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PubMed1981

[Comparative study of chromosome and plasmid transformation in Bacillus subtilis: the effect of lysozyme and polyethylene glycol].

The influence of lysozyme and polyethyleneglycol (PEG) on chromosomal and plasmid transformation was comparatively investigated. Treatment of competent cell culture with low concentrations of lysozyme which do not affect cell viability stimulates both types of transformation. When transforming DNA was treated with definite concentrations of PEG, inhibition of transformation using chromosomal DNA occurred. The similar treatment, however, stimulated transformation by oligomeric forms of plasmid DNA. Possibly, PEG alters the DNA conformation rendering it less sensitive to nuclease degradation. This promotes plasmid transformation but reduces the frequency of transformation by chromosomal DNA, because the changes resulting from nuclease action further DNA integration into the genome of a recipient.

Bacillus subtilis↗
PubMed1996

YAC cloning and identification of rice (Oriza sativa L.) genomic DNA.

Construction of the genomic library by using yeast artificial chromosomes (YAC) has been an approach to a map-based gene cloning strategy. We have obtained more than 2000 YAC clones for the construction of a rice (Oriza sativa L.) genomic library through a procedure that could be summarized as follows: a fractionate by the pulsed-field gel electrophoresis (PFGE), the rice nucleic high molecular weight (HMW) DNA which is partially digested with EcoRI, and recovered fragments larger than 200 kb; ligate the fragments to the EcoRI digested YAC vector pairs pJS97 and pJS98; transform competent spheroplasts prepared from yeast strain YPH252; and select transformants directly on Ura-Trp-double selective media. Southern hybridization results indicated that the sizes of inserts were in the range of 200-820 kb.

Chromosomes, Artificial, Yeast↗
PubMed1969

The integration of donor and recipient deoxyribonucleic acid during transformation of Bacillus subtilis.

1. DNA labelled with 5-bromo[(3)H]uracil was used to transform auxotrophic strains of Bacillus subtilis. 2. After various times of incubation, DNA was extracted from the transformed culture and subjected to equilibrium sedimentation in caesium chloride gradients. 3. In addition to heavy donor DNA and light recipient DNA, a component with an intermediate density was found and is believed to consist of a biological hybrid of donor and recipient. 4. The component of intermediate density was isolated and found to possess activity in transformation derived from both donor and recipient strains. 5. Denaturation of the component of intermediate density followed by centrifugation gave only one component, indicating that integration had occurred in both strands of the recipient DNA. 6. No integrated band was observed after uptake by competent cells of B. subtilis of heavy DNA prepared from Escherichia coli.

Bacillus subtilis↗
PubMed1993

DNA excision repair as a component of adaptation to low doses of ionizing radiation in Escherichia coli.

In this study we examined whether or not DNA excision repair is a component of adaptation induced by very low-dose ionizing radiation in Escherichia coli, a well-characterized prokaryote, and investigated the relationship between enhanced excision repair and the SOS response. Competent E. coli cells were irradiated using low doses (0.1-10 Gy) of 137Cs gamma-rays, allowed to recover for 2 h and were then transformed using pUC18 DNA containing approximately 22 oxidized thymine residues (thymine glycols) per molecule. Successful transformants were identified by recovery of plasmid-borne ampicillin resistance and the resulting data (colony counts) were used to calculate ratios of plasmid recovery in irradiated to control cells. Results showed that cells irradiated with very low doses (0.1-0.5 Gy) were up to 30-40% more efficient at utilizing thymine glycol-containing pUC18 DNA. The enhanced excision repair by very low doses (< 0.5 Gy) of gamma-rays was shown to be independent of the recA-controlled SOS response in experiments using recA cells or cells carrying recA-lacZ gene fusions. The stimulating effect in AB1157 prototype cells were subsequently confirmed using a DNA precipitation assay in which DNA incision events were accumulated by inhibiting DNA ligation with ethidium bromide. Our data suggest that there seems to be narrow 'windows' of dose-effect for the induction of SOS-independent DNA excision repair. Being similar to mammalian cell studies, the dose range for this effect was about 200-fold less than D37 for radiation survival.

Adaptation, Physiological↗
PubMed2004

Biased distribution of DNA uptake sequences towards genome maintenance genes.

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H.influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions. These results imply that the high frequency of DUS in genome maintenance genes is conserved among phylogenetically divergent species and thus are of significant biological importance. Increased DUS density is expected to enhance DNA uptake and the over-representation of DUS in genome maintenance genes might reflect facilitated recovery of genome preserving functions. For example, transient and beneficial increase in genome instability can be allowed during pathogenesis simply through loss of antimutator genes, since these DUS-containing sequences will be preferentially recovered. Furthermore, uptake of such genes could provide a mechanism for facilitated recovery from DNA damage after genotoxic stress.

Bacterial Proteins↗
PubMed1973

Congression of unlinked markers and genetic mapping in the transformation of Bacillus subtilis 168.

A thorough examination of cotransformation of two unlinked genetic markers in Bacillus subtilis 168 shows that the two recombinational events do not occur randomly. The cotransformation frequency is dependent on the distance between the two markers as well as on the order in which they replicate in the competent cell. These results indicate that uptake and/or integration of DNA molecules bearing these genetic markers is enhanced at the time these markers replicate in the competent cell.

Bacillus subtilis↗
PubMed1980

Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis.

The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmid-free recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per microgram of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.

Bacillus↗
PubMed1978

Transfection of Streptococcus pneumoniae with bacteriophage DNA.

It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966).

Bacteriophages↗
PubMed2001

[Production of phage-displayed anti-idiotypic antibody single chain variable fragments to MG7 monoclonal antibody directed against gastric carcinoma].

OBJECTIVE: To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) MG7 to monoclonal antibody directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer. METHODS: Balb/c mice were immunized i.p. with purified MG7 monoclonal antibody conjugated with keyhole limpet hemocyanin. mRNA was isolated from the spleens of immunized mice. Heavy and light chain genes (VH and VL) of antibody were amplified separately and assembled into ScFv genes with a specially constructed linker DNA by RT-PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage, which displayed ScFv fragments as a fusion with gene 3 protein on the tips of M13 phage. After four rounds of panning with monoclonal antibody MG7, the MG7-positive clones were selected with the enzyme-linked immunosorbent assay (ELISA) from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were primarily identified by competition ELISA. RESULTS: The VH, VL and ScFv DNAs were about 340, 320 and 750 bp respectively. Twenty-four MG7-positive clones were selected from 40 enriched phage clones. Five of the 24 clones displayed beta or gamma type anti-Id ScFv. CONCLUSION: The anti-Id ScFv frangments to MG7 monoclonal antibody can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma using anti-Id ScFv.

Animals↗
PubMed1994

High-efficiency electrotransformation of the yeast Schwanniomyces occidentalis.

A method has been developed for introducing heterologous DNA rapidly and efficiently by electropermeabilization into the yeast Schwanniomyces occidentalis. A transformation efficiency as high as 2 x 10(5) transformants/microgram of plasmid DNA was obtained with a square-wave electric pulse of 2.17 kV/cm during 18 ms. Small quantities of DNA (5 ng) can be used to transform 3 x 10(8) cells. The main parameters which have been optimized are: presence of adenine in the culture medium, pretreatment of the cells with dithiothreitol during the exponential growth phase of the cells, amount of cells treated, and pulse-field strength and duration. Competent cells can be stored to allow electrotransformation whenever needed.

Adenine↗
PubMed1998

Pleiotropic mutations alter the kinetics of calcium transport, competence regulation, autolysis and experimental virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Animals↗
PubMed1981

Role of Streptococcus sanguis (strain Wicky) cell surface-located deoxyribonucleic acid-binding factor in transformation of a homologous strain.

In a previous report we demonstrated the presence of a factor binding deoxyribonucleic acid (DNA) in vitro (BF) in cell leakage fluids from transformable Streptococcus sanguis strains Wicky, Challis, and Blackburn. BF originating from strain Wicky was purified to homogeneity, and its properties are described. In this work, it was found that BF occurs at the surface of Wicky cells in two forms, loosely bound (LB-BF) and strongly bound to the cell envelope. It was demonstrated that LB-BF formed fast-sedimenting complexes with exogenous DNA at the surface of Wicky cells. About 10-fold-more DNA became associated as a fast-sedimenting complex in competent than in incompetent cells. Thus, LB-BF is a cell receptor for exogenous DNA. However, the comparison of the effects of some agents on the transformation yield and the formation of LB-BF-DNA complexes, showed that the influence of these agents on both observed phenomena is not parallel and may be even opposite. These results are interpreted to mean that the LB-BF-DNA complexes do not take part in transformation. The problem of participation of BF strongly bound with the cell membrane fraction remains to be elucidated.

Carrier Proteins↗
PubMed1990

Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D.

Cell cycle-regulated gene expression is essential for normal cell growth and development and loss of stringent growth control is associated with the acquisition of the transformed phenotype. The selective synthesis of histone proteins during the S phase of the cell cycle is required to render cells competent for the ordered packaging of replicating DNA into chromatin. Regulation of H4 histone gene transcription requires the proliferation-specific promoter binding factor HiNF-D. In normal diploid cells, HiNF-D binding activity is regulated during the cell cycle; nuclear protein extracts prepared from normal cells in S phase contain distinct and measurable HiNF-D binding activity, while this activity is barely detectable in G1 phase cells. In contrast, in tumor-derived or transformed cell lines, HiNF-D binding activity is constitutively elevated throughout the cell cycle and declines only with the onset of differentiation. The change from cell cycle-mediated to constitutive interaction of HiNF-D with the promoter of a cell growth-controlled gene is consistent with, and may be functionally related to, the loss of stringent cell growth regulation associated with neoplastic transformation.

Animals↗
PubMed1995

In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61.

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Persistence of transgenic and not transgenic extracellular DNA in soil and bacterial transformation.

The study of the fate of transgenic and not transgenic extracellular DNA in soil is of extreme relevance because the soil extracellular DNA pool represents a genetic reservoir that could be utilized as a source of food by any heterotrophic microorganism or genetic information by recipient eukaryotic and prokaryotic cells. Several data have clearly evidenced that extracellular DNA could persist in soil for long time maintaining a sufficient integrity of the molecule. Recent microcosm studies under laboratory conditions have evidenced that extracellular DNA molecule could be leached or raised up by capillarity. The persistence and movement of extracellular DNA molecule in soil suggest that the genetic information of extracellular DNA could be taken up by microorganisms temporarily and spatially separated. Several authors have studied the persistence and transformation efficiency of the extracellular DNA in soil demonstrating that there is a sharp discrepancy between its biological efficiency and its persistence; fragments of target DNA were detected after a long time in soil but no transformations were determined probably because the genetic information originally present in the complete DNA molecule could be lost by degradation. It is also important to underline that the frequency of gene transfer in soil is markedly limited by the few number of bacteria able to develop competence and that this physiological state is reached only under certain conditions. Furthermore the dilution of the transgene in the soil extracellular DNA pool drastically decreases chances for the uptake of the transgene. Anyway the importance of transformation in evolutionary terms, represents a valid reason to continue the investigation on the fate of extracellular DNA in soil.

DNA↗
PubMed1994

Expression and immunogenicity of tripeptide repeat region on P190 of Plasmodium falciparum.

A DNA fragment, designated as P190TR, encoding amino acid residues of the tripeptide region of the P190 antigen was amplified by polymerase chain reaction from genomic DNA of FCC1/HN Plasmodium falciparum isolated from Hainan Province, China. Upon comparison with the nucleotide sequences of MAD20 allele, it was found that there were five bases substitution in the P190TR which cause amino acid changes. The DNA fragment sequenced were ligated to BamHI and XbaI-digested pGEX-2T vector. Competent E. coli JM109 (DE3) were transformed with either parental or recombinant pGEX-2T for expression. Analysis of soluble cellular proteins revealed the high level expression of GST-P190TR as fusion proteins. Affinity purification of the fusion protein under nondenaturing condition resulted in the removal of almost all other E. coli proteins. The purified P190TR protein was highly immunogenic in rabbits. The antibodies against the recombinant protein recognized the malaria parasite with the titers at 1:320 measured by IFA and antisera from malarial patients reacted with the expressed protein in Western Blot.

Amino Acid Sequence↗
PubMed2002

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans.

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.

Bacillus↗