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PubMed2006

[Cloning human heat shock protein 90beta-cDNA and constructing its eukaryon vector].

The purpose of this study was to clone human HSP90beta cDNA and construct its eukaryote expression vector . The total RNA was isolated by TRIzol Reagent (Invitrogen) from human NPC and its cDNA was gained by RT-PCR. The purified RT-PCR products and PGEM-T Easy Vector were ligated and transformed into XL1-blue E. coli bacteria. The white clones were selected and the plasmid was purified, which was further identified by double enzyme digestion and sequenced. PGEM-hHSP90beta and pcDNA3.1(+) DNA were digested by AflII and Xbal respectively. After purification, the two fragments obtained were ligased by using T4 DNA ligase (Fermentas). This recombinant DNA was then transformed into E. coli Competent Cells XL1-blue and positive clones were selected on the LB agarose plate containing Ampr (100 microg/ml). Single clones were identified by double digestion with AflII and Xbal, and two fragments with the size 5.4 kb and 2.1 kb were produced as expected. The hHSP90beta gene was successfully inserted into the eukaryote expression vector pcDNA3.1(+) by the recombination technique in vitro.

Cloning, Molecular↗

PubMed2003

[Construction of eukaryote expression vector carrying human soluble interleukin-1 receptor gene].

OBJECTIVE: The purpose of this study was to construct a eukaryote expression vector carrying human sIL-1R gene. METHODS: Both sIL-1R gene and plasmid pcDNA 3.1(+) DNA were digested with KpnI and XhoI. After purification, the two fragments obtained were ligated by using TakaRa DNA Ligation Kit. This recombinant DNA was then transformed into E. coli Competent Cells JM109 and positive clones were selected on the LB agarose plate containing Ampicillin (80 micrograms/ml). RESULTS: Six single clones were identified by double digestion with KpnI and XhoI, and two fragments with the size of 5.4 kb and 1.0 kb were produced as expected. CONCLUSION: The sIL-1R gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3.1(+) by the recombination technique in vitro.

Cloning, Molecular↗

PubMed1988

Locus-specific vector/primer systems for rapid cloning of allelic variants.

We have developed a rapid cDNA cloning procedure which uses a single-stranded (ss) vector/primer in which the primer sequence is locus-specific. Vector/primers were constructed by substituting a specific oligodeoxynucleotide primer sequence in place of the polylinker in M13mp19. The ss vector/primer is linearized and used to prime cDNA synthesis. Recircularized DNA is then used directly to transform competent bacterial hosts. As no intermediate column purifications or extractions are necessary, the entire procedure is performed in a single tube, contributing to the overall simplicity of the protocol. The primary use for this kind of vector/primer system will be for cloning and sequencing multiple allelic variants of polymorphic loci which contain a conserved 3' sequence. The two vector/primers we report here are specific for HLA-DQ beta genes and for human Ig variable regions associated with IgM antibodies.

Alleles↗

PubMed1991

Recovery of an integration shuttle vector from tandem repeats in Methanococcus maripaludis.

Transformation of Methanococcus maripaludis by using an integration vector, pKAS102, is described. Selection and subsequent growth at high concentrations of puromycin caused pKAS102 to develop tandem repeats within the genome. As a result, total DNA isolated from the transformant could be used to recover the intact vector by direct transformation of competent Escherichia coli.

DNA, Bacterial↗

PubMedDate not supplied

Quick screening of plasmid deletion clones carrying inserts of desired sizes for DNA sequencing.

Plasmids pWQX001 and pWQX005, constructed from pGEM-4 with an insert of 6.5 kb, were unidirectionally digested with exonuclease III and exonuclease VII. The DNA digests were ligated and used to transform competent cells of Escherichia coli DH5-alpha. The size of the deletion plasmid carried by each transformant was estimated through agarose gel electrophoresis of crude lysates without any purification of the plasmid DNA. Colonies carrying plasmid DNAs with different deletions of the insert were grown and their DNAs were purified through a miniprocedure. The size of each purified plasmid DNA was determined accurately after linearization of the plasmid with an appropriate restriction endonuclease. The remainder of the DNA preparation was sufficiently pure to be sequenced using Sanger's dideoxynucleotide chain termination method. An easy, quick procedure is described for the preliminary selection of templates for DNA sequencing after construction of deletion clones of recombinant plasmid DNA using exonuclease III and exonuclease VII. This procedure permits a rapid screening of large numbers of colonies and selection of those carrying plasmid DNAs with inserts of the desired sizes for sequencing. This procedure does not require purification of the deletion plasmid DNA.

Base Sequence↗

PubMed1979

Preparation of competent single-cell suspensions of Mycoplasma hominis tets and Mycoplasma salivarium tets for genetic transformation to tetracycline resistance by DNA extracted from Mycoplamsa hominis tetr.

DNA extracted from Mycomplasma hominis (Sprott strain), resistant to 100 micrograms of tetracycline/ml transformed M. hominis strain H29 and Mycoplasma salivarium strain S9, which are sensitive to 2.5 and 5.0 micrograms of tetracycline/ml, respectively, to resistance. The transformants were selected on agar medium containing 10 micrograms of tetracycline/ml. Some transformants were resistant also to 20 micrograms of tetracycline/ml, a finding confirming that transformation occurred between homologous and heterologous species and that resistance is stepwise and controlled by several genetic loci. Medium containing 10 micrograms of tetracycline/ml was bacteriostatic. Prototype experiments employing mixtures of strains that were tetr and tets (tetracycline-resistant and tetracycline-sensitive, respectively) demonstrated that tetr mutants and transformants formed typical fried-egg colonies when mixtures containing not more than 10(9) mycoplasmas were spread on tetracycline agar plates. No mutants to tetracycline resistance were detected. Both M. hominis and M. salivarium were competent after treatment with MgCl2 and CaCl2, while Mycoplasma orale type 2 was inactivated. During DNA extraction different quantities of DNA formed insoluble precipitates with protein, thus preventing quantitative experiments.

Calcium Chloride↗

PubMed1985

Carcinogen-induced insertion mutations in E. coli.

Mutagenic effects of AAF have been examined by transforming competent E. coli cells with plasmid DNA which were regionally-modified with this carcinogen. Transposition of IS5 from chromosomal DNA to target sequences in the AAF-modified region was detected in six plasmids obtained from wild type and recA strains. A single GC addition was identified in an additional plasmid; no deletions were detected. The observed frequency of IS5 insertion, 2.5 X 10(-3), exceeds that reported for naturally-occurring IS5 transposition in E.coli. These results suggest that the frequency of recombinational events in E.coli can be increased by the presence of a DNA-carcinogen adduct.

Acetoxyacetylaminofluorene↗

PubMed1986

"In vitro" method of assembling a synthetic gene.

Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.

Base Sequence↗

PubMed1995

Malignant and nonmalignant brain tissues differ in their messenger RNA expression patterns for ERCC1 and ERCC2.

Perturbation of the DNA repair process appears to be responsible for the occurrence of a number of human diseases, which are usually associated with a propensity to develop internal malignancies and/or disorders of the central nervous system. We have been interested in the possibility that a subtle abnormality in DNA repair competency might be associated with the transformation of nonmalignant cells to the malignant state. To study this question, we assayed malignant and nonmalignant brain tissues from 19 individuals for mRNA expression levels of the human DNA repair genes ERCC1, ERCC2, and XPAC and for differential splicing of the ERCC1 transcript. We separately compared expression levels of these genes in the following situations: concordance of expression within malignant tissues; concordance of expression within nonmalignant tissues; concordance between malignant and nonmalignant tissues within individuals of the cohort; and concordance of gene expression between two nonmalignant tissue sites within a single individual. Linear regression analyses of mRNA values obtained suggested orderly concordance of these three DNA repair genes in nonmalignant tissues within the patient cohort and an excellent concordance of these genes between two separate biopsy sites from the same individual. In contrast, malignant tissues showed disruption of concordance between the full-length ERCC1 transcript and ERCC2, which have excision and helicase functions, respectively. Furthermore, within the same individuals, malignant tissues were discordant with nonmalignant tissues for ERCC1 and ERCC2, although concordance for XPAC was preserved. These data suggest that one molecular characteristic of human malignancy may be the disruption of the normal relationship between the excision and the helicase functions of the nucleotide excision repair pathway.

Brain↗

PubMed1966

Effect of Bromouracil-containing Deoxyribonucleic Acid on Bacillus subtilis.

Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366-374. 1966.-Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism.

Journal Article↗

PubMed2003

Construction of a genomic library of wild rice and Agrobacterium-mediated transformation of large insert DNA linked to BPH resistance locus.

Here we report the first genomic library of wild rice constructed on a plant-transformation-competent binary vector (BIBAC2) and transformation of the large insert DNA into rice via Agrobacterium. We selected Oryza officinalis for genomic library construction. The library consists of 55,296 clones and stored in one hundred forty-four 384-well plates. Random sampling of 140 clones indicated an average insert size of 71 Kb at a range of 15-235 Kb and 4.8% empty vectors. Four wheat chloroplast probes and four maize mitochondrial probes were hybridized separately to the library, showing that contamination with organellar DNAs is very low (0.61% and 0.04%, respectively). The binary bacterial artificial chromosome (BIBAC) library provides 5.3 haploid genome equivalents, implying a 99.5% probability of recovering any specific sequence of interest. A stability test indicated that the large DNA inserts were stable in this BIBAC vector both in host cells of Escherichia coli and Agrobacterium. Two restriction-fragment length polymorphism (RFLP) markers R288 and C820, which co-segregate with brown planthopper (BPH) resistance gene Qbp2, were used to screen the library, and identified seven and eight positive clones, respectively. The candidate clones of target gene isolated from the library are directly used to transform cultivated rice. After screening the Agrobacterium strains and helper plasmids, and using an improved procedure of transformation, a BIBAC clone with 120 Kb O. officinalis DNA insert was successfully transferred into the rice genome via Agrobacterium-mediated transformation. The system developed here should serve as source for gene discovery, gene cloning and genome-related research in wild rice.

Animals↗

PubMed1971

Mechanism of inactivation of transforming deoxyribonucleic acid by X rays.

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to X rays either in phosphate buffer or in 10% yeast extract. Relations between determinations of biological inactivation, DNA uptake by competent H. influenzae, integration of DNA into the competent cell genome, and induced single-and double-strand breaks indicate that transforming DNA is inactivated by the direct and the indirect effect of X radiation primarily because integration of DNA is prevented as a result of the production of double-strand breaks.

Buffers↗

PubMed1997

Molecular cloning of PCR fragments with cohesive ends.

Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids. Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized. The primers are chosen to span a known region of interest, and extended at their 5'-ends to include the desired restriction sites. Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers. A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s). Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize. Ligation products are used to transform competent bacteria. Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.

Cloning, Molecular↗

PubMed1989

Transformation of Xenorhabdus nematophilus.

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.

Animals↗

PubMed2004

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli.

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.

Colony Count, Microbial↗

PubMed1998

Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor.

Competence is a physiological state, distinct from sporulation and vegetative growth, that enables cells to bind and internalize transforming DNA. The transcriptional regulator ComK drives the development of competence in Bacillus subtilis. ComK is directly required for its own transcription as well as for the transcription of the genes that encode DNA transport proteins. When ComK is sequestered by binding to a complex of the proteins MecA and ClpC, the positive feedback loop leading to ComK synthesis is interrupted. The small protein ComS, produced as a result of signaling by a quorum-sensing two-component regulatory pathway, triggers the release of ComK from the complex, enabling comK transcription to occur. We show here, based on in vivo and in vitro experiments, that ComK accumulation is also regulated by proteolysis and that binding to MecA targets ComK for degradation by the ClpP protease in association with ClpC. The release of ComK from binding by MecA and ClpC, which occurs when ComS is synthesized, protects ComK from proteolysis. Following this release, the rates of MecA and ComS degradation by ClpCP are increased in our in vitro system. In this novel system, MecA serves to recruit ComK to the ClpCP protease and connects ComK degradation to the quorum-sensing signal-transduction pathway, thereby regulating a key developmental process. This is the first regulated degradation system in which a specific targeting molecule serves such a function.

Bacillus subtilis↗

PubMed2006

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N- and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

Bacterial Outer Membrane Proteins↗

PubMed1972

Competence of pneumococcal isolates and bacterial transformations in man.

A survey of pneumococci isolated from 19 healthy carriers and from 23 patients with pneumococcal disease showed that, for both groups, 25 to 30% of the isolates were competent for transformation by soluble deoxyribonucleic acid (DNA) in vitro. Untransformable type 3 and type 8 pneumococci, whose capsules had been hydrolyzed by the Bacillus palustris enzymes prior to exposure to DNA, remained untransformable. Thus, at least for these isolates, it was not the presence of capsule that prevented transformation. Type 9 pneumococci in a healthy human carrier were transformed by DNA released from living unencapsulated pneumococci sprayed onto the pharynx. The donor bacteria were resistant to 1,000 mug of streptomycin/ml. Two types of streptomycin-resistant bacteria were recovered from the carrier's pharynx: a type 9 pneumococcus and an alpha-hemolytic streptococcus. No streptomycin-resistant, gram-positive cocci were isolated from this individual prior to inoculation of the pharynx with the resistant organisms. It seems possible that transformations can occur in the natural environment of some gram-positive cocci.

Journal Article↗