Regulation of competence for genetic transformation in Streptococcus pneumoniae.
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ATP-Binding Cassette Transporters↗
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Results for “DNA Transformation Competence”
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Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells.
The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor exogenous DNA upon Ca2+ treatment. At saturating DNA concentrations the cells were capable to take up irreversibly about 6-10(8) dalton of donor DNA per cell (if to assume that all the cells are competent). About 75% of this DNA was found to be attached to the cytoplasmic membrane. Employing Poisson approximation for distribution of the donor-marker DNA molecules on recipient cells it was found that the efficiency of a marker recombination and expression is about 2-10(-5). The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor 0exogenous
Calcium↗
Genomic clone of hst with transforming activity from a patient with acute leukemia.
We have previously reported the identification of a novel transforming gene, hst, in DNA samples taken from human stomach cancers and a noncancerous portion of stomach. Five clones, containing the genomic hst gene, were isolated from a human cosmid library constructed from leukocyte DNA from a patient with acute leukemia. All clones possessed transforming activity when transfected to NIH3T3 cells. From one clone, an 8.7 kb BamHI fragment was subcloned into pBR322, and this subclone was active in transforming NIH3T3 cells. This is the first isolation of transformation-competent genomic hst clones directly from a human genomic library, that is, without prior passage through NIH3T3 cells.
Acute Disease↗
Control of transformation competence in Azotobacter vinelandii by nitrogen catabolite derepression.
Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.
Ammonia↗
Density-dependent responsiveness to autocrine growth factors of Epstein-Barr virus transformed human B lymphocytes.
Analysis of the growth requirements of Epstein-Barr virus (EBV)-transformed B lymphocytes shows that interleukin 1 and thioredoxin, a disulfide reducing enzyme, are able to induce a marked increase in DNA synthesis in the early phases of in vitro culture. By contrast, interleukin 6 induces a steady increase in DNA synthesis comparable to that observed with crude conditioned supernatant. Furthermore, EBV-transformed B cells exhibit a density-dependent responsiveness to autocrine growth factors, thus suggesting that growth regulation of EBV-transformed B cells might result from the interplay between different self-stimulating soluble factors and from the competence of the cells to respond to autocrine growth factors.
B-Lymphocytes↗
Platelet-derived growth factor-modulated proteins: constitutive synthesis by a transformed cell line.
Platelet-derived growth factor (PDGF) initiates replication of density-arrested BALB/c-3T3 mouse cells by rendering them "competent" to respond to factors contained in plasma. Treatment of quiescent cells with PDGF rapidly stimulates the preferential synthesis of several cytoplasmic proteins (molecular weights 29,000 to 70,000). Four of these proteins were noted within 1.5 hr of PDGF addition and one (pI) within 40 min. Inhibitors of RNA synthesis prevented the synthesis of these proteins. Both the synthesis of pI and the stimulation of DNA synthesis displayed a similar dose response to PDGF concentration. Pituitary fibroblast growth factor, which also induces competence, stimulated pI and pII synthesis. Plasma, epidermal growth factor, or insulin, which do not induce competence, did not stimulate selective synthesis of these proteins. A transformed variant of BALB/c-3T3 cells, which has retained the growth requirement for plasma factors but lost the requirement for PDGF, synthesizes these PDGF-modulated proteins constitutively.
Animals↗
Studies on transformation of Escherichia coli with plasmids.
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
Cations↗
Structural analysis of a Lotus japonicus genome. IV. Sequence features and mapping of seventy-three TAC clones which cover the 7.5 mb regions of the genome.
Using the sequence information of expressed sequences tags (ESTs), cDNAs and genes from Lotus japonicus and other legumes, 73 TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of L. japonicus accession MG-20, and their nucleotide sequences were determined. The length of the DNA sequenced in this study was 7,455,959 bp, and the total length of the DNA regions sequenced so far is 26,167,443 bp together with the nucleotide sequences of 183 TAC clones previously reported. By similarity searches against the sequences in protein and EST databases and prediction by computer programs, a total of 699 potential protein-encoding genes with known or predicted functions, 163 gene segments and 267 pseudogenes were assigned to the newly sequenced regions. Based oil the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was located onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.
Base Sequence↗
Oncogenes of DNA and RNA tumor viruses and the origin of cellular oncogenes.
Oncogenes were first described as the genes of DNA tumor viruses responsible for in vivo and in vitro neoplastic transformation induced by such viruses. The first oncogenes described were the oncogenes of polyoma, SV40 and papilloma viruses. The polyoma virus oncogene has been designated the 'middle' T (tumor) antigen, whereas the SV40 oncogene is designated the 'large' T antigen. The large T of the polyoma virus can also be considered an oncogene, because it is capable of 'immortalizing', but not transforming primary cell cultures. Other oncogenes of DNA tumor viruses are the E1A of adenoviruses and the E7 gene of papilloma viruses. It has been proposed that some of the oncogenes of DNA tumor viruses act by binding the products of tumor suppressor genes, such as the retinoblastoma gene product Rb. In general, the products of the oncogenes of DNA tumor viruses need the cooperation of other oncogenes to establish a completely transformed phenotype. Oncogenes have been discovered in all replication defective RNA tumor viruses, whereas they are not present in the genomes of replication competent RNA tumor viruses, which can also give rise to a neoplastic phenotype, in vivo, but by a different mechanism. The discovery of oncogenes in RNA tumor viruses has led to the concept that these oncogenes are actually present in the genomes of all eucaryotic organisms and have been transduced and, in some cases, modified by a retrovirus. The characterization of the oncogenes of acute transforming retroviruses has led to the present classification of oncogenes, which include genes coding for proteins having different functional properties, but all characterized by having some role in the pathway which regulates cell proliferation. The experimental approach of DNA transfection has led to the discoveries of some oncogenes already known from the genomes of retroviruses and also of some new oncogenes which had never been transduced by retroviruses. The knowledge derived from retrovirus research and from the use of the DNA transfection technique has opened a whole new field of science thereby contributing greatly to our understanding of the mechanisms underlying neoplastic transformation and also to the understanding of normal cell physiology.
Animals↗
Increased therapeutic index of antineoplastic drugs in combination with intracellular histamine antagonists.
L-Histidinol, a protein synthesis inhibitor and structural analogue of L-histidine, has been demonstrated in chemotherapy-treated mice to be cytoprotective to normal stem cells but to enhance cytotoxicity to tumor cells. N,N-Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine.HCl (DPPE) is an antagonist of recently described microsomal and nuclear intracellular histamine receptors implicated in the mediation of proliferation and modulation of prostaglandin synthesis. DPPE is cytotoxic to tumor cells in vitro and cytoprotective to the gut in vivo. Noting the similar pharmacologic profiles for histidinol and DPPE and the structural resemblance between histidinol and histamine, we tested 1) whether binding to intracellular histamine receptors may be important to the action of histidinol, 2) whether there exists a differential effect of DPPE and histidinol on proliferating normal and transformed or malignant cells, and 3) whether DPPE, like histidinol, protects host cells from the effects of chemotherapy while augmenting tumor cell kill in vivo. It was observed that histidinol does compete at intracellular histamine receptors in isolated microsomes and nuclei, but with significantly lower affinity than DPPE. Nevertheless, for each agent, potency at intracellular histamine receptors correlates with potency to inhibit DNA and protein synthesis, without cytotoxicity, in normal mitogen-stimulated murine lymphocytes and to kill transformed mouse lymphocytes or MCF-7 human breast cancer cells. As demonstrated previously for histidinol (1-2 g/kg), DPPE (4 mg/kg) protected murine bone marrow progenitors from doxorubicin or fluorouracil, while doses of 4-50 mg/kg significantly enhanced the antitumor activity of doxorubicin and daunorubicin in murine models of early cancer. One postulate to explain the effects of intracellular histamine receptor ligands is that intracellular histamine mediates DNA and protein synthesis, possibly through a downward modulation of growth-inhibitory prostaglandin levels. Antagonism of the intracellular action of histamine at intracellular histamine receptors by DPPE or histidinol may result in differential perturbations of growth/eicosanoid metabolism in normal and malignant cells, thus forming the basis of a new approach to chemotherapy.
Animals↗
Calcium regulation of growth and differentiation in Streptococcus pneumoniae.
Streptococcus pneumoniae requires 0.15 mM-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mM triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2',4'-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.
Amiloride↗
Interference in SV40 DNA infections: a possible basis for cellular competence.
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Base Sequence↗
[Construction of recombinant plasmid using Neo/E Technology].
A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Bacteriophage lambda↗
A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae.
We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type.
DNA↗
Functional analysis of the Bacillus subtilis y shD gene, a mutS paralogue.
In the course of the Bacillus subtilis genome sequencing project, an ORF called yshD was identified, and its product was classified as a mismatch repair protein. Further analysis of the YshD primary sequence showed that the protein belongs to the MutS2 protein family, sharing a high degree of identity with the Thermootoga Inaritima protein TM1278 (34%) and with the so-called MutS2 protein sl11772 of Synechocystis (32%). The COG1193 family of MutS-like proteins is made up of polypeptides that have been predicted from genomic sequencing data from various prokaryotes, but their biological role has not yet been analysed. The functional study of yshD revealed that the gene is constitutively transcribed during the life cycle of B. subtilis, and in minimal medium expression remains at appreciable levels until very late in stationary phase. Fluctuation tests with yshD knock-out mutants did not indicate any role for the protein in preventing the accumulation of spontaneous forward mutations to RifR, nor was any functional interaction with MutS or MutL suggested in fluctuation experiments with mutants lacking combinations of the three genes. Nevertheless, the mutation spectrum observed in the rpoB gene in the deltayshD strain has some characteristic features. The gene does not seem to be involved in the prevention of interspecific recombination in transformation-competent cells.
Adenosine Triphosphatases↗
Transformation of chromosomal and plasmid characters in Staphylococcus aureus.
Plasmid and probably also chromosomal characters have been genetically transformed in Staphylococcus aureus. Recipient cells show competence throughout the exponential growth phase with a maximum at early times.
Cadmium↗
Genetic transformation in Streptococcus mutans requires a peptide secretion-like apparatus.
Competence for genetic transformation in Streptococcus pneumoniae and Streptococcus gordonii involves the ComAB secretion apparatus, which is thought to export the competence-stimulating peptide. Homologous secretory systems are also used for the export of certain bacteriocins and bacteriocin-like peptides. In this study, a similar secretory apparatus was found in the Streptococcus mutans genome, and its role in transformation was investigated. Gene inactivation resulted in a mutant deficient in transformability. We suggest that secretion of a peptide, possibly the competence-stimulating peptide itself, is involved in competence induction also in S. mutans.
ATP-Binding Cassette Transporters↗
Transformation in pneumococcus: protein content of eclipse complex.
A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.
Bacterial Proteins↗