Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

Partial characterization of a small, multiple-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant

An organism classified as Streptomyces espinosus was found to carry an approx. 9.2-kb plasmid. This plasmid, designated pUC6, has a copy number of 30-40 per host genome equivalent. Plasmid pUC1061, a copy-number mutant of pUC6, was isolated after in vitro deletion of an approx. 2.0-kb XhoI restriction fragment. Plasmid pUC1061 has a copy number of 500-600. Plasmid pUC1061 appears to be incompatible with pUC6 and will transform a pUC6-containing culture at a frequency of approx. 1%. The sizes, restriction maps and copy numbers of plasmids pUC6 and pUC1061 indicate these may be valuable vectors for gene cloning Streptomyces.     

Mitochondrial DNA from lupine: restriction analysis and cloning of fragments coding for tRNA

Mitochondrial DNA (mtDNA) was isolated from lupine. Restriction analysis was used to estimate its size, which is about 180 kb. A BamHI bank of this mtDNA was constructed using plasmids pBR322 and pBR327 as vectors. Eight clones containing plasmids hybridizing to mitochondrial tRNA (mttRNA) were isolated. Restriction maps of these plasmids were determined. Six of these plasmids hybridized to unique fragments and two to two fragments of very similar size, all obtained by BamHI cleavage of mtDNA.     

Fractional precipitation of plasmid DNA from lysate by CTAB

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.     

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Isolation of a new plasmid pIRL19: its use in construction of small vectors and detection of specific sequence in a foreign DNA

A new plasmid, pIRL19, was constructed by ligating a 1875-bp HaeII fragment carrying the ampicillin-resistance (Apr) gene to a 370-bp HaeII fragment containing the replication origin of the plasmid pBR322. The plasmid essentially contains only the basic replicator and the Apr gene. This basic replicator provides a valuable initial building block for in vitro construction of other very small vectors with antibiotic-resistance determinants. To illustrate this potential, we have transferred the chloramphenicol-resistance (Cmr) gene and a part of the Apr gene from the plasmid pBR329 into pIRL19 such that the new plasmid pIRL20 acquired the Cmr gene and maintained the integrity of its Apr structural gene.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYCl, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.     

Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts

From a genomic library of the filamentous fungus Podospora anserina, we have cloned a 4.9-kb fragment which complements an Escherichia coli mutant strain deficient for orotidylic acid pyrophosphorylase (pyrE gene). The recombinant plasmid pPAura5 also transforms to prototrophy a mutant strain of P. anserina carrying a mutation in the ura5 gene and lacking OMPppase activity.     

Cloning of both ends and the thermo-inducible genes A and B of bacteriophage Mu on a multicopy plasmid

Employing the recombinant DNA technique, two hybrid plasmids were constructed carrying both ends of bacteriophage Mu DNA in two different orientations. The expression of the early Mu genes located on these plasmids is thermo-inducible.     

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.     

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region (tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.