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PubMed2003

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures.

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.

Alkaloids↗
PubMed2003

Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure.

The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.

Ascomycota↗
PubMed1997

Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles.

Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

DNA, Circular↗
PubMed1995

Expression of the mouse testis-determining gene Sry in male preimplantation embryos.

The testis-determining factor in the mouse is encoded by the Sry gene on the Y chromosome. Transcripts of this gene have been shown previously to be present in the genital ridge at the beginning of gonadal differentiation (11.5 days post coitum) and in adult testis. In this study, RNA transcripts of the Sry gene are also detected in male blastocyst-stage embryos (3.5 days post coitum) at approximately 40-100 copies per cell, long before overt sex differentiation. These results indicate that preimplantation mouse embryos have sexually dimorphic gene expression at least with respect to Sry transcripts. In addition, at least some of the Sry RNA transcripts in blastocysts are circular, as has been reported for Sry transcripts from adult testis. The appearance of Sry transcripts in blastocysts at this level raises the possibility that sex determination begins earlier during embryonic development than previously thought.

Animals↗
PubMed1993

Local domains of supercoiling activate a eukaryotic promoter in vivo.

Experiments correlating template topology with transcriptional activity suggest that DNA topology plays a role in eukaryotic gene expression. Linear templates transfected into cultured cells produce far fewer transcripts than do circular transcription templates, and no transcripts can be detected from linear templates injected into Xenopus oocytes. Further, when transcriptionally active circular templates in Xenopus oocytes are linearized by injection of a restriction enzyme, transcription dramatically decreases. Here we show that transcription by phage T7 RNA polymerase from a divergent promoter can partially replace the requirement for circular Xenopus ribosomal RNA transcription templates in Xenopus oocytes. Supercoiled domains can apparently be generated on short pieces of DNA having no known sequences that result in association with the nuclear architecture, suggesting that localized, transient domains of supercoiling fulfil the minimum topological needs for Xenopus rRNA transcription in vivo.

Animals↗
PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. II. Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex.

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations. The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical. The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell. Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses. Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure. The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Bacteriophage phi 6↗
PubMed1984

Effects of secondary structures in RNA on interlocking probabilities.

In 1967 Wang and Schwartz reported on the formation of interlocked rings between linear or circular DNA molecules by the enzyme topoisomerase. We propose viewing the secondary structured loop in RNA (or single stranded DNA) as analogous to a circular DNA molecule. Formation of a catenane between such an RNA loop with a DNA molecule may constitute a probe of the secondary and general three dimensional structure of the RNA molecule. The experimental results may be compared with the theoretical calculation. We suggest here a method for estimating linkage probabilities and calculate them for several cases for which secondary structures of the RNA have been proposed.

DNA Topoisomerases, Type I↗
PubMed2004

Delayed HIV-1 infection of CD4+ T lymphocytes from therapy-naïve patients demonstrated by quantification of HIV-1 DNA copy numbers.

Measuring the amount of HIV-1 DNA in infected cells is important to estimate the size of the viral reservoir in patients. However, the clinical impact of the intracellular viral DNA level remains unclear. The present study examines the clinical significance of the HIV-1 DNA level in peripheral CD4+ T lymphocytes from 21 therapy-naïve patients. HIV-1 DNA levels in purified peripheral CD4+ T lymphocytes were measured by the real-time PCR method using the Roche LightCycler system that can detect 200 copies/10(6) cells. We detected intracellular HIV-1 DNA in 15 (71.4%) of 21 patients at levels ranging from 270 to 98,120 copies/10(6) CD4+ cells, with a median of 2,220 copies/10(6) cells. We also found HIV-1 DNA that was below the detection limit in the remaining 6 patients, although 8,800-150,000 copies/ml of HIV-1 RNA were detected in plasma. Circular HIV-1 DNA was not detected in 5 of 6 cases, suggesting that reverse transcription in CD4+ T lymphocytes of these cases was not active. Thus, delayed HIV-1 infection of CD4+ T lymphocytes was demonstrated in these patients. The level of HIV-1 DNA in peripheral CD4+ T lymphocytes indicates the clinical status of therapy-naïve patients.

CD4-Positive T-Lymphocytes↗
PubMed2006

G-specific RNA-cleaving conjugates of short peptides and oligodeoxyribonucleotides.

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.

Base Pairing↗
PubMed1989

[Physicochemical parameters of rat liver chromatin during nonhormonal induction].

The changes of RNA-polymerase activity of hepatocytes nuclei, chromatin circular dichroism (CD) spectra and differential melting curves of hepatocytes of rats which were injected hydrocortisone or amino acid mixture intraperitoneally have been studied. Actinomycin D reduces essentially RNA-polymerase activity and maximum of CD spectra. Under the influence of hydrocortisone or amino acid mixture the inhibiting action of antibiotic is taken off but the RNA-polymerase activity and maximum of CD spectra don't achieve the meanings found under the influence of inductors only. The antibiotic injection changes essentially the chromatin melting parameters. A conclusion has been drawn that the amino acid mixture and hydrocortisone effect the transcription process affecting the different levels of chromatin structure.

Amino Acids↗
PubMed1999

A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses.

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

Badnavirus↗
PubMed1979

Nuclease activity associated with the Ustilago maydis virus induced killer proteins.

An in vitro nuclease activity was found to be associated with the purified killer proteins of Ustilago maydis. The proteins are effective against single stranded RNA, single and double stranded DNA. Endonucleolytic activity was confirmed by cleavage of circular molecules of 0x174 and PM2. Double stranded RNA did not appear to serve as a substrate.

Basidiomycota↗
PubMed1985

Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.

Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III.

Cellulose↗
PubMed2006

Hepatitis delta virus genetic variability: from genotypes I, II, III to eight major clades?

Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and propagation, and infects nearly 20 million people worldwide. The HDV genome is composed of a compact circular single-stranded negative RNA genome with extensive intramolecular complementarity. Along with epidemiological, geographic distribution and pathological patterns, the variability of HDV has been limited to three genotypes and two subtypes that have been characterized to date. Recently, extensive phylogenetic reconstructions based on the delta antigen gene and full-length genome sequence data, have shown a wide and probably ancient radiation of African lineages, suggesting that the genetic variability of HDV is much more complex than previously thought. Indeed, sequences previously affiliated with genotype IIb should now be considered as belonging to clade 4 (HDV-4) and African HDV sequences segregate within four additional clades: HDV-5, HDV-6, HDV-7 and HDV-8. These results bring the geographic distribution of HDV closer to the genetic variability of its helper HBV.

Amino Acid Sequence↗
PubMedDate not supplied

Structure, sequence and expression of the hepatitis delta (delta) viral genome.

Biochemical and electron microscopic data indicate that the human hepatitis delta viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis delta viral infections.

Base Sequence↗
PubMed1983

Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy.

The complete cDNA of hop stunt viroid (HSV) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170. (1982] and the complete nucleotide sequence has been established. The covalently closed circular single-stranded HSV RNA consists of 297 nucleotides. The secondary structure predicted for HSV contains 67% of its residues base-paired. The native HSV can possess an extended rod-like structure characteristic of viroids previously established. The central region of the native HSV has a similar structure to the conserved region found in all viroids sequenced so far except for avocado sunblotch viroid. The sequence homologous to the 5'-end of U1a RNA is also found in the sequence of HSV but not in the central conserved region.

Base Sequence↗
PubMed1995

Circulation of oligonucleotides by disulfide bridge formation.

An effective, convenient method for the circularization of oligonucleotides has been developed. This procedure involved preparation of an oligonucleotide with backbone-linked 5'- and 3'-terminal hexamethylenethiol groups, followed by oxidation of the thiol groups with air of oxygen to produce the corresponding circular sequence bridged via a bis(hexamethylene)-disulfide moiety. The method has been applied to the circularization of oligodeoxynucleotide sequences of varying lengths (5, 10, 15, 20, 30 and 40 bases), and the circularization process was highly efficient as shown by HPLC or gel electrophoresis of the crude reaction mixtures. Competing reactions such as dimerization were not significant except for the longer sequences (30 and 40 bases). The circularization of an eight base RNA sequence was also accomplished, as well as hexa-ethylene glycol bridged poly-T sequences capable of triplex formation.

Base Sequence↗