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At least 703 records · Page 39Linked to original sources
PubMed1998

A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake.

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.

DNA Transposable Elements↗
PubMed1969

Heat-sensitive step in deoxyribonucleic acid-mediated transformation of Bacillus subtilis.

Over 90% of the competent cells in a population of Bacillus subtilis lost their competence after being heated to 50 C for 5 min. There was only a slight loss in the number of transformants if the culture was heated for 5 min after the termination of transformation, but 90% of the transformants were lost after 1 hr at 50 C. The population as a whole grew at a slightly faster rate at 50 C than at 32 C. We postulate that a heat-labile factor is required for the uptake or retention (or both) of deoxyribonucleic acid (DNA) in the cell, since uptake of (32)P-DNA into a deoxyribonuclease-resistant form was inversely proportional to the time of exposure to heat. Cells that had lost competence after being heated did not regain their competence for at least several hours, although other cells in the population became competent. These data suggest that the heat-labile factor required for competence is synthesized only once during the period that a cell remains competent.

Bacillus subtilis↗
PubMed2002

[Inducible expression of non-structural protein 3 of hepatitis C virus in E. coli].

BACKGROUND: To express recombinant non-structural protein 3 of hepatitis C virus (HCV) in E. coli. METHODS: The non-structural 3 (NS3) region DNA fragment of HCV was amplified by polymerase chain reaction (PCR) and inserted into inducible proeukaryotic expressive vector pET 30C(+)at Bam H1/EcoR1 sites. The competent BL21 (DE3) E.coli was transformed, and then cultured and induced with IPTG. The expressed HCV NS3 protein was confirmed with ELISA and dot blot hybridization using HCV NS3-specific single chain Fv (ScFv) antibody. RESULTS: 1 893 bp DNA fragment of HCV NS3 coding region was amplified by PCR technique. HCV NS3 expressive vector pET-NS3 was constructed. After transformation with pET-NS3 and induction with IPTG, recombinant HCV NS3 protein was expressed and confirmed by specific ELISA and dot blot hybridization. CONCLUSIONS: The recombinant HCV NS3 can be expressed in E. coli.

Escherichia coli↗
PubMed1996

Who's competent and when: regulation of natural genetic competence in bacteria.

Natural genetic competence, the ability of cells to bind to and to take up exogenous DNA, is widespread among bacteria and might be an important mechanism for the horizontal transfer of genes. Competent cells express specialized proteins that assemble into a DNA-uptake complex. In many organisms, the development of competence and expression of the uptake machinery is regulated in response to cell-cell signaling and/or nutritional conditions. Exciting new progress has been made in characterizing the signals and pathways that regulate the development of competence.

Bacteria↗
PubMed1978

DNA synthesis in competent Bacillus subtilis cells.

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells.

Bacillus subtilis↗
PubMed1999

The effect of 17beta-estradiol-DNA adducts on the replication of exon # 5 of the human suppressor gene p53.

Using a PCR technique, exon # 5 of the human tumor suppressor gene p53 was amplified and ligated into the pCRII vector and transformed into Escherichia coli INV alphaF' competent cells. The cloned exon # 5 was 184 bp long. Evidence is presented to show that after dimethyldioxirane epoxidation, 17beta-estradiol was able to form 17beta-estradiol-DNA adducts and to strongly inhibit the replication of the cloned exon # 5 producing smaller sizes of DNA fragments and introducing errors of incorporation at the 3'-end of the terminating DNAs. The errors occurred mainly at the clusters of the complementary 'G' and 'A' bases on the template strand DNA, presumably, the major sites where the 17beta-estradiol-DNA adducts were formed.

Base Sequence↗
PubMed1983

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein.

In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.

Bacillus subtilis↗
PubMed2001

An integrated map of Arabidopsis thaliana for functional analysis of its genome sequence.

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.

Arabidopsis↗
PubMed1989

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media.

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at -85 degrees C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 x 10 transformants per mug of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.

Journal Article↗
PubMed2005

[Screening ubiquitin/ribosomal protein S27a by yeast two-hybridization system].

BACKGROUND & OBJECTIVE: Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession. Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth. In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library. METHODS: The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR). Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I. The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments. Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcript method was used to construct a cDNA library in yeast cells. Library plasmid was named AD plasmid. BD plasmid and AD plasmid were co-transformed into competent yeast AH109. Yeast two-hybridization was used to screen positive clones. RESULTS: Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened. DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%). CONCLUSION: Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells.

Base Sequence↗
PubMed2001

Natural genetic transformation of Streptococcus mutans growing in biofilms.

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.

Amino Acid Sequence↗
PubMed1988

Designed gene amplification on the Bacillus subtilis chromosome.

We previously reported the cloning of a 1.6 kb HindIII fragment (containing the junction of the repeating unit) from chromosomal DNA of Bacillus subtilis strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the B. subtilis chromosome, a gene amplification of the 22 kb repeating unit containing the alpha-amylase structural gene (amyE), the tunicamycin-resistance gene (tmrB) and the shikimate kinase structural gene (aroI). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a B. subtilis wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tmr) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tmr transformants showed high productivity of alpha-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10-20. This system may provide an effective means of amplifying long (greater than 20 kb) DNA regions on the chromosome.

Amylases↗
PubMed1989

Ha-Ras-1 oncogene dosage differentially affects Balb/3T3 cells' growth factor requirement and tumorigenicity.

The effect of EJ-ras oncogene dosage on the phenotype of Balb/3T3 transfectants was analyzed with respect to: a) peptide growth factors' requirement; b) relaxation of cell cycle control; c) tumorigenic potential. Mouse embryo-derived Balb/3T3 cells were transfected with the mutated form of the human c-Ha-ras-1 (EJ-ras) along with a genetic marker (neo gene). Transfectants displaying high EJ-ras expression presented a relaxed cell cycle control, required only insulin to initiate DNA synthesis and were highly tumorigenic. On the other hand, low expression EJ-ras transfectants required both competence (FGF) and progression factors (EGF and insulin) exactly like the parental cells. But, upon serial cultivation, these transfectants became fully transformed and highly tumorigenic without EJ-ras amplification and/or overexpression. Therefore, low EJ-ras expression primes the cells to become tumorigenic but neither overrides the cells' requirement for competence growth factor nor deregulates the cell cycle.

Animals↗
PubMed1984

Evidence for a transforming growth factor(s) in the serum-free conditioned medium of SV40-transformed 3T3 fibroblasts.

Unlike untransformed 3T3 fibroblasts, Simian Virus 40-transformed 3T3 cells (SV3T3) do not require the presence of exogenous polypeptide mitogens in order to become competent for the initiation of DNA synthesis. As an explanation for this state of perpetual competence, growth activity for 3T3 cells was found in the serum-free conditioned medium of SV3T3 cells. This activity, which is abolished by incubation with pepsin, but withstands to a fair degree denaturants (8 M urea and 4 M guanidinium HCl) and a reducing agent (65 mM dithioerythritol), enhances the growth of Swiss and BALB/c 3T3 cells in 1% (v/v) calf serum medium and ensures the complete survival of Swiss 3T3 cells for several days in the total absence of serum. The growth activity appears to be derived from the SV3T3 cells, since it is not found in the non-conditioned medium (and the insulin and transferrin components of this serum-free medium have only weak growth activity alone) and the chances of it being due to residual serum growth factors are slight because of the method of growing the cells and the collection of the conditioned medium. Moreover, in a separate experiment to test the possible involvement of cell attachment factors, rat fibronectin was found not to affect 3T3 growth in the standard growth assay. The conditioned medium growth activity apparently has the biological capabilities of transforming growth factors. After concentration by ammonium sulfate precipitation, it can stimulate DNA synthesis in confluent, quiescent Swiss 3T3 cells, in both the absence and presence of 10% depleted calf serum. It also permits the growth of large colonies of Swiss 3T3 cells in soft agar. As part of a preliminary characterization of the conditioned medium growth activity, gel filtration in 0.15 M NaCl, 0.001 M HCl resolves the activity into high (mol. wt. greater than or equal to 100,000) and low (mol. wt. 5,000-6,000) molecular weight factors. Since re-chromatography of the high molecular weight factor, which may still be in an aggregated form, does not generate the smaller, the latter is not a dissociation product of the former. The precise relationship of these SV3T3-conditioned medium growth factors to other growth factors, including known transforming growth factors, has not yet been determined.

Animals↗
PubMed1970

Defined nongrowth media for stage II development of competence in Haemophilus influenzae.

The composition of a defined nongrowth medium used in stage II development of competence of Haemophilus influenzae affects the course of this development. The development of competence in two nongrowth media, M-IV and M-V, is rapid, logarithmic, and independent of the cell concentration. This last property indicates that there is probably no transfer of a competence factor from competent to noncompetent cells, in contrast to results reported for other organisms. Levels of competence reached in these completely defined media are such that 1 to 5% of the cells are transformed in the presence of an excess of marked deoxyribonucleic acid. The method of evaluating competence, which depends on the frequency of multiple independent transformations, has been reexamined. This and other methods are compared on samples taken from a culture during development of competence.

Culture Media↗
PubMed1979

Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer.

Entrapment of pBR322 DNA within liposomes was demonstrated by (i) its comigration with liposomes on Sepharose 4B columns, (ii) resistance of its biological activity to DNase digestion, and (iii) identification of plasmid DNA on agarose gels after lipid extraction. The biological activity of the liposome-entrapped plasmid was determined by transformation assays. The incubation of intact liposomes, containing entrapped pBR322, with competent Escherichia coli cells in the standard transformation mixture resulted in the appearance of tetracycline-resistant colonies at a frequency of 1% of the control frequency. Importantly, this frequency was unaffected by the addition of DNase to the incubation mixture, whereas transformation by free pBR322 DNA was totally eliminated after treatment with DNase.

Cytoplasm↗