Isolation and characterization of lambda transducing phages for the E. coli genes ksgA and pdxA

Summary Lambda phages carrying the Escherichia coli genes ksgA and pdxA were isolated from secondary site lysogens in araB. 1) The phage genomes were characterized by genetic complementation tests, restriction endonuclease digestion and electron microscopy. 2) A 6.3 kilobasepair (kb) EcoRI restriction fragment carrying both ksgA and pdxA was cloned in a lambda vector; this fragment has proven useful in further characterization of the ksgA gene (Andrésson and Davies, 1980a, b). The ksgA and pdxA genes are about 14 and 12–13 kb, respectively, counterclockwise of the arabinose operon and 1.5 and 2.5–3.5 kb clockwise of folA.     

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.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Summary Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara⁺, plasmid containing strains. These studies demonstrated that strains containing (araO1BA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.     

Characterization of cloned rat ribosomal DNA fragments

Summary Two Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5-spacer regions were different.     

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.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

Origin of replication, oriC, of the Escherichia coli chromosome: mapping of genes relative to R.EcoRI cleavage sites in the oriC region.

Summary A precise genetic-physical map of the tnailv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F carrying the genes between aroE and ilv.A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30–45 kb counterclockwise of ilv.The pattern of R.EcolRI cleavage sites in the het region is identical with the pattern obtained by Marsh and worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.Abbreviations CCC covalently closed circular - kb kilobases - MD 10⁶ Daltons - mw molecular weight - R.EcoRI restriction endonuclease EcoRI New Genetic Symbols het heterogeneity of cell size distribution (this study) - maf maintenance of F (Wada et al., 1977) - oriC chromosomal origin of replication (Hiraga, 1976) - oriV origin of vegetative replication of F (Guyer et al., 1976) - oriT origin of transfer replication of F (Guyer et al., 1976) - poh permissive on Hfr (Hiraga, 1976)     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

Summary An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their -lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.     

tra cistrons and proteins encoded by the Escherichia coli antibiotic resistance plasmid R6-5

Summary Hybrid plasmids obtained by cloning individual EcoRI and HindIII fragments of the conjugative plasmid, R6-5, were analyzed for their ability to complement transfer-deficient point mutations of Flac. As a result, the locations of 10 tra cistrons were defined on the physical map of R6-5. Two cistrons, traE and traG, are interrupted by EcoRI restriction sites and one cistron, traC, probably contains a HindIII restriction site. The origin of DNA transfer, oriT, was also localized. Surprisingly the hybrid plasmid carrying oriT is mobilized by the F factor as well as by R6-5. The surface exclusion cistrons, traS and traT, were mapped and their biological expression analyzed. A total of 18 proteins encoded by cistrons within the tra region were detected by SDS polyacrylamide gel electrophoresis of proteins synthesized in minicells; they represent about 53% of the coding capacity of the cloned DNA. R6-5 DNA fragments containing the cistrons traC, traE, and traT directed the synthesis of proteins which comigrated during SDS gel electrophoresis with the F-coded proteins previously characterized as TraCp, TraEp, and TraTp. A further two proteins encoded by R6-5 comigrated with F-encoded (but genetically unidentified) proteins whose cistrons map in the corresponding part of the tra region. In contrast, no R6-5 proteins corresponding to F proteins TraAp, TraDp, TraJp, TraMp, 6a or 6c were detected. These results are discussed in relation to known DNA sequence homologies between the F and R6-5 plasmids. A preliminary physical map of the tra region of R6-5 is presented and compared with that of F.     

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.     

A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid

Summary A general method has been developed for the deletion of restriction endonuclease sites in bacterial plasmid DNA. The procedure involves partial digestion of the covalently closed circular plasmid DNA with an appropriate restriction endonuclease under conditions which allow accumulation of unit-length linear DNA molecules, controlled digestion of the exposed 5 ends with the 5-exonuclease, and in vivo recircularization of the resulting linear DNA in a bacterial host cell. The method has been used for the deletion of one of the two EcoRI sites in the plasmid pML2 (colE1-Km). Two of the resulting plasmids, pCR1 and pCR11, have a single EcoRI cleavage site, but retain genetic determinants specifying resistance to colicin E1 and kanamycin, and thus may be useful as vectors for the cloning and amplification of DNA in bacteria.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda

Summary The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage . The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of the targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin sensitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.