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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↗
PubMed1993

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Aprotinin↗
PubMed1984

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. II. Glutathione, cysteine and cysteamine.

This paper extends our earlier observations on the effects of the sulphydryl (SH)-containing compound dithiothreitol (DTT) on the radiation response of Bacillus subtilis transforming DNA to three other SH-containing compounds-cysteamine, cysteine and glutathione (GSH). In general, all four compounds protect transforming DNA in a manner which is dependent on gassing conditions. In O2, the protection is consistent with the scavenging of OH radicals by the SH compounds, but in N2 there is additional protection which may be due to hydrogen atom donation from the SH compound to radiation-induced DNA lesions, a process which is blocked by O2. This additional protection in N2 results in an increase in the ratio of inactivation in the absence and presence of oxygen with increasing SH concentration to a maximum followed by a decrease at high SH concentrations. The maximum value of the ratio and the SH concentration at which it occurs depend on the SH compound. In particular, GSH appears to be significantly less efficient in the hydrogen-donation repair reaction with transforming DNA than are the other three SH compounds. Furthermore, on the basis of our results, we postulate the existence of a damage fixation process which occurs in the absence of O2, in competition with damage repair by SH compounds, and that this anoxic damage fixation occurs at a rate not less than 300 s-1. We also demonstrate here that the damage fixing reaction of O2 with transforming DNA radicals proceeds 200-fold faster than the competing repair reaction by hydrogen-donation from DTT.

Bacillus subtilis↗
PubMed1980

Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells.

Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation. Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation. A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles. The results are consistent with the existence of two types of receptor sites on the membrane of competent B. subtilis cells.

Bacillus subtilis↗
PubMed1983

[Restriction-modification systems in Bacillus strains related to Bacillus subtilis].

127 strains of bacilli sensitive to different phages of Bacillus subtilis were isolated from the soil of Moscow and its country-side. In 6 strains, restriction and modification systems were discovered which differed from these previously described for Bac. subtilis BsuR system. Two strains has identical restriction-modification systems, and one strain possessed two different systems. Using DNA from all 6 strains, it was possible to transform competent cells of Bac. subtilis RUB834. Two of these 6 strains could serve as recipients in transformation and transfection experiments.

Bacillus subtilis↗
PubMed1990

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

Animals↗
PubMed1998

Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori.

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.

Conjugation, Genetic↗
PubMed1983

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

Bacillus subtilis↗
PubMed1980

New insights into how bacteria take up DNA during transformation.

Haemophilus influenzae possesses an efficient natural transformation system. Under appropriate growth conditions, every cell in a culture acquires the competence to take up several molecules of DNA. Only donor DNAs from Haemophilus species are taken up; foreign DNAs are excluded. Specific recognition is achieved through the interaction of a receptor protein present on the recipient cell membrane with an 11 base pair sequence present at high frequency in the donor DNA.

Animals↗
PubMed1981

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.

Calcium↗
PubMed1977

Genetic transformation in Methylobacterium organophilum.

Several mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0-5%. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of 100 mug ml-1.

DNA, Bacterial↗
PubMed1988

An enrichment selection for mutants resulting from oligonucleotide-directed mutagenesis of double-stranded DNA.

We report here a simple and rapid procedure for enrichment and selection of mutants from oligonucleotide-directed mutagenesis on double-stranded plasmid DNA. Mutagenic oligonucleotides were designed to insert or delete a unique restriction site with silent codon changes. After mutagenesis, plasmid DNA from all resulting colonies was pooled, restricted with the appropriate endonuclease, and the resulting unique form of DNA (linear or circular) was isolated and used for transformation of competent E. coli. These procedures provided an enrichment of mutant plasmid from the 4% obtained by more conventional techniques to greater than 65%.

Base Sequence↗
PubMed2003

Natural transformation of Campylobacter jejuni requires components of a type II secretion system.

The human pathogen Campylobacter jejuni is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes. However, in C. jejuni little is known about the genes involved in this process. We used random transposon mutagenesis to identify genes that are required for the transformation of this organism. We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane. Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species. Other genes identified in our screen encode proteins unique to C. jejuni or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria.

Amino Acid Sequence↗
PubMed2005

[Development of transformation system of rice based on transformation-competent artificial chromosome (TAC) vector].

The TAC clone (NK15) containing a ca.50 kb DNA insert was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. The NK15 was stable in Agrobacterium tumefaciens strain LBA4404 under kanamycin selection for many generations. The calli of mature embryo of Nongken58S were infected with the Agrobacterium tumefaciens strain LBA4404 carrying NK15. PCR and Southern analyses of transgenic plants indicated that the 50 kb of foreign DNA was transferred into the rice genome, and most of transgenic plants had one copy of the insertion. Genetic and PCR analyses of T1 progeny confirmed that the inserted forgein DNA was stably inherited.

Blotting, Southern↗
PubMed1985

Replicative functions of the SV40(cT)-3 mutant defective for nuclear transport of T antigen.

The SV40(cT)-3 mutant is defective in transport of SV40 large tumor antigen (T-ag) to the nucleus. Several properties of T-ag associated with SV40 lytic infection and attributed to its nuclear localization were examined to determine whether biologically significant levels of the mutant T-ag (cT-ag) that were immunologically undetectable were transported to the nucleus in SV40(cT)-3-infected TC-7 cells. SV40(cT)-3 was defective in regulation of T-ag synthesis and initiation of viral DNA synthesis. These defects were presumably due to the lack of nuclear transport of cT-ag, since cT-ag was capable of interacting with the SV40 origin of viral DNA synthesis in a solution binding assay. The level of fatty acid acylation, a modification specific for the cell surface associated T-ag, was not affected by the cT mutation. The cT mutation sufficiently suppressed the nuclear transport of wild-type (WT) T-ag in SV40(cT)-3-infected COS-1 cells to result in the cessation of WT-T-ag-stimulated SV40(cT)-3 viral DNA synthesis. These results are discussed with respect to the recent findings that SV40(cT)-3 is fully competent for the transformation of established cell lines and the induction of cellular DNA synthesis in quiescent cells.

Animals↗
PubMed1982

Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA.

This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.

Base Sequence↗
PubMed1983

Genetic transformation of Streptococcus pneumoniae by DNA cloned into the single-stranded bacteriophage f1.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.

Cloning, Molecular↗