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At least 1,297 records · Page 72Linked to original sources
PubMed1993

[Drosophila beta-heterochromatin: molecular organization and function. Cloning and molecular biological analysis of the lambda 20 DNA fragment from Drosophila melanogaster beta-heterochromatin].

To isolate the DNA sequences specific for the pericentric heterochromatin of Drosophila we used two CREST-autoimmune sera which bind in the Western-blot analysis the nuclear antigens of 30 kDa, 43 kDa and 45 kDa molecular weight. Cloning of the DNA fragments associated with these CREST-specific proteins of Drosophila resulted in obtaining 8 clones. One of them, lambda 20, hybridized mainly to the chromomcenter of polytene chromosomes. The further analysis indicated that the lambda 20 DNA might belong to the proximal beta-heterochromatin of the polytene chromosomes of D. melanogaster.

Animals↗
PubMed1996

Typing of nosocomial strains of Serratia marcescens: comparison of restriction enzyme cleaved genomic DNA fragment (PFGE) analysis with bacteriocin typing, biochemical profiles and serotyping.

Eighty-eight selected clinical isolates of Serratia marcescens, representing 27 putative outbreaks of nosocomial cross-infection encountered during 1980-1995, were tested comparatively by bacteriocin typing, carbon source assimilation tests, serotyping (O and H antigens), and restriction pattern (RFLP) analysis of restriction cleaved (SpeI, XbaI) genomic DNA fragments after pulsed-field gel electrophoresis (PFGE). Serotyping served as the "gold standard" of the phenotypic methods. One pseudo-outbreak (bacteriocin typing incriminated type 26) was uncovered through serotyping as well as the biochemical profile and confirmed by PFGE analysis of genomic DNA. Bacteriocin typing and determination of biochemical profiles disclosed several instances of phenotypic variation; serotyping revealed two episodes of shifts from motility (H12) to nonmotility. Resolution of restricted genomic DNA fragments with the PFGE procedure permitted detection of 27 PFGE patterns (A-M, N-1-N-3, O-1, O-2, P-1-P-3, Q-1-Q-3, R-1-R-3, S-1, and T). Based on the analysis of PFGE patterns against the background of epidemiological data, the number of nosocomically significant strains of S. marcescens could be reduced to 16 (PFGE patterns A-M, N-2, O-1, P-2, and T). It was concluded that PFGE analysis of restricted genomic DNA of S. marcescens was superior to the three phenotypic methods.

Animals↗
PubMed1989

Zn++ inhibits both tumor necrosis factor-mediated DNA fragmentation and cytolysis.

The cellular events involved in direct tumor-cell destruction by tumor necrosis factor (TNF) are still incompletely understood, but a role of endonucleases has been suggested. In the present study we have analyzed in detail the effect of Zn++, an inhibitor of endonucleases, on TNF-mediated DNA-fragmentation and on cytolysis in Actinomycin-D-pre-treated WEHII 64-S cells. Beginning 2 hr after addition of TNF, a rapid degradation of cellular DNA is observed, as evidenced by release of 3H-Thymidine (TdR) label from nuclei into cytoplasm. TNF-mediated lysis of WEHII64-S cells begins at 3 hr and reaches plateau levels at 7 hr. Addition of Zn++ to TNF-treated WEHII64 cells completely abrogates DNA fragmentation at ImM. Of greater importance is the fact that Zn++ treatment also completely blocks TNF-mediated cytolysis of the target cells. Concentrations-between 0.1 and I mM ZnSO4 prevent cell death, as assessed by chromium-release and Trypan blue dye exclusion. In addition, ZnCl2, but not other divalent cations like CaCl2, MgSO4 and CuSO4 in the same concentration range, prevents cell death as well, demonstrating that the effect in fact is mediated by Zn++. Zn++ added 2 hr after TNF treatment, still effectively inhibits cell lysis, indicating that it acts at a late stage after binding of TNF to its receptor. Our data suggest that activation of endonucleases is not an accompanying effect but an essential step in TNF-mediated tumor-cell destruction.

Animals↗
PubMed1997

Constitutive endonuclease to induce high molecular weight or internucleosomal DNA fragmentation in freshly isolated leukemia cells.

Using an autodigestion method, we investigated endogenous endonuclease(s) in leukemia cells freshly obtained from pediatric patients with various types of leukemia. Endonucleolytic activity was found to cause both high molecular weight and internucleosomal DNA fragmentation at a neutral pH in whole cell lysates of all common acute lymphoblastic leukemia (cALL) blasts, which was Mg2+-dependent and Ca2+-independent. Whole lysates from most acute myeloblastic leukemia (AML) cells possessed similar endonuclease activity, but both Mg2+ and Ca2+ were required for the activity. Our results suggest that leukemia cells of different lineages have distinct constitutive endonucleases, which may play a role in the occurrence of apoptosis in these cells.

Apoptosis↗
PubMed1987

A general method for cloning DNA fragments in multiple copies.

A general method for the cloning of DNA fragments in tandem arrays is presented. Synthetic directional adapters are attached to the fragment ends to establish complete control over fragment orientation during ligation. Use of these directional adapters thereby ensures the production of direct repeats, an arrangement essential for clone stability. The technique involves a protocol that is both less complex and less time-consuming than previous methods. Using this technique, a 10-min ligation has yielded a plasmid containing 20 copies of a 151-bp fragment.

Cloning, Molecular↗
PubMed1990

Agents that elevate cAMP stimulate DNA fragmentation in thymocytes.

Increases in the cAMP level are often inhibitory in mature T lymphocytes and may be involved in the development of tolerance to self Ag. In this report, agents inducing an increase in the cAMP level by independent mechanisms were found to stimulate DNA fragmentation, characteristic of a suicide program known as apoptosis, in isolated thymocytes. Data obtained with cAMP analogs known to act synergistically to stimulate protein kinase A suggested that the latter directly mediated endonuclease activation. Agents previously shown to stimulate protein kinase C and to inhibit Ca2(+)-dependent, TCR-mediated thymocyte apoptosis, including IL-1, also blocked both DNA fragmentation and cell death in response to cAMP, suggesting interactions ("cross-talk") between the two protein kinase systems. As it has been proposed that apoptosis mediates negative cell selection in the thymus, our results indicate that cAMP may play a role in the development of functional mature T lymphocytes.

Animals↗
PubMed1997

Recent advances in capillary electrophoresis of DNA fragments and PCR products in poly(n-substituted acrylamides).

In this article, capillary electrophoresis of DNA fragments and polymerase chain reaction products in sieving liquid polymers is reviewed. The first part of the review is dedicated to the development of N-substituted acrylamides, which in general guarantee a greater resistance to hydrolytic attack. Among the various monomers developed, an outstanding molecule appears to be N-acryloyl aminopropanol (AAP), which combines an extreme resistance to alkaline hydrolysis (500 times greater than plain acrylamide) with a substantially higher hydrophilicity. Poly(AAP) matrices exhibit a unique behavior in DNA separations, especially at high temperatures (60 degrees C), where most other matrices fail. In separation of antisense oligonucleotides, isoelectric buffers allow very high field strengths (up to 800 V/cm) due to their extremely low conductivity, thus permitting separations on the order of a few minutes. Among such buffers, histidine (pH = pI = 7.47) and lysine (pH = pI = 9.74) appear to be the best ones. Detection of DNA point mutations is accomplished with a peculiar nonisocratic capillary zone electrophoresis (CZE), consisting of creating temporal thermal gradients, analogous to temperature-programmed gas chromatography. A unique approach is described, consisting of creating temperature gradients not with external thermostats, but with internal Joule effects produced by voltage programming (which results in temperature ramps). The review ends with some unique applications of CZE: the possibility of precise gene dosage, relying on on-line detection and quantitation of the separated peaks. Examples of such gene dosages are given, such as assessment of Down's syndrome and determination of homozygous and heterozygous states of RhD blood groups.

Acrylamides↗
PubMed2006

P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.

Animals↗
PubMed1995

Regulation of signal transduction and DNA fragmentation in thymocytes by ethanol.

We demonstrated previously that ethanol enhances apoptosis of murine thymocytes. In this report, we determined intracellular cAMP and cytosolic free calcium ([Ca2+]i) levels in mouse thymocytes following acute exposure to ethanol and investigated the involvement of cAMP, [Ca2+]i, protein kinase A (PKA), and protein kinase C (PKC) in thymocyte apoptotic death induced by ethanol. It was found that ethanol did not alter basal cAMP levels, but produced a dose-dependent, prolonged small [Ca2+]i increase within thymocytes. This dose dependence of [Ca2+]i increase was paralleled by the magnitude of DNA fragmentation induced by ethanol at various concentrations. Additionally, the ethanol-enhanced DNA fragmentation was blocked by H7, a PKC inhibitor, but not by potent PKA inhibitors having little or no effect on PKC. These data suggest that both [Ca2+]i increase and PKC activation triggered by ethanol may belong to the signal pathway(s) leading to thymocyte programmed death.

Animals↗
PubMed1993

Outbreak of nosocomial infections with two different MRSA-strains involved: significance of genomic DNA fragment patterns in strains otherwise difficult to type.

Methicillin-resistant Staphylococcus aureus isolates from an outbreak of 17 cases of wound infection in a municipal hospital were typed by conventional methods, phage typing by three sets of phages, reverse phage typing and plasmid profiles, as well as by genomic DNA fragment patterns obtained after Sma-I digestion and pulsed-field electrophoresis. These isolates were non-typable by phages, only some were typable by reverse phage typing and were not uniform in plasmid profile. Only the genomic DNA fragment patterns resulted in a clear discrimination of 2 strains (12 isolates for the first and 7 isolates for the second). Both strains were disseminated in different wards of the same hospital and one strain had obviously spread to another clinic in the same city.

Bacterial Typing Techniques↗
PubMed1993

[Construction of clone about major DNA fragment coding for 150kd phosphoprotein of human cytomegalovirus and its restriction site analysis].

Human cytomegalovirus (HCMV) DNA EcoRI-Y fragment, which is major DNA fragment coding for 150kd phosphoprotein (pp150) of HCMV, was separated and recovered from recombinant cosmid pCM1015. The recombinant plasmids pHCY1 and pHCY2, which contain major DNA fragment coding for pp150, were constructed successfully, using the plasmid pUC19 as vector. The recombinant plasmids were analysed by restriction endonucleases and determined by Southern hybridization. The restriction sites of DNA encoding region for immunodominant epitopes of HCMV pp150 were analysed by GENEPRO system. The region could be digested by 76 kinds of restriction endonucleases. The sizes of cleaved fragments were between 4-2667 bp.

Cloning, Molecular↗
PubMed2006

[Function of promoter DNA fragments from halophilic archaea in Escherichia coli].

RM07 and RM13 DNA fragments could function as promoter in Escherichia coli, which were isolated from an archaeon Halobacterium halobium R1. In the present study, promoter activities of these two fragments were confirmed by beta-galactosidase activity analysis and microcalorimetric studies. They were cloned into promoter-probe vector pYLZ-2 respectively. Four recombinant strains TE07, TE07-2, TE131 and TE132 were obtained, and all fragments were found to be active in E. coli DH5alpha. The beta-galactosidase activity of TE132 was higher than that of TE07-2. Both TE07 and TE131 had weak beta-galactosidase activity. Then the heat output of E. coli DH5alpha and its transformants had been detected by a microcalorimetric method at 37 degrees C. Compared with E. coli DH5alpha, the growth rate constant of E. coli T2 (pYLZ-2), TE07, TE07-2, TE131 and TE132 strain was reduced 6.5%, 11%, 41.1%, 47.5% and 42.7% respectively. When IPTG was added to LB medium, beta-galactosidase activity and heat output had been enhanced slightly in all strains. The results suggested that there was close correspondence between promoter activity and microcalorimetric results, and the heat output of growth was mainly affected by gene expression in E. coli. The higher beta-galactosidase activity of E. coli was, the lower its growth rate constant was. At the meantime, Microcalorimetric studies implied that 700bps of RM13 ( RM131) fragment would have stronger promoter activity than RM13. Microcalorimetry may be used as a new approach for analyzing the regulation of foreign gene expression.

DNA, Archaeal↗
PubMed1980

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector.

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.

Cloning, Molecular↗
PubMed2001

[Separation of DNA fragments from halobacterium halobium by capillary electrophoresis with non-gel sieving media].

The optimum electrophoresis separation conditions of Lambda DNA/EcoR I + Hind III fragments were investigated using the capillary column DB-1 coated with polysiloxane. When hydroxyethylcellulose (HEC, 1.0%, mass percentage) was used as the only non-gel sieving medium solution, some Lambda DNA fragments could not be separated. After polyvinylpyrrolidone (PVP, 2%, mass percentage) being added into the medium solution, PVP and HEC formed a network colloidal solution in buffer, and changed the network hole size of sieving media. In addition, the results showed that PVP could restrain the adsorption of capillary to DNA, reduced electroosmotic flow, and improved the selectivity of separation. For the first time, all fragments of Lambda DNA marker was separated completely under the same condition of mixed sieving medium solution. The method was applied to separate two groups of DNA fragments of halobacterium halobium, and the base pair number was conjectured.

Cellulose↗
PubMed1998

Sequence analysis of a DNA fragment from buchnera aphidicola (Aphid endosymbiont) containing the genes dapD-htrA-ilvI-ilvH-ftsL-ftsI-murE

Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum. One of the endosymbiont's functions is the synthesis of branched-chain amino acids. A 9.7-kilobase B. aphidicola chromosomal DNA fragment was cloned and sequenced and found to contain genes encoding acetohydroxy acid synthase (ilvIH), the first enzyme of the parallel pathway of isoleucine and valine biosynthesis. Previously we have detected ilvC and ilvD, encoding the two other enzymes of this pathway. In addition the DNA fragment contained genes for cell division (ftsL, ftsI), murein biosynthesis (murE), lysine biosynthesis (dapD) and a periplasmic protease (htrA). In these properties B. aphidicola resembles free-living bacteria.

Journal Article↗
PubMed1983

Neoplastic transformation by a cloned human cytomegalovirus DNA fragment uniquely homologous to one of the transforming regions of herpes simplex virus type 2.

Specific DNA fragments of human cytomegalovirus strain Towne exhibited sequence homology to the transforming regions of herpes simplex virus type 2 (HSV-2) when examined by nitrocellulose filter hybridization under nonstringent conditions. Cloned Towne Xba I fragments B and C were homologous to both Bgl II transforming fragments N and C of HSV-2 DNA, whereas cloned Towne Xba I fragment E was uniquely homologous to HSV-2 Bgl II fragment C. Furthermore, Towne Xba I fragment E exhibited homology to a unique fragment of cytomegalovirus strain AD169 but lacked homology to the recently identified Xba I transforming (focus-forming) fragment N. Normal diploid Syrian hamster embryo cells transfected with cloned Towne Xba I fragment E displayed colonies of refractile, rapidly dividing cells which escaped senescence to form immortal cell lines. At early passages, these lines exhibited growth in 2% serum and formed small (less than 0.1 mm) colonies in 0.3% agarose. Serial passaging resulted in the appearance of large (greater than 0.25 mm) colonies in agarose, indicating the involvement of more than one step in Towne Xba I fragment E-induced transformation of the diploid hamster embryo cells. NIH 3T3 cells transfected with Towne Xba I fragment E rapidly displayed large colonies in agarose and tumors in vivo.

Base Sequence↗
PubMed1992

Sequences of DNA fragments contacting the nuclear lamina in vivo.

To study the DNA sequences contacting the nuclear lamina (NL) in vivo, Ehrlich ascites tumor cells were UV-irradiated. The NL was purified, and the DNA fragments covalently linked to the lamina proteins in vivo were cloned and sequenced. Although heterogeneous in length and composition, the sequences displayed homology to the introns and/or flanking regions of different genes, suggesting that functionally distinct regions are organized in a topologically defined manner at the nuclear periphery.

Animals↗