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PubMed1994

Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification.

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.

Animals↗
PubMed2001

Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803.

The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium Synechocystis: sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pili in a Gram-negative bacterium Pseudomonas aeruginosa. It was found that motility of Synechocystis cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pili on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P: aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.

Amino Acid Sequence↗
PubMed1979

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.

Bacteriophages↗
PubMed2001

Preparation of single chain variable fragment of MG(7) mAb by phage display technology.

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Animals↗
PubMed2006

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Antibodies, Monoclonal↗
PubMed2006

[Cloning of Human beta2-microglobulin gene and efficient expression in Escherichia coli].

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.

Cloning, Molecular↗
PubMed2006

[Prevalence of Leptotrichia amnionii sp. nov. in pregnant women].

INTRODUCTION: Leptotrichia bacteria belong to the group of Gram-negative anaerobes most frequently colonizing the oral cavity and reproductive organs. Leptotrichia sanquigenes constitute one of the bacterial factors in postpartum bacteremia of mothers and newborns, whilst Leptotrichia amnionii sp. nov., described for the first time in 2002, has turned out to be the etiopathogenetic factor in missed abortion in the second trimester of pregnancy. AIM: The purpose of this study was evaluation of the frequency of occurrence of Leptotrichia amnionii sp. nov. in patients in the 1st, 2nd and 3rd trimester of pregnancy. MATERIALS AND METHODS: The study involved 69 patients. Amplification reactions detecting the presence of Leptotrichia amnionii so. RNA in the smears were performed using specific starters complementary to 16SrRNA Leptotrichia amnionii--primer 1 Lam upper and primer 2 Lsp lower--complementary to Leptotrichia 16SrRNA. After amplification, the specimens were placed on 1% agarose gel in the presence of MassRuler marker (Fermentas). Next, PCR products were ligated to pGEM-T Easy Victor and then competent E. coli DH5 alpha cells were transformed with pGEM vector with an insert. Plasmid DNA isolation was performed using Plasmid Mini kit from A&A Biotechnology. Sequencing of inserts from the purified plasmids was performer in Molecular Biology Techniques Laboratory in the Department of Biology at Adam Mickiewicz University in Poznań. RESULTS: In the group of 69 pregnant patients Leptotrichia amnionii sp. nov. was identified in 8 women, which is 11.6%. CONCLUSION: Recently identified Leptorichia amnionii sp. nov. is a bacterie, which relatively frequently occurs in pregnant women.

Adult↗
PubMed1984

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants.

We investigate the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2 -gpt. pSV2 -gpt DNA transformants arise with a frequency between 10(-5) and 10(-4) per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process.

Ataxia Telangiectasia↗
PubMed2005

DNA binding-uptake system: a link between cell-to-cell communication and biofilm formation.

DNA has recently been described as a major structural component of the extracellular matrix in biofilms. In streptococci, the competence-stimulating peptide (CSP) cell-to-cell signal is involved in competence for genetic transformation, biofilm formation, and autolysis. Among the genes regulated in response to the CSP are those involved in binding and uptake of extracellular DNA. We show in this study that a functional DNA binding-uptake system is involved in biofilm formation. A comGB mutant of Streptococcus mutans deficient in DNA binding and uptake, but unaffected in signaling, showed reduced biofilm formation. During growth in the presence of DNase I, biofilm was reduced in the wild type to levels similar to those found with the comGB mutant, suggesting that DNA plays an important role in the wild-type biofilm formation. We also showed that growth in the presence of synthetic CSP promoted significant release of DNA, with similar levels in the wild type and in the comGB mutant. The importance of the DNA binding-uptake system in biofilm formation points to possible novel targets to fight infections.

Bacterial Proteins↗
PubMed2004

[The construction and screening of genomic library based on transformable artificial chromosome (TAC) vector in Lotus japonicus].

Many vectors, especially artificial chromosome vectors, have been developed for genome-scale mapping and sequencing. As the first artificial chromosome, YAC has been extensively used in the genomic library construction and mapping. Shizuya et al. (1992) devised the bacterial artificial chromosome (BAC) originated from F factor of E.coli, which is much easy to handle and isolated with large harboring capacity. Liu et al. (1999) designed the transformation-competent artificial chromosome (TAC) vector, derived from PAC vector. TAC could shuttle a large-scale DNA fragment between bacteria and Agrobaceria. Further, it could integrate a large targetted DNA fragment into plant genome, as being documented in Arabidopsis and rice genome research (Liu et al. 2000, 2002). The time-saving virtue of TAC should be significant in genomics research in Lotus japonicus, a model plant of legume. Using a nuclei-based method of Liu and Whittier, high molecular weight DNA was isolated from Lotus japonicus (Gifu ecotype). The DNA was digested partially with Hind III and size-fractionated in the 10- to 20-kb size range as described (Liu and Whittier 1994). The partially digested and size selected DNA fragments were ligated with Hind III-digested pYLTAC7 and then used for transformation of E. coli DH10B by electroporation. Transformants carrying inserts were selected on LB agar plates containing 25 mg/L kanamycin and 5% sucrose. The library, 6 haploid genome equivalents, was pooled in 12 96-well microtiter plates at about 150 transformants per well. The TAC library was then arrayed in nylon membranes and subjected to screening. The probe, a homolog fragment of CEN gene controlling the structure of inflorescence Antirrhinum, was used for screening. 0.5 muL solution from the positive pool was titered and the secondary screening was conducted in the same way. Finally, these positive clonies were confirmed by Southern blot. These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.

Chromosomes, Artificial, Bacterial↗
PubMed2006

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

Adhesins, Bacterial↗
PubMed2005

Prooxidant activity of resveratrol in the presence of copper ions: mutagenicity in plasmid DNA.

Resveratrol, a polyphenolic compound of plant origin, has been of much interest to researchers because of its anticancer and cardiovascular properties. Although antioxidant action of this compound is believed responsible for its reported properties, it has also been shown to exhibit prooxidant properties, especially in the presence of copper ions. Here we report the mutagenicity of resveratrol in plasmid DNA. Plasmid bluescript SK(+) DNA was treated with increasing concentrations of resveratrol in the presence and absence of copper ions, transformed into competent DH5alpha cells and sequenced. We looked for mutations caused by resveratrol treatment by comparing the sequences of treated plasmids versus control (untreated plasmid). The results show a decrease in the transformation efficiency of the plasmid after resveratrol treatment, and although all types of mutations were recorded, point mutations (deletions/substitutions) were found to be the predominant ones. Resveratrol alone resulted in deletion of mainly guanine bases. Since copper ions are known to be found in the nucleus, bound to guanine bases in chromatin, our results suggest mobilization of such endogenous copper by resveratrol resulting in prooxidant DNA cleavage at the site. Concentration of copper is reported to be elevated in various malignancies and the present studies might explain the reported anticancer activity of resveratrol in various cancer cell lines.

Antineoplastic Agents, Phytogenic↗
PubMed1993

Genetic transformation in Helicobacter pylori.

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.

Chromosomes, Bacterial↗
PubMed1968

Transformability of streptomycin-resistant group H streptococci.

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.

DNA, Bacterial↗
PubMed1999

Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation.

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.

Bacterial Proteins↗
PubMed1984

Cytoplasmic transformation of Saccharomyces cerevisiae using purified mtDNA.

Transformation of cytoplasmic mutants of Saccharomyces cerevisiae proved to be successful using plasmid or intact mitochondria, but not when mtDNA was used as the transforming principle. The sphaeroplasts of two cytoplasmic mutants, respiratory deficient and erythromycin sensitive cells, were transformed using purified mtDNA from respiratory competent or erythromycin resistant donor strains.

Cytoplasm↗