN346 CSase serum of the enzyme produced, it was confirmed that the hybrid plasmid (pTY71) carries the relevant chromosomal cephalosporinase gene from C. freundii GN346. A restriction endonuclease cleavage map of cloned EcoRI fragments was constructed, and the structural gene of the cephalosporinase could be limited in the 1.5 kb BamHI fragment. The cloned gene(s) was expressed at an extremely low level in Escherichia coli. Furthermore, its expression was constitutive in E. coli, although inducible in its own cytoplasm.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

Transfer of the chromosomal bla gene from Enterobacter cloacae to Escherichia coli by RP4::mini-Mu

The resistance gene for beta-lactamase-stable cephalosporins from Enterobacter cloacae was transferred to Escherichia coli by the aid of RP4::mini-Mu. The R-prime plasmids generated carried 60 to 80 kilobases (kb) of E. cloacae DNA and coded for the chromosomal E. cloacae beta-lactamase. The gene was fully expressed in the recipient. Restriction endonuclease EcoRI fragments of the R-prime plasmid pBP100 were cloned into the vector pBP328, yielding the plasmid pBP102 with a size of 14 kb. A restriction map of this plasmid was constructed. By digesting pBP102 into seven PstI fragments, ligating the fragments, and looking for the smallest plasmid generated, pBP103 was isolated. It consisted of three PstI fragments, two of them (together 4.2 kb) necessary for resistance. During the experiment (performed in a recA+ background) the largest PstI fragment had undergone a substitution of a 0.3-kb segment of pBP102 by a 0.7-kb segment in pBP103 (as deduced by heteroduplex analysis). The bla gene of resistant E. cloacae strains was dominant over the gene of susceptible organisms.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2.

The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, BamH-I, SalI and HpaI. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.     

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.     

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti.

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.

The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

Physical and genetic analyses of streptococcal plasmid pAM beta 1 and cloning of its replication region.

Plasmid pAM beta 1, originally isolated from Streptococcus faecalis DS5, mediates resistance to the MLS (macrolide, lincosamide, and streptogramin B alpha) group of antibiotics. A restriction endonuclease map of the 26.5-kilobase (kb) pAM beta 1 molecule was constructed by using the enzymes AvaI, HpaII, EcoRI, PvuII, Kpn1, BstEII, HpaI, HhaI, and HindIII. A comparison of this map to those of four independently isolated deletion derivatives of pAM beta 1 located the MLS resistance determinant within a 2-kb DNA segment and at least one conjugative function within an 8-kb region. The 5.0-kb EcoRI-B fragment from pAM beta 1 was ligated onto the 4.0-kb Escherichia coli plasmid vector, pACKC1, and used to transform E. coli HB101. The 9.0-kb chimeric plasmid was then used to transform Streptococcus sanguis Challis with concurrent expression of the E. coli kanamycin resistance determinant. The 5.0-kb EcoRI-B fragment from pAM beta 1 was subsequently used as a vector to clone a streptomycin resistance determinant from a strain of Streptococcus mutans containing no detectable plasmid DNA. Subcloning experiments, using a HindIII partial digest of pAM beta 1 DNA, narrowed the replication region of this plasmid to a 2.95-kb fragment.     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.