Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

A functional map of plasmid ColE1

Summary Examination of the properties of ColE1 derivatives containing either deletions or insertions of transposable genetic elements, has enabled a functional map of plasmid ColE1 to be constructed.     

Intrastrand base pairing in single-stranded deoxyribonucleic acid from ColE1-derived plasmid pCR1.

Single strands of EcoRI-cleaved pCR1 deoxyribonucleic acid were examined by electron microscopy for intrastrand base pairing by using partial denaturing conditions. The locations of three stem and loop structures were mapped relative to the inverted repeat of Tn903. Potential roles and origins of these loops are discussed.     

Excision of a DNA sequence determining kanamycin resistance from a ColE1-Km recombinant plasmid

Summary A 4.8×10⁶ dalton ECoRI-generated fragment of the R-factor R6-5 carrying the gene for kanamycin resistance (Km) was joined in vitro to ECoRI-treated ColE1 plasmid DNA. Transformation ofE. coli with the ColE1-Km recombinant plasmid yielded clones, which were immune to colicin E1, resistant to kanamycin and failed to produce colicin E1. During multiplication of this recombinant plasmid in the presence of chloramphenicol, cells expressed an increased resistance to kanamycin. Transformation studies with the recombinant DNA molecule showed very frequent loss of Km resistance in those cells harbouring a preexisting F'gal plasmid. Since colicin immunity is not affected and the col^- phenotype is still present, one has to test for a remaining DNA sequence further existing in ColE1 DNA by cleaving the plasmid DNA with the ECoRI restriction endonuclease. The full length of ColE1 DNA (6.2 kb) was restored, which confirmed that no deletion of ColE1 DNA sequences had occured. The remaining DNA sequence was identified as a 2.0 or 2.2 kb segment. On the basis of the length of the excised fragment it is proposed that the insertion sequence IS1 and a part of the inverted repeat sequence with coordinates 21.0 to 22.0 of the R6-5 DNA are recognised by a nucleolytic function.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.     

Cloning of the lacI gene into A ColE1 plasmid.

The binding of lac repressor to lac operator was utilized to isolate an EcoRI fragment of lambda h80dlac (i+ as well as iq) that contains the lacI gene and lac promoter-operator regions. Ligation of this fragment into EcoRI cleaved pMB9 yielded chimeric DNA molecules of mol. w.t. 9.6 . 10(-6) and 1.5 . 10(7) daltons. Transformed strains containing the plasmids were analyzed for repressor production in vivo and in vitro. Repressor production in one plasmid strain is 7-fold greater than that in heat-inducible lambda h80dlac(iq)lysogens.     

Denaturation map of polyoma DNA.

A denaturation map of polyoma DNA cleaved by Eco R1 to form linear molecules was established by electron microscopy. Partial denaturation, under the same conditions, of fragments obtained by Haemophilus influenzae restriction enzymes allowed us to align the denaturation map with the already established physical map of polyoma DNA (Griffin et al., 1974).     

Structural-functional analysis of the par-region of ColE1 plasmid

The DNA fragment identical to the right shoulder of the inverted repeat from the par-region of ColE1 plasmid has been synthesized chemically. It is shown to participate in the plasmid multimers resolution and to define the stable inheritance of the plasmid pKS1 containing the fragment in Escherichia coli C600 cells as well as in the multirecombinogenic strain Escherichia coli JC8679. The efficiency of the fragments functioning in Escherichia coli JC8679 is not enough for resolution of all forms of oligomeric pKS1 DNA. The site for recombinase action is found to be located in the synthesized oligonucleotide. However, some extra sequences of DNA located within the region of inverted repeat are necessary for maximally efficient functioning of the recombinase, the enzyme participating in plasmid multimers resolution.     

A ColE1-encoded gene directs entry exclusion of the plasmid.

To detect entry exclusion of the ColE1 plasmid, we established an assay system that was not influenced by incompatibility of extant plasmids in the recipient cells or by the viability of the cells due to the killing action of colicin E1 protein. The assay revealed that exc1 and exc2, assigned as genes directing entry exclusion, had no exclusion activity. Instead, mbeD, which had been characterized as a gene for plasmid mobilization, directed the exclusion activity. MbeD was overexpressed and identified as a 35S-labeled protein, which was recovered in both the soluble and membrane fractions, particularly in the inner membrane fraction. An amphipathic helical structure was predicted in the N-terminal region of MbeD as well as in the corresponding homologous proteins of ColA and ColK. These proteins may bind to the inner membrane via the N-terminal amphipathic helix and function in entry exclusion.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Functional relationship between parts of the replication region of plasmid ColE1.

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.     

Effect of altering GATC sequences in the plasmid ColE1 primer promoter.

Plasmid ColE1 has three recognition sites for the Escherichia coli DNA adenine methylase in the immediate upstream region of the primer promoter. Two of these sites are conserved among all plasmid relatives of ColE1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. Recent experiments have indicated that hemimethylated ColE1-type plasmids are inefficiently replicated after transformation (D. W. Russell and N. Zinder, Cell 50:1071-1079, 1987). By mutating the three methylation sites, we found that disruption of all three GATC sites was necessary for complete relief of the hemimethylation-mediated inhibition of replication in vivo. We also found that these three methylation sites acted in a position-specific manner. The putative cruciform, if present, did not play a regulatory role in the hemimethylation-mediated inhibition of replication.     

Control of ColE1 plasmid replication by antisense RNA

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.     

Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale

Plasmid pCR1 is a cryptic plasmid harboured by Corynebacterium renale. It is the smallest corynebacterial plasmid known to date. Although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. It can also replicate in Escherichia coli, in which it is stably maintained. The copy number of pCR1 in this host is higher than that of pUC19, with which it shows unidirectional incompatibility. It is also incompatible with pBK2, a plasmid bearing the common corynebacterial replicon pBL1. Its size is 1488bp, as revealed by DNA sequencing. A total of eight open reading frames (ORF) were detected in this plasmid, the largest of which codes for a putative Rep protein of predicted molecular mass of 21kDa. The plasmid pCR1 can be mobilized by the plasmid R6K from E. coli to other corynebacteria. Sequence analysis revealed the presence of an oriT homologous to that of R64. An E. coli plasmid pKL1 shows more than 90% identity with pCR1. Like many coryenbacterial plasmids, pCR1 also replicates by rolling circle mode.     

Raman spectroscopy of supercoiled and nicked ColE1 plasmid

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.