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PubMed2001

Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis.

Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.

Bacillus subtilis↗
PubMed1978

Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites.

We have analyzed the DNA from 15 clones of avian sarcoma virus (ASV)-transformed rat cells with restriction endonucleases and molecular hybridization techniques to determine the location and structure of proviral DNA. All twenty units of proviral DNA identified in these 15 clones appear to be inserted at different sites in host DNA. In each of the ten cases that could be sufficiently well mapped, entirely different regions of cellular DNA were involved. Thus ASV DNA can be accommodated at many positions in cellular DNA, but the existence of preferred sites has not been excluded. Six of the 15 clones carry only one normal provirus, two contain two normal proviruses, and seven harbor either one or two proviruses that appear anomalous in physical mapping tests. Both ends of at least 18 proviruses, however, were found to contain sequences specific to both the 3' and 5' termini of viral RNA. The organization of these terminally redundant sequences appeared identical to that of the 300 base pair (bp) repeats found at the ends of unintegrated linear DNA (Shank et al., 1978). Proviral DNA is therefore co-extensive, or nearly co-extensive, with unintegrated linear DNA and has a structure we denote as CELL DNA-3'5'----------3'5'-CELL DNA. Three of the four anomalous proviruses which were fully analyzed were deletion mutants lacking 25--65% of the genetic content of ASV; the fourth provirus had a novel site for cleavage by Eco RI but was otherwise normal. Tests for the biological competence of proviral DNA, based upon rescue of transforming virus after fusion with chicken cells, were generally consistent with the physical mapping studies.

Avian Sarcoma Viruses↗
PubMed2003

Natural genetic transformation in monoculture Acinetobacter sp. strain BD413 biofilms.

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.

Acinetobacter↗
PubMed2004

Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation.

This study aimed to evaluate the possibility of DNA sequences from genetically modified plants to persist in the gastrointestinal (GI) tract. PCR analysis and transformation assays were used to study DNA persistence and integrity in various ex vivo and in vivo systems using gnotobiotic rats. DNA studied was either plasmid DNA, naked plant DNA or plant DNA embedded in maize flour. Ex vivo experiments performed by incubating plant DNA in intestinal samples, showed that DNA is rapidly degraded in the upper part of the GI tract whereas degradation is less severe in the lower part. In contrast, plasmid DNA could be recovered throughout the GI tract when intestinal samples were taken up to 5 h after feeding rats with plasmid. Furthermore, DNA isolated from these intestinal samples was able to transform electro-competent Escherichia coli, showing that the plasmid was still biologically active. The results indicate that ingested DNA may persist in the GI tract and consequently may be present for uptake by intestinal bacteria.

Animals↗
PubMed1993

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Viral↗
PubMed1996

[Sequence analyses of Epstein-Barr virus (EBV) Bam HI F DNA fragment in EBV associated diseases].

In order to compare nucleotide differences of Epstein-Barr virus (EBV) Bam HI F DNA fragment from various EBV associated diseases, polymerase chain reactions (PCR) were used to amplify a subfragment (nucleotides 55,381-56,020) of the F fragment from different tissue DNAs, including 20 NPCs, 2 B-cell lymphomas, 2 T-cell lymphomas, 3 infectious mononucleosis (IM), and 3 normal controls. DNA sequences were determined by PCR direct sequencing or sequencing after DNA cloning. The PCR products were cloned into pGEM-3Z vector, then the resulting recombinant plasmids were used to transform DH5 alpha competent cells. Plasmid DNAs from the correct transformants were prepared for DNA sequencing. The results showed that the proportion of the f variant in NPCs, B-cell lymphomas, T-cell lymphomas, IMs, and normals were 40%, 0%, 0%, 33%, and 33%, respectively. Because the f variant was not specifically more prevalent in NPC tissues compared to the non-tumor tissues, we speculate that there is no strong association between the f variant and NPC. These results were different from other reports. Coinfection of the F strain and the f variant was found both in some NPC patients and normal individuals. Analyses of Bam HI F subfragments of 35 EBV isolates from the 30 tissue DNAs revealed that there were changes at four corresponding positions of the B95-8 strain. They were nucleotide T at 55,473 replaced by G, an insertion of TGT after nucleotide 55,543, nucleotide A at 55,564 replaced by G, and nucleotide T at 55,958 replaced by C. These 4 nucleotide changes may confer a character of Taiwan strains. The nucleotides of the F strain at coordinates 55,519, 55,596, 55,680, 55,703, and 55,895 were T, T, A, A, and C, and those for the f variant were C, C, C, C, and T. These two patterns were not correlated with types A and B of EBV.

Base Sequence↗
PubMed1988

Differential effects of DNA gyrase inhibitors on the genetic transformation of Neisseria gonorrhoeae.

Inhibitors of DNA gyrase in Escherichia coli exerted differential effects on the genetic transformation of Neisseria gonorrhoeae. When competent cells of the gonococcus were exposed to novobiocin before the uptake of transforming antibiotic resistance DNA, there was a 50 to 60% reduction in the number of transformants compared with the number of control untreated cells. Norfloxacin, a more potent inhibitor of DNA gyrase and an analog of nalidixic acid, nearly abolished the production of transformants by recipient cells. On the contrary, exposure of competent cells to nalidixic acid had no effect on transformant yield. The target of these inhibitors appears to be at the level of recombination. Possible mechanisms are discussed.

Nalidixic Acid↗
PubMed1980

Stimulation of Bacillus subtilis transformation by spermidine.

Addition of spermidine in millimolar concentrations to Bacillus subtilis cells curing competence development increases transformability. The spermidine must be added at least 30 min before DNA for maximum stimulation. An incubation period of about 30 minutes is also required for the maximum uptake of labeled spermidine. The amount of DNA initially attached and the rate of DNA uptake are increased to the same extent as transformation. The rate of protein synthesis is also equivalently increased. These observations are consistent with an increase in the number of competent cells in the cell population; this increase is mediated by a spermidine-stimulated protein synthesis.

Bacillus subtilis↗
PubMed2005

[Cloning, expression and purification of His-tagged mouse mitochondrial transcription factor A and preparation of its polyclonal antibody].

OBJECTIVE: To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody. METHODS: The total RNA was extracted from mouse liver cells, and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was then cloned into the prokaryotic expression vector pET14b. After enzyme digestion and DNA sequencing, the plasmid was transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of His-tagged mtTFA. The expressed fusion protein was purified with Ni(2+)-NTA His-bind resin and quantified with Bradford method. RESULTS: The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing, and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG. The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained. CONCLUSIONS: The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained, which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.

Animals↗
PubMed1987

Combined genetic transformation and nutritional assay for identification of Moraxella nonliquefaciens.

A combined genetic transformation and nutritional assay is described that permits definitive identification of clinically isolated strains of Moraxella nonliquefaciens. Crude DNA preparations of strains of various Moraxella species were used to transform nutritional mutants of a stably competent strain of M. nonliquefaciens for ability to grow on a defined medium (Mn-B). DNA samples from 24 independently isolated strains of M. nonliquefaciens all resulted in massive (4+) transformation of each of two mutant assay strains. DNA samples from strains of M. bovis and M. lacunata frequently gave weak (1+) transformation of one of the mutant assay strains (Mn64) but almost always failed to transform another assay strain (Mn136). DNA samples from eight other Moraxella species failed completely to transform either of the mutant assay strains. When streaked on the defined medium used for the transformation assay (Mn-B), 23 of the 24 strains of M. nonliquefaciens grew well, but all strains of M. bovis and M. lacunata failed to grow on this medium.

Culture Media↗
PubMed1978

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

Bacteriophages↗
PubMed1980

Lack of SOS repair in Streptococcus pneumoniae.

Wild-type strains of Streptococcus pneumoniae were non-mutable by UV radiation and thymidine starvation. Moreover, UV-irradiated pneumococcal omega 2 phages were not reactivated in an irradiated host. This suggests that, in pneumoococcus, there is no efficient inducible repair process similar to the SOS repair described in detail for E. coli. We also report that mutations cannot be induced by a process thought to be linked to competence during transformation with isogenic wild-type DNA either on wild-type strains or in strains in which the hex function of excision and repair of mismatched bases is inactive.

Bacteriophages↗
PubMed1994

Nuclear assembly is independent of linker histones.

The role of linker histones in the assembly of functional nuclei was examined with the use of a cell-free extract of Xenopus eggs that transforms condensed sperm chromatin into DNA-replication-competent pronuclei. When linker histones were removed from the extract, the resultant pronuclei were indistinguishable from those formed in the complete extract. The assembly of functional nuclear membrane, nuclear lamina, and prereplication centers allowed identical DNA replication efficiencies. Thus, linker histones are not required for the assembly of morphologically normal nuclei capable of DNA replication.

Animals↗
PubMed1972

Repair of pyrimidine dimer damage induced in yeast by ultraviolet light.

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.

Animals↗
PubMed2006

Conjugative transfer of chromosomally encoded antibiotic resistance from Helicobacter pylori to Campylobacter jejuni.

Many strains of Helicobacter pylori are naturally competent for transformation and able to transfer chromosomal DNA among different isolates using a conjugation-like mechanism. In this study, we sought to determine whether H. pylori can transfer DNA into Campylobacter jejuni, a closely related species of the Campylobacterales group. To monitor the transfer, a chromosomally encoded streptomycin resistance cassette prearranged by a specific mutation in the rpsL gene of H. pylori was used. Mating of the bacteria on plates or in liquid broth medium produced C. jejuni progeny containing the streptomycin marker. DNA transfer was unidirectional, from H. pylori to C. jejuni, and the progeny were genetically identical to C. jejuni recipient strains. DNase I treatment reduced but did not eliminate transfer, and DNase I-treated cell supernatants did not transform, ruling out phage transduction. Recombinants also did not occur when the mating bacteria were separated by a membrane, suggesting that DNA transfer requires cell-to-cell contact. Transfer of the streptomycin marker was independent of the H. pylori comB transformation system, the cag pathogenicity island, and another type IV secretion system called tfs3. These findings indicated that a DNase I-resistant, conjugation-like mechanism may contribute to horizontal DNA transfer between different members of the Campylobacteriales group. The significance of this DNA uptake by C. jejuni in the context of acquiring antibiotic resistance is discussed.

Anti-Bacterial Agents↗
PubMed1984

Repair of DNA-protein cross-links in an excision repair-deficient human cell line and its simian virus 40-transformed derivative.

DNA-protein cross-links are induced in mammalian cells by X-rays, ultraviolet light, fluorescent light, and numerous chemical carcinogens. Others have shown that these cross-links are repaired by normal cells but that excision repair-deficient xeroderma pigmentosum (XP) Group A cells, XP12BE, are deficient in repair of these bulky adducts. This paper compares the DNA-protein cross-link repair competency of another XP Group A strain, XP20S, with its more rapidly proliferating simian virus 40-transformed derivative line and with normal human skin fibroblasts. DNA-protein cross-links were induced with 20 microM transplatinum(II)diamminedichloride and assayed by the membrane alkaline elution procedure of Kohn. Treated and untreated cells are lysed on a polycarbonate membrane filter, and the coelution rates of the DNA at pH 12.2 are compared; DNA-protein cross-links retard elution of DNA. The repair competency of XP20S cells for trans-platinum(II)diamminedichloride-induced DNA-protein cross-links was similar to that of XP12BE cells, but the competency of the simian virus 40-transformed XP20S cells was nearly equal to that of normal human skin fibroblasts. These results suggest that either cell cycling compensates for the genetic deficiency present in the nucleotide excision process of XP Group A cells or that a process other than nucleotide excision can repair these lesions; this process requires cell cycling or activation by the virus.

Cell Line↗
PubMed1990

Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids.

A shuttle plasmid for Acinetobacter calcoaceticus and Escherichia coli has been constructed from a cryptic A. calcoaceticus lwoffi plasmid and pBR322. It is transformed to A. calcoaceticus BD413 by natural competency, yielding about 10(6) transformants per microgram of plasmid DNA. The ApR and TcR genes of pBR322 are functional in A. calcoaceticus. A gene bank was constructed from chromosomal A. calcoaceticus DNA and the shuttle plasmid. Direct transformation to A. calcoaceticus yielded about 95% recombinants, indicating a sixfold enrichment of recombinant plasmids compared to E. coli. One clone complementing a trpE mutation carried a 20-kb insertion and transformed with a 30-fold higher efficiency when compared to the vector. A deletion analysis of the shuttle plasmid indicates that 2.2 kb is necessary for autonomous replication and stable maintenance in A. calcoaceticus. No rearrangements of the DNA or loss of plasmids are found in that organism, even in the absence of selective pressure, when this sequence is present. A further insertional inactivation analysis creating lacZ transcriptional fusions suggests that the origin of replication (ori) is contained within about 1350 bp. Analysis of beta-galactosidase production in A. calcoaceticus indicates that only a weak promoter activity is directed out of one end of this ori. Its sequence contains A + T-rich regions, an 18-bp element with nearly perfect palindromic symmetry and eleven repeats of the consensus sequence, AAAAAATAT, eight of which are clustered within 360 bp. However, no open reading frames or significant homologies to other ori were found.

Acinetobacter↗
PubMed2004

Exploring permeability of Escherichia coli competence using quantum dots as fluorescent probes.

Though people had recognized the pivotal function of CaCl(2) during DNA transformation into Escherichia coli, the mechanism of divalent Ca(2+) cation inducing E. coli competence development is still unknowable. Quantum dots (QDs), as a new fluorescent probe, being applied in biology research, had aroused great interest. We explored the penetrability of E. coli competent cells membrane using QDs and proved directly that competent cells were more permeable than that of noncompetent. The results are significant on understanding the problems of the microbiological genetics.

Calcium↗