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PubMed1995

Native DNA fragments photocrosslinked to psoralen binds to anti-B and anti-Z DNA antibodies.

Calf thymus DNA fragments of varying size (100-600 bp) were covalently linked to psoralen in the presence of 365 nm UV light. The crosslinked species were separated from the monoadducts by hydroxyapatite column chromatography. The DNA-psoralen crosslink showed binding with naturally occurring anti-DNA autoantibodies. This was substantiated by the recognition of affinity purified IgG by DNA-psoralen crosslinks of varying DNA size. The photo-crosslinks exhibited considerable recognition of both monoclonal and polyclonal anti-Z-DNA antibodies in inhibition ELISA and band shift assay.

Animals↗
PubMed1991

Cloning, restriction digestion and DNA labeling of large DNA fragments (greater than or equal to 1 kb) in the presence of remelted SeaPlaque GTG agarose gels.

Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.

Cloning, Molecular↗
PubMed1999

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments.

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2 X or 4 X 48 base pair repeats) of the dopamine D4 receptor gene (D4DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.

DNA↗
PubMed1998

Separation of oligonucleosomal DNA fragments from apoptotic animal cells using a triad of Sephacryl columns.

The degradation of DNA, resulting in information of oligonucleosomal fragments, is a characteristic feature of apoptosis, i.e., the process of programmed cell death. In this work we have developed a method for exact determination of the proportion of fragmented DNA in an apoptotic cell population. To this end we employed Sephacryl gel chromatography matrices and UV detection of DNA concentration. The disturbing effect of low-molecular-mass UV-absorbing contaminants was eliminated by insertion of a Sephacryl S-200 HR pre-column. Optimum resolution of DNA samples isolated from apoptotic cells was achieved using a triad of Sephacryl S-200 HR, Sephacryl S-500 HR and Sephacryl S-1000 SF columns.

Animals↗
PubMed2003

Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids.

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.

Colloids↗
PubMed2006

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors.

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.

Cloning, Molecular↗
PubMedDate not supplied

[Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites].

The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.

Adenovirus E1A Proteins↗
PubMed1999

Early decrease in apurinic/apyrimidinic endonuclease is followed by DNA fragmentation after cold injury-induced brain trauma in mice.

Apurinic/apyrimidinic endonuclease, a multifunctional protein in the DNA base excision repair pathway, plays a central role in repairing DNA damage caused by reactive oxygen species. We examined protein expression of apurinic/apyrimidinic endonuclease before and after cold injury-induced brain trauma in mice, where we have previously shown reactive oxygen species to participate. Immunohistochemistry showed the nuclear expression of apurinic/apyrimidinic endonuclease in the entire region of control brains. One hour after cold injury-induced brain trauma, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, whereas there was a slight increase in the outer boundary area. Four hours after cold injury-induced brain trauma, nuclear immunoreactivity was almost absent in the entire lesion, and remained so until 24 h. At this time, a marked increase in apurinic/apyrimidinic endonuclease immunoreactivity was seen in the outer boundary zone. Western blot analysis of the sample from the non-ischemic area showed a characteristic 37,000 mol. wt band, which decreased markedly 24 h after cold injury-induced brain trauma. A time-dependent increase in DNA fragmentation was also observed after cold injury-induced brain trauma. Our data provide the first evidence that apurinic/apyrimidinic endonuclease decreased rapidly in the lesion after cold injury-induced brain trauma, whereas it was significantly increased at the outer boundary zone. Although further examination is necessary to elucidate the direct relationship between apurinic/apyrimidinic endonuclease alteration and the pathogenesis of cold injury-induced brain trauma, our results suggest the possibility that an early decrease in apurinic/apyrimidinic endonuclease and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after cold injury-induced brain trauma.

Animals↗
PubMed1998

The mechanism of thioacetamide-induced apoptosis in the L37 albumin-SV40 T-antigen transgenic rat hepatocyte-derived cell line occurs without DNA fragmentation.

The hepatotoxicant thioacetamide (TH) has classically been used as a model to study hepatic necrosis; however, recent studies have shown that TH can also induce apoptosis. In this report we demonstrate that 2.68+/-0.54% of the albumin-SV40 T-antigen transgenic rat hepatocytes undergo TH-induced apoptosis, a level comparable to other in vivo models of liver apoptosis. In addition, TH could induce apoptosis and necrosis in the L37 albumin-SV40 T-antigen transgenic rat liver-derived cell line. Examination of dying L37 cells treated with 100 mM TH by electron microscopy revealed distinct morphological characteristics that could be attributed to apoptosis. Quantitation of apoptosis by FACS analysis 24 h after treatment with 100 mM TH revealed that 81.3+/-1.6% of the cells were undergoing apoptosis. In contrast, when L37 cells were treated with 250 mM TH, cells exhibited characteristics consistent with necrotic cell death. DNA fragmentation ladders were produced by growth factor withdrawal-induced apoptosis; however, in 100 mM TH-induced apoptosis, DNA fragmentation ladders were not observed. Analysis of endonuclease activity in L37 cells revealed that the enzymes were not inactivated in the presence of 100 mM TH. The data presented in this report indicate that the L37 cell line could be used to study the mechanism of TH-induced apoptosis that was not mediated through a mechanism requiring DNA fragmentation.

Albumins↗
PubMed2004

Separation of double-stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH.

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.

DNA↗
PubMed1975

Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments.

Adenovirus 2 RNA synthesized early in productive infection was analyzed by RNA-DNA hybridization. Hybridization experiments were performed with adenovirus 2 DNA and wit, the six adenovirus 2 DNA fragments generated 0y digestion with the restriction endonuclease Eco R.R1. Duplex formation between RNA and -32P-labeled viral DNA was assayed by S(1) nuclease digestion. RNA from the cytoplasm annealed 12 percent of the total viral DNA and the following percentage of each of the R.R1 fragments: 6 percent of R1-A, 24 percent of R1-B, 0 percent of R1-F, 40 percent of R1-D, 13 percent of R1-E, and 22 percent of R1-C. The early cytoplasmic RNA is composed of two sequence classes: class I, present in greatly reduced quantities at late times in infection (18 h), and class II, which remains at high concentrations at 18 h. In hybridization-inhibition experiments, hybridization of class II RNA is inhibited by late cytoplasmic RNA, whereas hybridization of class I RNA is not blocked by late cytoplasmic RNA (J. J. Lucas and H. S. Ginsberg, 1971; E.A. Craig and H. J. Raskas, 1971). To determine the location of class I and II sequences on the genome, membrane bound DNA fragments were used in hybridization-inhibition experiments. These studies demonstrated that the early cytoplasmic transcripts of R1-D belong to class II, whereas R1-C transcripts are class I sequences. The cytoplasmic RNAs transcribed from fragments A and B contain both class I and class II sequences. Analysis of cytoplasmic RNA fractionated by size demonstrated that the class I sequences include a 19 S RNA transcribed from R1-B and class II sequences include a 20S RNA derived from R1-D. Nuclear RNA purified from cultures early in infection was annealed with -32P-labeled R1 fragments. With all six fragments the nuclear RNA annealed as much or more of the DNA than did cytoplasmic RNA. Eco R1-F annealed at least 25 percent with early nuclear RNA, whereas no sequences homologous to R1-F were detected in early cytoplasmic RNA. When cultures were labeled from 2 to 6 h after infection, at least 5 percent of the -3H-labeled early nuclear viral RNA annealed to Eco R1-F. Some of these nuclear transcripts from R1-F appear to be covalently linked to sequences transcribed from a contiguous region of the genome (Eco R1-B). 8.4 percent of the RNA selected by hybridization of R1-F reannealed to R1-B, whereas no more than 1.5 percent reannealed to R1 fragments A, D, E, or C.

Adenoviridae↗
PubMed1997

Combination therapy with 5-fluorouracil and IFN-alpha2a induces a nonrandom increase in DNA fragments of less than 3 megabases in HT29 colon carcinoma cells.

We have used pulsed-field gel electrophoresis to examine 5-fluorouracil (5FU)-induced DNA double-strand breaks (DSBs), both with and without modulation by IFN-alpha2a (IFNalpha), in HT29 human colon adenocarcinoma cells. Although 24-h treatment with either 10 microM 5FU or 500 units/ml IFNalpha did not result in significant DNA fragmentation, the combination of 5FU + IFNalpha resulted in a significant increase in DNA DSBs versus either drug alone (P < 0.05). The pattern of fragmentation induced by treatment with 5FU + IFNalpha was compared to that induced by gamma-radiation, which generates lesions at random sites, digestion with NotI restriction endonuclease, which cleaves at the specific sequence 5' ellipsis GCGGCCGCellipsis 3', and HhaI restriction endonuclease, which cleaves at the specific sequence 5'ellipsis GCGCellipsis 3'. 5FU + IFNalpha resulted in a specific pattern characterized by the accumulation of fragments of <3 Mb in the absence of fragments of >3 Mb, which differed from that of gamma-radiation and restriction endonuclease digestion. Because neither morphological nor DNA fragmentation characteristic of apoptosis was observed after 5FU + IFNalpha treatment, the nonrandom pattern of DSBs that was observed did not appear to be the result of the initiation of programmed cell death within these cells.

Apoptosis↗
PubMed1989

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA.

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.

Circular Dichroism↗
PubMed2002

Among substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetraones, the lead antitumor triptycene bisquinone TT24 blocks nucleoside transport, induces apoptotic DNA fragmentation and decreases the viability of L1210 leukemic cells in the nanomolar range of daunorubicin in vitro.

In contrast to their inactive parent compound triptycene (code name TT0), several new synthetic analogs (TT code number) have antileukemic activities and remain effective in daunorubicin (DAU)-resistant tumor sublines in vitro. Among variously substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetraones, a total of six lead antitumor compounds have been identified, and their code names are TT2, TT13, TT16, TT19, TT21 and TT24. These active antitumor triptych structures have bisquinone functionality, and various bromo, methoxy, methylamino and/or dimethylamino substitutions with or without longer alkyl chains on the amino function. Like the anthracycline quinone antibiotic DAU, these triptycene (TT) bisquinones also inhibit DNA synthesis and induce DNA cleavage in relation with their cytotoxic activities, but have the additional advantage of blocking the cellular transport of purine and pyrimidine nucleosides, an effect which DAU cannot do. As demonstrated by intact chromatin precipitation and agarose gel electrophoresis, the ability of TT bisquinones and DAU to induce DNA fragmentation is biphasic with a peak that shifts to lower concentrations with increasing times of drug exposure. The most effective lead antitumor compound, TT24, induces DNA cleavage in the same concentration-dependent manner as DAU at 24 h (similar peak in response to 1.6 microM) and is nearly equipotent to DAU against L1210 tumor cell viability at day 4 (IC50 values of TT24 and DAU: 48 and 25 nM, respectively). The mechanism by which TT24 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO4, suggesting that TT bisquinones trigger apoptosis by caspase and endonuclease activation. Since TT24 is cytotoxic in the nanomolar range of DAU, but might have a more versatile mechanism of action than DAU in wild-type and multidrug-resistant tumor cells, this new class of DNA-damaging quinone antitumor drugs inhibiting nucleoside transport might be valuable to develop new means of polychemotherapy.

Animals↗
PubMed1997

[Plasmid DNA fragments from Halobacterium halobium active as eubacteria promoters in Escherichia coli].

In this paper; a promoter-probe plasmid pKK232-8 was used as a vector which functioned in E. coli. The plasmid pHH205 of Halobacterium halobium J7 was digested by two groups of restriction endonucleases BamHI-SalI and HindIII -SalI, recombined in vitro and transformed into E. coli. The transformants were selected on resistance plates to contain ampicillin (Am) and chloramphenicol (Cm). From random-selected 20 strains of transformants we obtained transformants T1 and T2, whose level of Cm resistance got to 110 micrograms/ml. The recombinant plasmids from T1 and T2 were named pJH and pJB, respectively. The resultes of analysis by restriction endonucleases digestion and molecular hybridization showed that recombinant plasmid pJH carried a inserted fragment with size of 800bp from plasmid pHH205. Experimental results of retransfomation proved further that the DNA fragment had promoter function in E. coli. Thus, this indicated that DNA fragments from plasmid of archaebaeteria (H halobium) may function as eubacteria (E. coil) promoters.

Escherichia coli↗
PubMed1989

Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum.

The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the pcbC and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin producing strain (P. chrysogenum Wis 54-1255) and two high producing strains (AS-P-78 and P2) showed hybridizing fragments of identical sizes in their chromosomes. Dot-blot hybridization of serial dilutions of the total DNA of the three strains showed that the intensity of all the hybridizing bands was much higher in strains AS-P-78 and P2 than in Wis 54-1255. Hybridization of total DNA digestions with probes corresponding to fragments which mapped upstream or downstream of the pcbC-penDE region revealed that a fragment of at least 35 kb DNA has been amplified 9 to 14 fold in the high penicillin producing strains. The amplified region did not include the previously cloned pyrG gene that encodes OMP-decarboxylase, an enzyme involved in pyrimidine biosynthesis.

Acyltransferases↗
PubMed1998

Genotyping by mass spectrometric analysis of short DNA fragments.

A method has been developed to produce small DNA fragments from PCR products for analysis of defined DNA variations by mass spectrometry. The genomic region to be analyzed is PCR-amplified with primers containing a sequence for the type IIS restriction endonuclease Bpml. Bpml digestion of the resultant PCR products yields fragments as small as seven bases, which are then analyzed by electrospray ionization mass spectrometry. The approach was validated using seven different variants within the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands were analyzed independently, and several variants can be analyzed simultaneously. These results provide the basis for a generally applicable and highly accurate method that directly queries the mass of variant DNA sequences.

Base Sequence↗
PubMed1984

Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment.

Four early transcripts and the polypeptides they encode have been mapped to the vaccinia virus EcoRI F DNA fragment, which spans the vaccinia HindIII J and H fragments. In addition, two transcripts for which no encoded polypeptides have been identified have also been mapped. Elucidation of the organization of these six transcripts by hybrid selection, S1 nuclease mapping, translation of size-fractionated RNA, and by filter hybridization demonstrates that approximately 90% of this DNA fragment is transcribed during early infection. All but one of these RNAs (1.35 kilobases [kb]) are transcribed in a rightward direction. The leftmost transcript of 0.6 to 0.7 kb encodes a 19,000-dalton (19K) polypeptide that has been determined to be thymidine kinase (D.E. Hruby and L.A. Ball, J. Virol. 43:403-409, 1982; G. Bajszar et al., J. Virol. 45:62-72, 1983). Immediately following the 3' end of this RNA is the 1.7-kb transcript encoding a 36K polypeptide. The 5' end of a 1.25-kb RNA encoding a 22K polypeptide is downstream of the 5' end of the 1.7-kb RNA, and its 3' end may be coterminal with the 3' end of the 1.7-kb RNA. The 2.45-kb transcript is coterminal at its 5' end with the message encoding thymidine kinase and is coterminal at its 3' end with the adjacent 1.7-kb mRNA. This RNA was not demonstrated to encode a polypeptide. Approximately 300 nucleotides from the 3' end of the 1.7-kb RNA is a 3.6-kb transcript encoding a 110K polypeptide. The 3' end of this large RNA lies several hundred nucleotides from the 3' end of the 1.35-kb RNA which is transcribed in the opposite direction. No splicing of RNA has been detected with the S1 nuclease mapping technique of Berk and Sharp. The organization of three late messages, which overlap these early transcripts, has been determined, and these results are discussed in the accompanying paper.

Animals↗