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At least 1,099 records · Page 61Linked to original sources
PubMed1981

Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites.

A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site. Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase. Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP. A plasmid vehicle, pAO1, containing a single PstI site has been constructed. Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP. When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired. Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules.

Base Sequence↗
PubMed1999

Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line.

1. Using an in vitro model of shear stress-induced cell injury we demonstrate that application of shear to differentiated human SH-SY5Y cells leads to cell death characterized by DNA fragmentation. Controlled shear stress was applied to cells via a modified cone and plate viscometer. 2. We show that pulsatile shear stress leads to DNA fragmentation, as determined via flow cytometry of fluorescein-12-dUTP nick-end labelled cells, in 45 +/- 4 % of cells. No lactate dehydrogenase (LDH) release was observed immediately after injury; however, 24 h after injury significant LDH release was observed. 3. Nitric oxide production by cells subjected to pulsatile shear increased two- to threefold over that in unsheared control cells. 4. Inhibition of protein synthesis, nitric oxide production, Ca2+ entry into cells, and pertussis toxin-sensitive G protein activation attenuated the shear stress-induced cell injury. 5. Our results show for the first time that application of pulsatile shear stress to a neuron-like cell in vitro leads to nitric oxide-dependent cell death.

Calcium↗
PubMed1999

[Gene detection and localization by FISH with short DNA fragment cloned in plasmid].

OBJECTIVE: To establish a method of detecting and localizing newly cloned gene by FISH using short DNA fragment. METHODS: Bio-14-dATP was incorporated into the target gene cloned in plasmid by nick translation followed by conventional FISH with some modification. By Means of this method, c-met, c-erbB2, 1A6, APC and Rb genes were examined in ovarian and gastric cancer cell lines. RESULTS: 1A6, a newly isolated gene was located at the long arm of one of the C group chromosomes. Oncogene c-met, c-erbB2 and trmor suppressor gene APC, Rb were shown to produce clear-cut hybridization signals in ovarian and gastric cancer cells. CONCLUSION: Short DNA fragment cloned in plasmid is useful for studies of gene localization, gene deletion and amplification by FISH.

Chromosome Mapping↗
PubMed1994

DNA fragment size determination on agarose gel by using the application GEL.

We have developed an application to calculate double-stranded DNA fragment size on an agarose gel. This application is named GEL and is implemented for two different platform: MS-DOS and Macintosh. Both implementations allow entry of the relative migration distance of a DNA fragment of an agarose gel containing up to 20 different wells and up to 20 bands per well. Up to three different standards can be present on the gel, and the application will use the closest standard to calculate the fragment size. Both applications have a familiar user interface: the Turbo Vision interface for the MS-DOS implementation and the familiar Macintosh interface for the Macintosh implementation. In addition, the MS-DOS implementation has an extensive context on-line help.

Algorithms↗
PubMed1997

In situ DNA fragmentation assay for detection of apoptosis in paraffin-embedded tissue sections. Technical considerations.

Detection, by light microscopy, of cells in situ undergoing apoptosis has been improved by use of an in situ apoptosis (DNA fragmentation) assay on formalin-fixed and paraffin-embedded tissue sections. We studied conditions of tissue preparation and fixation that may affect the test results. In this study, we intended to determine whether archival tissues prepared under unknown conditions can be used for the in situ apoptosis assay. All tissue sections were pretreated with Proteinase K, followed by incubation with biotinylated 11-deoxyuridine triphosphate in terminal deoxynucleotidyl transferase and then avidin-biotin-peroxidase complex. The following formalin-fixed and paraffin-embedded histologic sections were tested: (1) normal tissues from surgically resected specimens fixed immediately or stored at 4 degrees C and then fixed after 1, 2, 4, 6, or 24 hours; (2) archival autopsy material from histologically normal tissues; and (3) freshly prepared normal tissues from C57 mice. We observed that fixation- and prefixation-elapsed times do not adversely affect the results of the assay. Similar, if not identical results were seen in archival human tissues stored for up to 25 years, the normal tissues freshly prepared from surgical specimens, and the tissues from C57 mice. We conclude that the in situ assay of DNA fragmentation is rapid, sensitive, and reproducible. The use of formalin-fixed and paraffin-embedded archival material as old as 25 years opens the way for a variety of studies of apoptosis in diverse pathologic states.

Animals↗
PubMed1985

Viscometric detection of liver DNA fragmentation in rats treated with ten aromatic amines. Discrepancies with results provided by the alkaline elution technique.

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA reduced viscosity, has been used to evaluate DNA fragmentation in liver of rats treated with single i.p. doses of ten aromatic amines. Persistent and dose-dependent changes of DNA viscometric parameters, which are considered indicative of DNA fragmentation, were produced by six hepatocarcinogenic aromatic amines: 2-naphthylamine, benzidine, 2,4-diaminotoluene, auramine O, 4-aminoazobenzene, and 4-dimethylaminoazobenzene. In contrast, changes of liver DNA viscometric parameters were minimal and transient or practically absent in rats treated with aniline, 1-naphthylamine, 4,4'-oxydianiline and 2,4-diaminoanisole, all of which are devoid of hepatocarcinogenic activity. The comparison with data previously obtained with the alkaline elution technique demonstrates that the two methods can give different results, and that viscometrically-detected DNA damage is better correlated with carcinogenic activity than DNA damage detected by alkaline elution.

Amines↗
PubMed2003

Selective alteration of DNA fragmentation and caspase activity in the spinal cord of aged rats and effect of dietary restriction.

Age-related alterations in CNS may preferentially affect spinal cord. We report here that in spinal cord of aged (30-month-old) rats, a significant increase of DNA fragmentation accompanied by an increased activity of caspase-1, but not of caspase-3, can be measured. The increase in caspase-1 activation, but not the increased level of DNA fragmentation, was substantially reverted by long-lasting dietary restriction.

Aging↗
PubMed1990

pUC18rspL: a plasmid vector for indirect selection and direct sequencing of promoter-bearing DNA fragments.

Plasmid pUC18rspL is a 3.788-kilobase pair vector, derived from pUC18, pKK232-8 and pHSG664, which identifies promoter-bearing DNA fragments functional in StrA E. coli by activation of a promoterless streptomycin-sensitive gene cartridge (rspL). Expression of the plasmid-borne rspL gene leads to sensitivity dominance and death of the cell. Promoter-bearing DNA fragments can be cloned within a synthetic polylinker containing 12 unique restriction nuclease target sequences. After transformation of StrA E. coli TB1, ampicillin-resistant transformants are replica plated on medium containing ampicillin and streptomycin to identify promoters cloned in their functional orientation. These elements can be sequenced without additional subcloning steps from the M13 universal forward primer hybridization site located 5' of the polylinker in the pUC18 contribution. Transcriptional terminators are cloned 3' of the rspL gene to maintain a balance between transcription and replication when high signal strength promoters such as the E. coli Tac promoter are analyzed. pUC18rspL is used to clone transcriptional promoters from the extreme thermophile T. aquaticus. Promoter signal strength can be estimated by determining the extent of sensitivity dominance conferred to the host.

Ampicillin↗
PubMed1984

Direct calculation of the sizes of DNA fragments separated by gel electrophoresis using programmes written for a pocket calculator.

In order to facilitate the direct computation of the sizes of DNA fragments separated by gel electrophoresis, we have written and evaluated programmes for the Hewlett-Packard 41C programmable calculator. The sizes estimated for DNA fragments of known length using some of these programmes were found to be more accurate than the estimates obtained by conventional graphical procedures. These programmes should be adaptable to other programmable calculators.

Computers↗
PubMed1986

Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper.

A solid-phase method for simultaneous sequencing of ten or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173-1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO4 (T greater than Pu) and NH2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation.

Anions↗
PubMed1979

Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12.

Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.

Adenoviruses, Human↗
PubMed1993

Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas campestris DNA fragment with the lux transposon Tn4431 using the delivery plasmid pDS1, thermosensitive in replication.

A system allowing transposon mutagenesis of cloned DNA fragments in Escherichia coli with Tn4431, which carries the promotorless luciferase (lux) operon of Vibrio fischeri, has been developed. The transposon delivery plasmid, pDS1, based on an IncF replicon, is thermosensitive in replication and mobilizable to many Gram-negative bacteria. We used pDS1 for Tn4431-saturation mutagenesis of a 10-kb DNA fragment of Xanthomonas campestris pv. campestris (X.c.c.) in E. coli and showed that the expression of the lux operon was dependent on orientation and location of the transposon. Transfer of a specific Tn4431 insertion to X.c.c. allowed the determination of the bioluminescence phenotype in planta.

Cloning, Molecular↗
PubMed2005

Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice.

Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.

Animals↗
PubMed1993

Interaction of the trp repressor with trp operator DNA fragments.

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor L-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpOc, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the 31P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.

Base Sequence↗
PubMed2004

Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF.

The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.

3' Untranslated Regions↗
PubMed1984

A method for recovering strand-specific probes from nick-translated DNA fragments.

A method of preparing strand-specific probes for DNA X DNA or DNA X RNA hybridizations is described. Double-stranded DNA fragments are first isolated from any recombinant DNA clone containing the desired sequence, and then labeled in vitro by nick-translation (T. Maniatis, A. Jeffrey, and D. G. Kleid (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188; P. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg (1977) J. Mol. Biol. 113, 237-251). Sequences homologous to the desired strand are captured by annealing the denatured nick-translate to viral strands of an appropriate M13 clone, and recovered by elution of the resulting hybrids from a column of agarose A50M (Bio-Rad). By this method, separate probes with specificity to either strand, as well as the double-stranded probe, may conveniently be prepared from a single nick-translation reaction. Probes may be obtained which are homologous either to the full length of the cloned region or to selected portions thereof by selecting appropriate M13 clones for annealing. The probe is recovered as a population of fragments several hundred bases or less in length, which have been found ideal for saturating liquid hybridizations, and should be similarly well suited for in situ hybridizations to cytological preparations.

Bacteriophages↗
PubMed2003

Mutation analysis of the RET gene in total intestinal aganglionosis by wave DNA fragment analysis system.

BACKGROUND/PURPOSE: Total intestinal aganglionosis (TIA) extending from the duodenum to the rectum is the most rare form of Hirschprung's disease (HSCR) and usually is fatal. RET is the major gene associated with HSCR, and germline mutations of this gene account for up 50% of familial and up to 15 to 20% of sporadic cases in HSCR. The aim of this study was to investigate DNA variants in the RET gene in TIA patients using the WAVE DNA Fragment Analysis System. METHODS: Genomic DNA was extracted from whole blood samples from 6 patients with TIA. Polymerase chain reaction (PCR) amplification of the 21 exons of RET was performed using published oligonucleotide primers. Heteroduplexes were followed by the WAVE DNA Fragment Analysis System with the DNASep cartridge. RESULTS: WAVE system technology detected 16 variants in the RET gene in the 6 patients with TIA. Three patients had a significant mutation in exon 8, 11, and 15, respectively. Thirteen RET polymorphic variants also were detected in the 6 patients, with L746L variant in exon 13 occurring in 4 patients. CONCLUSIONS: WAVE system technology is an efficient method for the detection of DNA sequence variants. Our findings suggest that not only RET mutations but also RET polymorphic variants may contribute to the occurrence of TIA.

Amino Acid Substitution↗
PubMed1994

[An approach for PCR amplification of long DNA fragments].

The polymerase chain reaction (PCR) technique, evolved as a standard laboratory technique, has a major limitation: its inability to efficiently amplify fragments higher than 1.5 kb. In this paper, PCR conditions allowing the efficient amplification of long DNA fragments, especially a new proper buffer system, have been developed. The difference between the new PCR reaction buffer system and standard reaction system is that no KCl is included in the former. The results indicated that specificity and reproducibility for amplifying long DNA fragments by using new buffer system are satisfactory.

Animals↗