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PubMed1988

Structural interconversion of alternating purine-pyrimidine inverted repeats cloned in supercoiled plasmids.

Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.

5-Methylcytosine↗

PubMed1983

Structural analysis of RNA molecules involved in plasmid copy number control.

DNA replication of the plasmid ColE1 and its relatives is controlled mainly by a small plasmid-encoded transcript known as RNA11. We have studied the conformation of RNA1 molecules from two related but compatible plasmids, ColE1 and RSF1030, using T1 ribonuclease, S1 nuclease, cobra venom nuclease, and diethyl pyrocarbonate modification as probes for secondary structure. Both RNA1 molecules contain three double-stranded stems, three single-stranded loops, and an exposed 5' tail. All loops in both molecules show similar sensitivities to the probes used, as do stems 1 and 3. The region comprising stem 2 of each RNA1 molecule contains the most sequence differences as well as the most structural changes of any region in the two molecules. The structure of the RNA1 molecule encoded by a recessive high copy number mutant of ColE1 was also investigated. Structural alterations involving the first stem and loop of the molecule are proposed to be responsible for the inability of the mutant RNA1 to function in vivo.

Base Sequence↗

PubMed1990

The virulent bacteriophage IRA of Salmonella typhimurium: cloning of phage genes which are potentially lethal for the host cell.

Morphological and biological properties of Salmonella typhimurium phage IRA were studied. The phage is a member of the Styloviridae family and exhibits a very wide spectrum of lytic activity. Molecular cloning of phage genes whose expression is lethal for the host cell has been performed and consequences of gene expression have been investigated. Expression of recombinant plasmid pKI71 causes structural changes of the cell wall and also degradation of host DNA while plasmid DNA remains intact. Expression of pKI72 blocks normal cell division. The possibility of applying such recombinant clones in marking pathways of microbial contamination in water areas is proposed.

Bacteriolysis↗

PubMed1999

Experimental microcosms as models of natural ecosystems for monitoring survival of genetically modified microorganism.

An experimental approach for investigation of genetically modified microorganisms (GMMO) introduced into model ecosystems to evaluate potential risk of propagation of recombinant plasmids in surrounding medium has been developed. The object of modeling was Escherichia coli Z905 strain with a recombinant plasmid with bacterial luminescence genes, which was introduced into water microcosms of different structure. The approach involves comprehensive investigation of GMMO at four hierarchical levels: molecular (retaining the structure of the plasmid and expression of cloned genes); cellular (variation of metabolic activity); population (competitive power and metabolic interactions of GMMO with indigenous microflora, migration of recombinant and natural plasmids); ecosystem (effect of GMMO and cloned genes on ecosystem parameters). The experimental evidence and theoretical estimates are intended to form grounds to develop a basic version of an ecological certificate for different GMMO variants.

Adaptation, Biological↗

PubMed1986

Consecutive A X T pairs can adopt a left-handed DNA structure.

The capacity of six sequences with different numbers and orientations of A.T pairs flanked by alternating C.G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (approximately 2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by two-dimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive T.A base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G)n (n = 3-5). Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations.

Adenine↗

PubMed1999

TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli.

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402-410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

Agrobacterium tumefaciens↗

PubMed2001

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites.

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Animals↗

PubMed1988

Multiple DNA secondary structures in perfect inverted repeat inserts in plasmids. Right-handed B-DNA, cruciforms, and left-handed Z-DNA.

The capabilities of five recombinant plasmids, containing relatively long (approximately 60-100 base pairs) perfect inverted repeat (IR) inserts, to support supercoil stabilized non-B-DNA structures were studied in vitro. The IRs were also alternating purine-pyrimidine sequences, thus, each could form either left-handed Z-DNA or cruciforms. Single-strand specific endonucleases, restriction endonucleases and methylases, and OsO4 modifications were used to characterize the DNA structures. Two-dimensional gel electrophoretic studies indicated that three of the five IRs formed both cruciforms and Z-DNA. (C-G) containing inserts preferred to form Z-DNA, whereas (T-G) sequences favored cruciforms. In vivo supercoil relaxation experiments demonstrated the existence of cruciforms in Escherichia coli. The physiological significance of these structures is discussed.

Base Sequence↗

PubMed1993

Immunochemical probing of DNA structure with monoclonal antibody to OsO4/2,2'-bipyridine adduct.

Chemical modification with osmium tetroxide (OsO4) in the presence of 2,2'-bipyridine was employed for selective labeling of certain non-B DNA structures. A murine monoclonal antibody (IgG1) specific for deoxythymidine adduct of the modification was produced. It was demonstrated that the level of single-stranded DNA can be evaluated by enzyme-linked immunosorbent assay using the modification and monoclonal antibody. Site-specific OsO4-bipyridine modification followed by dot-blotting was used for probing of some unusual conformations occurring in supercoiled plasmids. The cruciform structures, B-Z junctions, and intramolecular triplex and long (A-T)n inserts were targets for the modification and small quantities of the labeled plasmids can be immunodetected. The method enabled to catch certain structural transitions within the same plasmid in vitro and to detect the left-handed Z DNA labeled inside bacteria. The antibody was also shown to recognize specifically the in situ modified DNA in cell nuclei and polytene chromosomes isolated from Drosophila melanogaster. Preferentially S1-nuclease-sensitive (single-stranded) regions of the nuclear and chromosomal DNA were stained by indirect immunofluorescence after the modification.

2,2'-Dipyridyl↗

PubMed2006

Autonomous cytoplasmic linear plasmid pPac1-1 of Pichia acaciae: molecular structure and expression studies.

The genome organization of the linear DNA-element pPac1-1 from Pichia acaciae was determined. It turned out to be the smallest autonomous cytoplasmic yeast plasmid known so far, consisting of only 12 646 bp, carrying the shortest terminal inverted repeats yet found (138 bp). As for other cytoplasmic linear yeast plasmids, it is characterized by a strikingly high A + T content (75.35%). Ten putative genes (open reading frames, ORFs) reside on the element, leaving only 2.9% of the sequence outside a coding region. Highest similarities of the predicted proteins were obtained for proteins encoded by the three hitherto known autonomous cytoplasmic linear yeast plasmids. Amino acid sequences correspond to predicted polypeptides encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis, pSKL of Saccharomyces kluyveri and pPE1B of Pichia etchellsii. As for the latter, ORF1 existing on the two other plasmids is lacking on pPac1-1. Consistent with cytoplasmic localization, a cytoplasmic promoter termed upstream conserved sequence (UCS) is located in front of each reading frame. RT-PCR transcript analyses for ORFs 8, 9 and 11 proved expression of such genes but functions could not be attributed. The genome organization of pPac1-1 and other autonomous linear elements was found to be almost congruent, irrespective of the accompanying smaller elements, which may or may not encode their own element-specific DNA polymerases.

Amino Acid Sequence↗

PubMed1985

Cloning and expression of human interleukin 2 in Streptomyces lividans using the Escherichia coli consensus promoter.

Streptomyces lividans was transformed with a plasmid containing the structural gene that specifies human interleukin 2. The expression of interleukin 2 in this plasmid is controlled by both the consensus promoter and a consensus ribosome binding site characteristic of Escherichia coli. We have detected production of active human interleukin 2 in liquid cultures of the transformed Streptomyces by both, biological assay and immuno-blotting analysis.

Base Sequence↗

PubMed1990

Expression of a foreign KmR gene in linear killer DNA plasmids in yeast.

The killer plasmids of the yeast Kluyveromyces lactis, pGKL1 and 2 (k1 and k2 for short), are linear double-stranded DNAs. The expression of genes of these plasmids is thought to depend on their own transcription system. Cloning the plasmid genes in conventional circular vectors is therefore not suitable for transcriptional studies, because such vectors use the host nuclear transcription system. In vitro modification of the linear plasmid genomes in order to introduce transcription reporter genes has been difficult because the structure of the plasmids, with covalently bound terminal proteins, does not allow their manipulation in vitro and amplification in Escherichia coli. We introduced the kanamycin/G418 resistance gene, KmR, into the k1 plasmid in vivo, by transforming the yeast with the linearized KmR gene bordered with short k1 sequences (part of the region encoding the toxin) to allow homologous recombination with the resident k1. In the linear recombinants obtained, however, the KmR was not expressed, while it was expressed if carried on circularized plasmids. By replacing the native promoter of KmR by the ORF1 promoter from k1, the KmR gene could be expressed in linear recombinants and conferred on the host a high level of resistance to the drug. All the linear recombinant plasmids were extremely stable under nonselective conditions. As a rare event, the integration of KmR produced a palindromic rearrangement of the k1 plasmid.

DNA, Fungal↗

PubMed1986

Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture.

We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein. PRV-10 produced no detectable gIII-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture.

Animals↗

PubMed1986

Structural analysis of an RNA molecule involved in replication control of plasmid R1.

The replication control circuit of the FII plasmids R1, R100 and R6-5 involves a small untranslated RNA molecule of about 93 nucleotides. This RNA, designated CopA RNA in plasmid R1, acts in vivo as a post-transcriptional inhibitor of the expression of the RepA protein whose synthesis is required for each round of plasmid replication. We have studied the structure of CopA RNA in solution by probing with single-strand- as well as double-strand-specific nucleases. Our data show that CopA RNA has two stem-loop structures connected by a long spacer region and a single-stranded 3'-tail. Enzyme reactions were performed under buffer conditions where we find strong and specific binding of CopA RNA to its target RNA (to be published elsewhere), and the patterns were compared to digests in two other buffer systems. All partial cleavages supported the proposed secondary structure. We also analyzed the stem-loop regions of CopA RNAs from two R1 copy number mutants. The data show that both RNAs have altered loop structures. The implications of these findings are discussed.

DNA Replication↗

PubMed1995

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa.

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 bp in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1-2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.

Base Sequence↗

PubMed2006

Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli.

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225'-232'), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.

Animals↗

PubMed1981

Plasmid vectors capable of transferring large DNA fragments to yeast.

We have constructed several cloning vectors which can be used in vitro packaging and yeast transformation. These plasmids have been designed for the convenient cloning of large segments of DNA and their transfer to yeast. They contain bacterial plasmid DNA sequences for replication and selection in Escherichia coli, yeast 2-microns plasmid DNA sequences or chromosomal replicators and yeast markers necessary for replication and selection in yeast, and the cohesive ends of bacteriophage lambda which allow packaging of recombinant molecules into lambda phage heads. Large fragments (22-38 kb) of Klebsiella pneumoniae and Zea mays DNA were ligated into plasmid vector pBTI-1 to make complete genome libraries. One clone from the K. pneumoniae library was amplified in E. coli and the purified DNA used to transform yeast cells. Transformation of yeast by large DNA fragments occurred at high frequencies. The recombinant plasmid was stably maintained in yeast, provided selective pressure for Leu+ transformants was maintained. The structurally complete recombinant plasmid can be recovered from yeast by transforming E. coli to ampicillin resistance. Fewer than 5% of the recovered plasmids had undergone recombination with endogenous yeast 2-microns plasmid.

Bacteriophage lambda↗

PubMed2003

Characterization of SrgA, a Salmonella enterica serovar Typhimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA, essential for biogenesis of plasmid-encoded fimbriae.

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.

Amino Acid Sequence↗