Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element

Summary Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield trp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of trp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of -transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final ::Tn409 or ::Tn410 product.Abbreviations p.f.u. plaque-forming units - MIC minimal inhibitory concentration - LFT low frequency transducing - HFT high frequency transducing     

Molecular cloning of the fnr gene of Escherichia coli K12

Summary Mutations in the fnr gene of Escherichia coli have pleiotropic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and, as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization.     

Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

HindIII/Eco RI RFLP of the human immunoglobulin IGHE gene detected with a Cɛ probe in the French and Tunisian populations

Summary Three differentHindIII/EcoRI restriction fragment alleles of the human immunoglobulin IGHE gene have been detected with a C probe in the Tunisian population. Two of these alleles can be explained by a Negroid contribution in their gene pool.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

Fertility functions of resistance factors

Summary The isolation of transducing phages carrying the tolPAB cluster is described. These genes map between gltA and gal in Escherichia coli, and thus are relatively close to att. To isolate these transducing phages, it was necessary to use a strain deleted of most of the intervening genes (nadA to chlD) between tolPAB and att. Using a lysogen of such a deletion strain, several defective dtol phages were isolated that carry different amounts of the tolPAB cluster.All of these dtolPAB phages were defective in both lysogenization and vegetative growth, and in this respect were similar to dgal transducing phages.The usefulness of such specialized transducing phages in studying the cell surface is discussed.Research Fellow of the National Cancer Institute of Canada.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region

Summary The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of -lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that 1.9x10⁶ daltons of the 6.0x10⁶ dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.