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PubMed1984

Recombination efficiency is a quadratic function of the length of homology during plasmid transformation of Bacillus subtilis protoplasts and Escherichia coli competent cells.

Recombination efficiency between transforming and resident plasmids was 100 times higher in Bacillus subtilis protoplasts than in Escherichia coli competent cells. In both systems it varied with the square of the length of the sequences common to two plasmids within the studied range 0.96-8.8 kb in B. subtilis, 0.72-3.9 kb in E. coli. The observed exponential dependence may be characteristic of recombination occurring within a relatively short interval bordered by relatively long heterologous regions.

Bacillus subtilis↗

PubMed2004

ComX activity of Streptococcus mutans growing in biofilms.

In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene. In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing. The quorum sensing system responsible for genetic competence induction in S. mutans has been linked to biofilm formation and the acid tolerance response. To examine the response of comX to CSP in S. mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S. mutans UA159 comC-, which is unable to produce endogenous CSP. CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal beta-gal activity 5 and 10 min after CSP addition. The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h. To determine the temporal pattern of transformation frequency, pMA2, a Spr shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h. Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population. These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms.

Adaptation, Physiological↗

PubMed1993

Mitogenic and receptor activities of human growth hormone 108-129.

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.

3T3 Cells↗

PubMed1999

The antiapoptotic decoy receptor TRID/TRAIL-R3 is a p53-regulated DNA damage-inducible gene that is overexpressed in primary tumors of the gastrointestinal tract.

Both DR4 and DR5 have recently been identified as membrane death receptors that are activated by their ligand TRAIL to engage the intracellular apoptotic machinery. TRID (also named as TRAIL-R3) is an antagonist decoy receptor and lacks the cytoplasmic death domain. TRID protects from TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL. TRID has been shown to be overexpressed in normal human tissues but not in malignantly transformed cell lines. DR5 is a p53-regulated gene and we have recently reported that DR5 expression is induced in response to genotoxic stress in both a p53-dependent and independent manner (Sheikh et al., 1998). In the current study, we demonstrate that TRID gene expression is also induced by the genotoxic agents ionizing radiation and methyl methanesulfonate (MMS) in predominantly p53 wild-type cells, whereas UV-irradiation does not induce TRID gene expression. Consistent with these results, exogenous wild-type p53 also upregulates the expression of endogenous TRID in p53-null cells. Thus, TRID appears to be a p53 target gene that is regulated by genotoxic stress in a p53-dependent manner. Using primary gastrointestinal tract (GIT) tumors and their matching normal tissue, we also demonstrate for the first time that TRID expression is enhanced in primary tumors of the GIT. It is, therefore, possible that TRID overexpressing GIT tumors may gain a selective growth advantage by escaping from TRAIL-induced apoptosis.

Adenocarcinoma↗

PubMed2002

Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes.

We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.

Bacillus↗

PubMed1978

[Competence in Escherichia coli cells. V. Interaction of the competence factor with the cells].

Some trends of the interaction of the competence factor with the cells are described on the model of phage lambda transfection. It is shown that this process depends on the composition of the recipients growth media, the temperature regime in the interaction processand on the concentration of the competence factor and the recipient cells. The preliminary treatment of the recipient by EDTA (0.0001 M), CaCl2 (less than 0.02 M), MgCl2 (less than 0.01 M), and NaCl (less than 0.01 M) resulted in the increaze of their sensitivity to subsequent treatment by the factor. Data are obtained demonstrating that after the treatment of cells by the competence factor their sensitivity to decreased CaCl2 concentration is increasing.

Calcium Chloride↗

PubMed1996

Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone.

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Havarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.

Alleles↗

PubMed1992

Sharp anterior boundary of homeotic gene expression conferred by the fushi tarazu protein.

Parasegmental boundaries in the Drosophila embryo are delimited by the products of the fushi tarazu (ftz) and even-skipped (eve) genes. We show here that these act through particular key control regions of the homeotic gene Ultrabithorax (Ubx) to generate ftz- or eve-like stripe patterns of beta-galactosidase expression. Footprint analysis and tests in transformed embryos of constructs bearing mutated footprint regions suggest that ftz protein acts directly as a transcriptional activator of Ubx. Its activity outside the Ubx expression domain is suppressed by hunchback (hb), a repressor of Ubx. Some DNA binding sites for ftz protein are adjacent to, others overlap binding sites for hb protein, and we provide evidence that ftz protein competes with hb protein for DNA binding and/or for transcriptional activation. This competition mechanism results in a sharp anterior expression boundary. Direct activation of homeotic gene control regions by ftz (or eve) protein may be a regulatory step which is generally used to align expression of homeotic genes with parasegmental boundaries.

Animals↗

PubMed2006

Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.

A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveria bassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at -76 degrees C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.

Aminobutyrates↗

PubMed1991

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.

Animals↗

PubMed1989

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes.

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.

Bacillus subtilis↗

PubMedDate not supplied

[Anti-idiotypic and natural catalytically active antibodies].

The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.

Antibodies, Anti-Idiotypic↗

PubMed2002

Nuclear exclusion of Smad2 is a mechanism leading to loss of competence.

Controlling the duration of a signalling process in development by loss of competence is important because too strong an induction can change cell fate. To understand some of the mechanisms that underlie loss of competence, we have analysed the transduction of transforming growth factor-beta (TGF-beta) signalling during mesoderm formation, which is thought to be induced by TGF-beta-like signalling, in embryos of the frog Xenopus laevis. Here we show that gastrula ectoderm has the ability to express mesodermal marker genes in response to the TGF-beta signalling molecule activin for many hours, but then loses this ability within 1 h for all mesodermal genes tested. This loss of mesodermal competence correlates with the inability of Smad2, the principal intracellular signal transducer of activin, to accumulate in the nucleus. Mutating three phosphorylation sites within Smad2 abrogates the temporal restriction of Smad2 to accumulate in the nucleus. Overexpression of this mutant form of Smad2 can prolong the competence of endogenous mesodermal genes to respond to activin signalling. Thus, restricting the subcellular localization of an intracellular signal transducer constitutes a mechanism that leads to loss of mesodermal competence. This mechanism operates within less than an hour, and is therefore well suited to control an orderly sequence of inductions.

Active Transport, Cell Nucleus↗

PubMed2005

Experimental methods for assaying natural transformation and inferring horizontal gene transfer.

The observation of frequent lateral acquisitions of genes in sequenced bacterial genomes has spurred experimental investigations to elucidate the factors governing ongoing gene transfer processes in bacteria. The uptake of naked DNA by natural transformation is known to occur in a wide range of bacterial species and in some archaea. We describe a series of protocols designed to dissect the natural genetic transformability of individual bacterial strains under conditions that progress from standard in vitro conditions to purely in situ, or natural, conditions. One of the most important factors in ensuring the success of any transformation assay system is the use of a sensitive, effective, and distinguishable selection regimen. Detailed template protocols for assaying bacterial transformation in vitro are presented using the naturally competent bacterium Acinetobacter baylyi strain BD413 as a model. Factors increasing the complexity of the assay systems are included in the following section describing the incorporation of components of natural systems to the in vitro models, such as in soil and water microcosm experiments. We then present template protocols for the transformation of bacteria in modified natural systems, such as in the presence of host tissues and extracts or in the greenhouse. Clear and ecologically meaningful demonstrations of in situ natural transformation are most desirable but are also the most complex and challenging. Because of the highly variable nature of these experiments, we include a discussion of important factors that should be considered when designing such experiments. Some advantages and disadvantages of the experimental systems with regard to resolving the hypotheses tested are included in each section.

Acinetobacter↗

PubMed1987

Regulation of growth and differentiation of human keratinocytes by type beta transforming growth factor and epidermal growth factor.

The role of type beta transforming growth factor (TGF beta) and epidermal growth factor (EGF) as regulators of the growth and differentiation of cultured human neonatal epidermal cells and squamous carcinoma cells was investigated in postconfluent cultures. Neither cell proliferation nor DNA synthesis was affected by treatment with TGF beta alone; however, EGF significantly stimulated cell growth, and this process was specifically antagonized by TGF beta. In addition, TGF beta inhibited the maturation of human foreskin-derived epidermal cells, as measured by their competence to synthesize involucrin and to form cornified cell envelopes, in a dose-dependent manner. Although treatment with EGF did not affect the maturation of human foreskin-derived epidermal cells, the combination of a low concentration of TGF beta with EGF resulted in significant enhancement of the maturation of these normal keratinocytes. Growth of three of four squamous carcinomas in the presence of EGF was not inhibited by TGF beta. In addition, all four carcinomas were either totally or partially resistant to the induction of maturation by the combination of TGF beta and EGF. This resistance of squamous carcinomas to TGF beta was paralleled by an increased sensitivity to the antikeratinizing effects of EGF. Thus, TGF beta inhibited the mitogenic stimulation of keratinocytes by EGF and induces cell maturation.

Cell Differentiation↗

PubMed2003

Transforming growth factor alpha binds to Trypanosoma cruzi amastigotes to induce signaling and cellular proliferation.

Macrophages secrete transforming growth factor alpha (TGF-alpha) to trigger proliferation of cancer cells. Here, we report a new role for TGF-alpha in modulating the direct cellular proliferation of a parasitic protozoan, Trypanosoma cruzi. Amastigotes present two classes of receptors for TGF-alpha with different binding affinities. (125)I-TGF-alpha binding was competed by an excess of cold epidermal growth factor and TGF-alpha but not by an irrelevant molecule. Upon binding of TGF-alpha to amastigotes, the ligand is internalized, inducing trypanosome tyrosine phosphorylation of 90- and 87-kDa proteins and increasing DNA synthesis and proliferation of amastigotes. Furthermore, exposure of macrophages to TGF-alpha induced increased amastigote proliferation. These results describe a novel mechanism used by amastigotes to regulate their proliferation mediated by a TGF-alpha-dependent signal transduction pathway.

Animals↗

PubMed1985

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.

Antigens, Polyomavirus Transforming↗

PubMed1996

N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N'-diacetic acid as a new iron chelator with potential medicinal applications against oxidative stress.

N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N,-diacetic acid dihydrochloride (OR10141) is a member of a recently described series of "oxidative stress activatable iron chelators." These chelators have a relatively low affinity for iron but can be site-specifically oxidized, in situations mimicking oxidative stress in vitro, into species with strong iron-binding capacity. It is hoped that this local activation process will minimise toxicity compared to strong iron chelators that may interfere with iron metabolism. The present paper describes the results of experiments aimed at characterising oxidative reactions between iron-OR10141 complexes and hydrogen peroxide. Incubation of ascorbate and hydrogen peroxide with the ferric chelate of OR10141 in neutral aqueous solution yields a purple solution with a chromophore at 560 nm, which is consistent with an o-hydroxylation of one of the trimethoxybenzyl rings. Oxidation of OR10141 also takes place, although more slowly, by incubating hydrogen peroxide with ferric OR10141 complex in the absence of reductant. HPLC analysis shows that OR10141 is consumed during the reaction and transformed principally into N-(2-hydroxy 3,4,5-trimethoxybenzyl) N'-(3,4,5-trimethoxybenzyl) ethylenediamine N,N'-diacetic acid. Minor products are also formed, some of which were identified by mass spectrometry. The protective effect of OR10141 in vitro against DNA single strand breaks, protein damage, and lipid peroxidation induced by Fenton chemistry suggests that this compound is able to compete for iron with biological molecules and, thus, that this strategy of protection against oxidative stress is feasible. In addition, preliminary results showing protective effects of OR10141 dimethyl ester against toxicity induced by hydrogen peroxide in cell culture are described. It is concluded that OR10141 and related prodrugs might be useful in vivo in chronic situations involving oxidative stress.

Ascorbic Acid↗