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PubMed1990

Drug binding by branched DNA: selective interaction of tetrapyridyl porphyrins with an immobile junction.

The differential binding of a number of water-soluble cationic porphyrins to a branched DNA molecule is reported. Tetrakis(4-N-methylpyridiniumyl)porphine (H2TMpyP-4) interacts near the branch point with an immobile DNA junction formed from four 16-mer strands. Its Cu(II) and Ni(II) derivatives show stronger preferential binding in the neighborhood of the branch point. Axially liganded derivatives, Zn, Co, and Mn, also interact near this branch point, but in a different way. We use the reagents methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(OP)2Cu(I)] to cleave complexes of DNA duplex controls and the junction with these porphyrins. The resulting cleavage patterns are consistent with previous evidence that the branch point provides a strong site for intercalative binding agents, which is not available in unbranched duplexes of identical sequence. The preferential scission by (OP)2Cu(I) in the presence of Ni and Cu porphyrins near the branch point exceeds that seen for any agents we have studied. This hyperreactivity is not seen in the case of porphyrins with axial ligands, ZnTMpyP-4, CoTMpyP-4, and MnTMpyP-4, although these also interact near the branch point. The Zn derivative tends to protect sites close to the branch point from cutting, while the Co and Mn porphyrins moderately enhance cleavage of sites in this region.

Base Sequence↗
PubMed1992

Three-dimensional heteronuclear NMR studies of RNA.

Multidimensional heteronuclear NMR has revolutionized solution structure determinations of proteins. But this technique has not been applied to nucleic acids because of difficulties in the synthesis of isotopically (13C and/or 15N) labelled molecules. Here we report the application of three-dimensional heteronuclear NMR to the study of a uniformly 13C/15N or 15N-labelled RNA duplex of defined sequence. These experiments simplify resonance assignment and the analysis of proton-proton nuclear Overhauser effects (and therefore distance information) in the molecule. Our results show that it is now possible to determine the structures of larger and more complex RNAs using multidimensional heteronuclear NMR.

Base Composition↗
PubMed2005

Dual modes of RNA-silencing suppression by Flock House virus protein B2.

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Crystallography↗
PubMedDate not supplied

Vectors for RNAi technology in poplar.

The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar. A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the beta-glucuronidase (GUS) reporter gene system. These gene cassettes are driven by the CaMV-35S promoter. To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants. Agrobacterium-mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene. From these results we conclude that RNAi is also functional in poplar.

Base Sequence↗
PubMed1983

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.

Adenine↗
PubMed1995

Occurrence of three-stranded DNA within a RecA protein filament.

A RecA protein-generated triple-stranded DNA species can be observed by electron microscopy, within narrowly defined conditions. Three-stranded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrate, when ATP is hydrolyzed, and when the DNA is cross-linked with a psoralen derivative prior to removal of RecA filaments. When adenosine 5'-O-(thiotriphosphate) is used, only the product hybrid duplex DNA can be cross-linked within the RecA filament. The third strand is either displaced or interwound in a conformation that does not permit cross-linking. When ATP is hydrolyzed by RecA, all three strands are cross-linked within the filament in a complex pattern that suggests a dynamic structure. This structure is altered when RecA protein is removed before cross-linking. Hsieh et al. (1990) and Rao et al. (1991, 1993) have proposed, on the basis of nuclease protection and chemical modification studies, that a stable triple-stranded DNA species can persist after removal of RecA protein. We have been unable to visualize these triple-stranded structures by the methods used in the present investigation. When RecA removal was followed immediately by interstrand cross-linking, only the two strands of the hybrid duplex DNA were cross-linked.

Adenosine Triphosphate↗
PubMed1989

Interactions between a symmetrical minor groove binding compound and DNA oligonucleotides: 1H and 19F NMR studies.

High-resolution NMR techniques (proton and 19F) have been used to study the interactions between several DNA oligonucleotides with varying length of AT base pairs and the synthetic pyrrole-containing compound (P1-F4S-P1), which has properties similar to the DNA minor groove binding drug distamycin A. When this two-fold symmetrical DNA binding molecule is added to the self-complementary DNA oligomers, the resulting complex exhibits an NMR spectrum without any doubling of individual resonances, consistent with a two-fold symmetry of the complex. This is in contrast to all other complexes studied so far. The minimum length of an AT stretch for specific ligand binding is judged to be greater than 4 base pairs. Inter-molecular proton nuclear Overhauser effects between the ligand molecule and a DNA dodecamer d(CGCAAATTTGCG) provide evidence that P1-F4S-P1 binds DNA in the minor groove and interacts with the middle AT base pairs. The presence of a specific interaction between P1-F4S-P1 and DNA is conclusively demonstrated by 19F NMR studies, in which four previously chemically equivalent fluorine nuclei in the free molecule become two non-equivalent pairs (yielding an AB quartet pattern) upon the binding of P1-F4S-P1 to DNA duplex. A sequence-dependent binding behavior of P1-F4S-P1 is evident by comparing the 19F NMR spectra of the complexes between P1-F4S-P1 and two different but related DNA dodecamers, d(CGCAAATTTGCG) and d(CGCTTTAAAGCG). P1-F4S-P1 binds more strongly to the former dodecamer with an association constant of approximately 1 X 10(3) M-1.

Base Sequence↗
PubMed2000

Masked antisense: a molecular configuration for discriminating similar RNA targets.

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These 'masked' antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR-ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.

Autoradiography↗
PubMed1983

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.

Bacteriophage lambda↗
PubMed1985

Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol.

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.

Adenosine Triphosphate↗
PubMed1987

Structure of the human oestrogen-responsive gene pS2.

The human pS2 gene, whose expression is restricted to breast cancer cells, and whose transcription is induced by oestrogen in the human breast cancer cell line MCF-7, has been cloned from both placental and MCF-7 cell DNA. The exon-intron organization has been established by electron microscopy using genomic DNA-cDNA or -mRNA hybrid duplexes and by sequencing the exons and exon-intron junctions. The overall organization within and around the pS2 gene is the same in placental and MCF-7 cell DNA and the exonic sequences are identical to those previously determined from the cDNA. The 5'-flanking region of the pS2 gene is also identical (with the exception of two base transitions) in the two tissues. Thus no gene rearrangement nor sequence modification has occurred in the pS2 gene of the malignant and polyploid MCF-7 cells. A TATA-box, a CAAT-box and a GC-rich motif are present in the 5'-flanking region of the pS2 gene, but the latter motif is unusually located between the TATA-box and the capsite. No significant homology could be detected between the 5' flanking sequences of the pS2 gene and those of other oestrogen-responsive genes from different species.

Amino Acid Sequence↗
PubMed1988

Evidence for a gapped linear duplex DNA intermediate in the replicative cycle of human and simian spumaviruses.

Two forms of linear DNAs have been found in simian (SFV1) and human (HSRV) spumaviruses: a linear duplex unsensitive to nuclease S1 and a sensitive structure with a single-stranded gap. Two nuclease S1 sensitive sites, mapping at the same position for both viruses, have been identified in the gapped structure. Using different molecular subgenomic clones of HSRV as probes in Southern blot analysis, one S1 site was localized in the 3'LTR and the other near the middle of the molecule at about 6.5 kbp from the 5' end of the viral genome. The latter site was shown to correspond to a single stranded region within the linear duplex DNA. Nucleotide sequence analysis revealed that the polypurine tract (PPT) usually found at the 5' boundary of the 3'LTR of retroviruses, is duplicated in HSRV at the 3' end of the pol gene, near the gap. This suggests that the synthesis of plus strand DNA is discontinuous, generating the gap.

Animals↗
PubMed2000

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

Alkaloids↗
PubMed1995

Enhanced and coordinated processing of synapsed viral DNA ends by retroviral integrases in vitro.

We have designed novel substrates to investigate the first step in retroviral integration: the site-specific processing of two nucleotides from the 3' ends of viral DNA. The substrates consist of short duplex oligodeoxynucleotides whose sequences match those of the U3 and U5 ends of viral DNA but are covalently synapsed across the termini by short, single-strand nucleotide linkers. We show here that the optimal separation between termini in a synapsed-end substrate for avian sarcoma/leukosis virus (ASV) IN is 2 nucleotides. This places the two conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation equal to the staggered cut in target DNA produced by this enzyme during the subsequent joining reaction. Based on estimates of initial reaction rates, this synapsed-end substrate is processed by IN at > 10-fold higher efficiency than observed with an equivalent mixture of U3 and U5 single-end (uncoupled) substrates. Enhanced processing is maintained at low IN concentrations, suggesting that the synapsed-end substrate may facilitate enzyme multimerization. Enhanced processing by HIV-1 IN, which produces a 5-bp stagger during integration, was observed with a synapsed-end substrate in which the separation between processing sites was 5 nucleotides. These observations provide estimates of the distances between active sites in the multimeric IN-DNA complexes of ASV and HIV-1. Our results also show that processing of paired U3 and U5 ends need not be coupled temporally. Finally, we observed that substrates that paired a wild-type with a mutated terminus were cleaved poorly at both ends. Thus, in vitro processing of the synapsed-end substrates requires specific recognition of the sequences at both ends. These findings provide new insights into the mechanism of integrative recombination by retroviral integrases and, by extension, other prokaryotic and eukaryotic transposases that are related to the viral enzymes.

Avian Sarcoma Viruses↗
PubMed1998

Magnetic resonance angiographic and clinical features of extracranial vertebral artery dissection.

OBJECTIVES: Clinical data and neuroradiological findings of 19 patients with 20 vertebral artery dissections were analysed to describe the features of time of flight magnetic resonance angiography (MRA) for the diagnosis and follow up of this vascular disorder. METHODS: All patients underwent a combined MRI and MRA protocol with 1.5 T scanners, using a three dimensional flow compensated gradient echo sequence for MRA. Duplex sonography was performed on all patients and selective angiography was available from 17 vertebral artery dissections. RESULTS: MRI showed ischaemic lesions of the brain in 18 of 19 patients (95%). In the acute and subacute stage, MRA detected signal abnormalities within the dissected vertebral artery in 94% (16/17) and MRI was specific for a dissection in 29% (5/17). Sensitivity of selective angiography was 100% and specificity was 35% (6/17). Combination of the results of both methods increased the specificity to 50%. Duplex sonography was sensitive in 79% (15/19), but lacked specific results. Follow up magnetic resonance in 16 patients showed recanalisation of the dissected vessel in 10 (63%), persistent occlusion in five (31%), and a dissecting aneurysm in one (6%) patient. CONCLUSIONS: Magnetic resonance improves the triage for selective angiography and discloses complementary information for the diagnosis of vertebral artery dissection. If magnetic resonance identifies a double lumen or a mural haematoma with a stenosis or aneurysmal dilatation, invasive procedures can be omitted.

Adult↗
PubMed1983

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase.

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.

Base Sequence↗
PubMed1993

[Intensification of genomic dactyloscopy by means of hybridization with modified oligonucleotides, forming duplexes with increased stability].

The modified oligodeoxyribonucleotide (m5C-n2A-m5C)5 containing 5-methylcytosine and 2-aminoadenine instead of cytosine and adenine residues, respectively has been used as a hybridisation probe in DNA fingerprinting. The oligonucleotide, due to the substitutions forms more stable duplexes with complementary sequence in DNA than the corresponding nonmodified pentadecanucleotide. The comparison with its natural counterpart displays considerably increased intensity of bands in patterns obtained with modified analog. The use of such analogues can increase sensitivity and shorten time of DNA fingerprinting.

2-Aminopurine↗