Circular dichroism evidence for G-U and G-T base pairing in poly[r(G-U)] and poly[d(G-T)].
Explore the source record for details and available documents.
Binding Sites↗
SEARCH · Search PubMed
Results for “Poly G”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Interaction of antimalarial drug quinacrine with nucleic acids of variable sequence studied by spectroscopic methods.
The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV-visible absorption, fluorescence and surface-enhanced Raman spectroscopy (SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.
Animals↗
A family of cold-regulated RNA-binding protein genes in the cyanobacterium Anabaena variabilis M3.
I previously found a cold-regulated RNA-binding protein gene rbpA (now named rbpA1) in Anabaena variabilis M3 [Sato, N. (1994) Plant Mol. Biol. 24, 819-823]. I show here that this gene is a member of a gene family containing at least eight members as evidenced by Southern blot and immunoblot analyses. I have isolated three additional genes (rbpB, rbpC and rbpD) in this family. Of these, rbpB was 100% identical to the rbpB gene of Anabaena 7120 reported previously. Another gene named rbpA in Anabaena 7120 was also found to exist in A.variabilis M3 with identical sequence and named rbpA2. The amino acid sequences of these gene products were highly conserved, except that the RbpD protein lacked glycine-rich C-terminal domain present in all other known members of the gene family. RNA blot and immunoblot analyses showed that the expression of rbpA1, rbpA2, rbpB, rbpC and rbpD, as well as uncloned rbp genes was regulated by cold, though the exact time-course and extent of response to cold were different among these genes. Gel-filtration assay showed that all of the Rbp proteins have higher affinities to poly(G) and poly(U) than to poly(A) and poly(C).
Amino Acid Sequence↗
Wheat germ tRNAs containing uridine in place of ribothymidine: a characterization of an unusual class of eukaryotic tRNAs.
An unusual class of wheat germ tRNAs has been isolated which completely lacks ribothymidine (rT) and contains an unmodified uridine in its place. We discuss here the isolation, identification and properties of these tRNAs. The rT-lacking tRNAs of wheat germ are essentially limited to the glycine isoacceptors (a minimum of five identifiable species), three threonine and at least, one tyrosine tRNA. All tRNAs were obtained 70-100% pure by chromatographic methods, and were detected by their ability to be methylated by E. coli rT-forming uracil methyltransferase with methyl-labeled S-adenosyl-L-methionine (SAM) as the methyl donor. In vitro methylation of each of the tRNAs resulted in the formation of 1 mole of rT per mole of tRNA. In the one case analyzed in detail (tRNA1Gly), all of the rT was found to be located at the 23rd position from the 3' end of the tRNA molecule. Following complete digestion of four highly purified glycine isoacceptors (tRNAGly1,4,5,6) to nucleosides and subsequent periodate oxidation and 3H potassium borohydride reduction, all were found to contain an unusually high level of 5-methylcytidine (m5C) (3-4 residues per molecule), and all contained no rT. The possible correlation between the presence of m5C and the absence of rT is discussed. All of the chromatographically purified glycine tRNAs function in a wheat germ cell-free protein synthesizing system and polymerize glycine in response to either poly G or poly (G, U).
Base Sequence↗
[Effect of the combined use of dextran sulfate and double-stranded RNA on interferon production].
The studies were performed on CBA mice. It was shown that polynucleotide inductors (poly I-poly C, RFf2 and poly G-poly C) stimulated production of interferon with a peak attained 4-6 or 24 hours after the induction. Intraperitoneal injection of dextran sulfate in a dose of 200 micrograms/m induced production of interferon in small amounts (80-120 units). The production peak was observed in 12 hours. The combined use of dextran sulfate and the polynucleotide inductors provoked an increase in the interferon production. The effect was of a synergistic nature. The cells of the immunocompetent system were the target of the dextran sulfate effect in the host, which was shown experimentally with a culture of mouse lymphocytes.
Animals↗
Laser-induced modification of DNA and Poly[A,G] at guanine moiety using acetone as photosensitizer.
The interactions of triplet acetone with polyadenylic acid (Poly[A]), polyguanylic acid (Poly[G]), polyadenylic-guanylic acid (Poly[A,G]) and single-stranded DNA (ssDNA) were investigated in neutral aqueous solution using KrF (248 nm) laser flash photolysis. The transient absorption spectra and kinetics of DNA and polynucleotides obtained under acetone sensitization demonstrated that the predominant transient species was guanine radical. These novel findings have offered time-resolved evidence for photochemical modification of DNA and Poly[A,G] at guanine moiety.
Acetone↗
Inhibition of human neutrophil elastase by polyguanylic acid and other synthetic RNA homopolymers.
Human neutrophils contain large amounts of a neutral serine protease, human neutrophil elastase (HNE), which has been implicated as a mediator of acute and chronic lung injury. We found that this enzyme is effectively inhibited, at physiological ionic strength, by several synthetic non-base-paired polyribonucleotides. Among the most active of these is polyguanylic acid (poly G). Inhibitory activity is greatest with high-molecular-weight poly G fractions, but poly G fractions even as low as 60K Mr (app) are effective. Both amidolysis of synthetic elastase substrates, such as succinyl-ala-ala-ala-p-nitroanilide, and proteolysis of elastin are blocked. Poly G inhibits elastin proteolysis even when subsequently added to mixtures of elastin and HNE that have first been preincubated together for 10 min. Under these conditions, polyribosylribitol phosphate, a polyanion derived from Haemophilus influenzae capsular polysaccharide, is not inhibitory. Complex formation between HNE and poly G is dependent on ionic rather than covalent interactions, since it is blocked by 0.6 M NaCl but not by inactivation of the enzyme's catalytic-site serine residue with diisopropylfluorophosphate. However, nonspecific ionic interactions alone cannot explain complex formation, since pancreatic elastase and cathepsin G, an even more basic serine protease from human neutrophils, do not form complexes with poly G, even at low ionic strength. Moreover, in the presence of the amphiphiles taurocholic acid and glycocholic acid, HNE is much less effectively blocked by poly G. Peptide chloromethyl ketone-inactivate HNE (which has its extended substrate-binding pocket occupied by the peptidyl inactivator) also fails to form complexes with poly G. These results indicate that HNE may utilize both hydrophobic and ionic binding sites to couple with poly G, and suggest that these sites may be close to or within the extended substrate-binding pocket of the enzyme.
Amino Acid Chloromethyl Ketones↗
Immunochemical similarities between monoclonal antibacterial Waldenstrom's macroglobulins and monoclonal anti-DNA lupus autoantibodies.
Six monoclonal IgM from patients with Waldenstrom's macroglobulinemia that react with Klebsiella polysaccharides were tested for their ability to bind to nucleic acid antigens. One of the macroglobulins bound to the polynucleotide poly(G), and one bound to poly(G), poly(I), and single-stranded DNA. The reaction with the polynucleotides was specifically inhibited by the Klebsiella polysaccharide K30. A monoclonal lupus anti-DNA antibody (16/6) was found to react weakly with the Klebsiella polysaccharides K30 and K21. Five of the Waldenstrom macroglobulins shared an idiotypic determinant with the 16/6 anti-DNA antibody. The reaction between the macroglobulins and the antiidiotype serum was specifically inhibited by Klebsiella polysaccharides, an indication that the idiotypic marker was in the antigen-binding site of the macroglobulins. These results indicate the existence of widely dispersed conserved variable region genes that encode idiotypically related immunoglobulins with the capacity to bind to both bacterial polysaccharides and nucleic acids. Such genes can be expressed by patients with either Waldenstrom's macroglobulinemia or systemic lupus erythematosus.
Antibodies, Bacterial↗
Role of V protein RNA binding in inhibition of measles virus minigenome replication.
Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein-RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3' terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein-RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238-266 were necessary for efficient RNA binding and overlapped with residues (238-278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238-247) that blocked RNA binding and repression indicating that these two activities were related.
Amino Acid Substitution↗
Mitomycin C binding to poly[d(G-m5C)].
Poly[d(G-m5C)] was modified by reductively activated mitomycin C, an anti-tumour drug, under buffer conditions which are known to favour either the B or the Z conformations of DNA. C.d. and 31P-n.m.r. were used to characterize the poly[d(G-m5C)]-mitomycin cross-linked complexes, as well as the effects on the equilibrium between the B and Z forms of the polynucleotide. Mitomycin C appears to inhibit the B-->Z transition, even in the presence of 3 mM MgCl2, while the Z-form of poly[d(G-m5C)] does not interact significantly with the drug under bifunctionally activating conditions; thus no reversion from the Z-form to the B-form of the polynucleotide can be observed under the salt conditions which are required for the Z-form to exist.
Circular Dichroism↗
Intracellular alginolytic enzymes of the marine bacterium Pseudoalteromonas citrea KMM 3297.
The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus. When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII. Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII. These enzymes were separated using anion-exchange chromatography. The alginate lyase AlI completely retains its activity at 35 degrees C, AlII and AlIII being stable at 45 degrees C. The alginate lyases exhibit maximal activities in the range of pH 7-8. The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively. All the investigated enzymes are endo-type alginate lyases. They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 > or = n > or = 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus. A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate. The Km values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 micro g/ml, respectively. Alginate lyase AlIII exhibits less affinity to poly-M (Km = 130.0 microg/ml) than to poly-G (Km = 40.0 microg/ml). NaCl (0.2 M), MgCl2 and MgSO4 (0.01 M) activate all three enzymes more than twofold. The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P. citrea KMM 3297.
Alginates↗
Mercury-induced transitions between right-handed and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)].
Poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)], each dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.8, experience inversion of their circular dichroism (CD) spectrum subsequent to the addition of Hg(ClO4)2. Let r identical to [Hg(ClO4)2]added/[DNA-P]. The spectrum of the right-handed form of poly[d(A-T).d(A-T)] turns into that of a seemingly left-handed structure at r greater than or equal to 0.05 while a similar transition is noted with poly[d(G-C).(G-C)] at r greater than or equal to 0.12. The spectral changes are highly cooperative in the long-wavelength region above 250 nm. At r = 1.0, the spectra of the two polymers are more or less mirror images of their CD at r = 0. While most CD bands experience red-shifts upon the addition of Hg(ClO4)2, there are some that are blue-shifted. The CD changes are totally reversible when Hg(II) is removed from the nucleic acids by the addition of a strong complexing agent such as NaCN. This demonstrates that mercury keeps all base pairs in register.
Circular Dichroism↗
Protonated forms of poly[d(G-C)] and poly(dG).poly(dC) and their interaction with berberine.
The pH -induced structural changes on the conformation of homo- and hetero-polymers of guanosine-citydine (G.C) sequences were investigated using spectrophotometric and circular dichroic techniques. At pH 3.40, 10 mM [Na(+)] and 10 degrees C both polynucleotides adopted a unique and stable structural conformation different from their respective B-form structures. The protonated hetero-polymer is established as left-handed structure with Hoogsteen base pairing (H(L)-form) while the homo-polymer favored Watson-Crick base pairing with different stacking arrangements from that of B-form structure as evident from thermal melting and circular dichroic studies. The interaction of berberine, a naturally occurring protoberberine group of plant alkaloid, with the protonated structures was studied using various biophysical techniques. Binding of berberine to the H(L)-form structure resulted in intrinsic circular dichroic changes and generation of extrinsic circular dichroic bands with opposite sign and magnitude compared to its B-form structure while with the homo-polymer of G.C no such reversal of extrinsic circular dichroic bands was seen indicating different stacking arrangement of berberine at the interaction site. Scatchard analysis of the binding data, however, indicated non-cooperative binding to both the protonated forms similar to that of their respective B-form structure. Fluorescence spectral studies, on the other hand, showed remarkable increase in the intrinsic fluorescence of the alkaloid in presence of the protonated forms compared to their respective B-form structure. These results suggest that berberine could be used as a probe to detect the alteration of structural handedness due to protonation and may potentiate its use in regulatory roles for biological functions.
Berberine↗
Poly(7-deazaguanylic acid), the homopolynucleotide of the parent nucleoside of queuosine.
Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.
Base Composition↗
[Structure of polyriboguanylic acid in solution].
The electron microscopic data and the CD spectra of poly(G) have shown that the transition of a freeze-dried preparation of poly(G) from a "metastable" to a "stable" form is the transition of the four-stranded poly(G) from the globular form to the linear one. The observed phenomenon of the formation of long four-stranded poly(G) fibers (4--5 nm in diameter and 200-2000 nm in length) is suggested to be a result of a joint of the initial poly(G) molecules (50--80 nm in length) due to noncovalent binding ("sticking") of their free one-, double- or three-stranded ends. This phenomenon has permitted us not only to compare the thickness of low molecular weight poly (G) preparations under different conditions but to observe directly the existence of a large number of double-stranded (2--3 nm in diameter) regions ("defects") in four -stranded poly(G) fibers. The data obtained have shown that under certain conditions (high temperature, low pH, etc.) the four-stranded poly(G) fibers break down with the formation of two-stranded poly(G) fibers whose hydrogen bond systems depend on the conditions of their formation.
Chemical Phenomena↗
DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides.
A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.
Cell Nucleus↗
Reovirus-specific enzyme(s) associated with subviral particles responds in vitro to polyribocytidylate to yield double-stranded polyribocytidylate-polyriboguanylate.
In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C).polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300S to 550S. Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30 degrees C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2'-O-methyl-poly(C). Synthesis was optimal in the presence of Mn(2+) as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg(2+). The size of the poly(C).poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The T(m) of the product was between 100 and 130 degrees C. Hydrolysis of the product labeled in [(32)P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.
Cell-Free System↗
Oligonucleotides tethered to a short polyguanylic acid stretch are targeted to macrophages: enhanced antiviral activity of a vesicular stomatitis virus-specific antisense oligonucleotide.
The poor membrane permeability of oligonucleotides is one of the major problems of antisense technology. Here we report the construction of designer oligonucleotides for targeted delivery to macrophages. The oligonucleotides tethered to a 10-mer poly(G) sequence at their 3' ends were recognized by scavenger receptors on macrophages and were taken up about 8- to 10-fold as efficiently as those oligonucleotides that either lacked a poly(G) tail or that contained a 10-mer poly(C) tail instead of the poly(G) tail. The enhanced uptake of poly(G) constructs was inhibited in the presence of poly(G) and other known ligands of the scavenger receptor. The bioefficacy of poly(G)-mediated targeting of antisense oligonucleotides (ANS) was demonstrated by using vesicular stomatitis virus (VSV) as a model system. The ability of ANS directed against the translation initiation site of N protein mRNA of VSV to inhibit virus replication was assessed. The ANS with the 10-mer poly(G) sequences (ANS-G) brought about significant inhibition of VSV replication in J774E cells (a murine monocyte/macrophage cell line) and Chinese hamster ovary (CHO) cell transfectants expressing scavenger receptors. The ANS lacking a 10-mer poly(G) stretch were ineffective. The inhibition of VSV replication due to ANS-G was completely abrogated in the presence of 10-mer poly(G), indicating that the antisense effect of the ANS-G molecule was a consequence of scavenger receptor-mediated enhanced uptake. Importantly, antisense molecules linked exclusively by natural phosphodiester bonds were as bioeffective as those synthesized with a mixed backbone of phosphodiester and phosphorothioate. Taken together, these results suggest that macrophage-directed designer ANS against infective agents may simply be obtained by adding a short stretch of guanylic acid sequence to the desired specific ANS during solid-phase synthesis. This nucleic acid-based strategy, which utilizes homogeneous preparation of ANS, may find applications in directed manipulation of macrophage metabolism for a variety of purposes as well as in therapy of a broad spectrum of macrophage-related disorders amenable to the antisense approach.
Animals↗