PubMed Health⌕ Search

Biomedical subjects

C C Hardin

Publications and source records attributed to C C Hardin.

31 records · Page 2Linked to original sources
PubMed1988

Characterization of anti-Z-RNA polyclonal antibodies: epitope properties and recognition of Z-DNA.

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Binding of ethidium ion to left-handed Z-RNA induces a cooperative transition to right-handed RNA at the intercalation site.

The equilibrium binding of the ethidium cation (Etd+) to the right-handed A-form of poly-[r(C-G)], the B-form of poly[d(C-G)], and the left-handed Z-forms of Br-poly[r(C-G)] and Br-poly[d(C-G)] was investigated in 0.22 M NaCl by optical methods. Scatchard analysis indicates that Etd+ intercalates into right-handed forms of poly[r(C-G)] and poly[d(C-G)] in a noncooperative manner. Correlation of Etd+ absorbance binding isotherms and polynucleotide circular dichroism data indicates that drug binding to Br-poly[r(C-G) and Br-poly[d(C-G)] results in cooperative conversion from left-handed Z-forms to right-handed intercalated conformations. Approximate stoichiometries necessary to induce the left- to right-handed transitions are 1 Etd+/9 base pairs (bp) for Z-RNA and 1 Etd+/6 bp for Z-DNA. The apparent limiting binding stoichiometries are approximately 1 Etd+/3 bp for RNA and 1 Etd+/2 bp for DNA. The equilibrium binding constants for binding to the right-handed forms decrease in the order Br-poly[d(C-G)], Br-poly[r(C-G)], poly[d(C-G)], and poly[r(C-G)]. Thermodynamic parameters are obtained by van't Hoff analysis of Etd+ absorbance thermal dissociation data. Enthalpy values for all four polynucleotides are negative and of similar magnitude. Negative entropy values indicate that the binding processes are primarily enthalpically driven.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1988

Partial assignment of resonances in the 19F nuclear magnetic resonance spectra of 5-fluorouracil-substituted transfer RNAs.

Features of the 19F nuclear magnetic resonance (NMR) spectra of three purified 5-fluorouracil-(FUra-) substituted Escherichia coli tRNAs, tRNA(1Val), tRNA(mMet), and tRNA(fMet), are compared. Each of the tRNA species can be resolved into two isoaccepting forms, A and B, whose 19F NMR spectra differ in the shift of one peak from the 4.5 to 4.8 parts per million (ppm) range (FUra = O) in the spectrum of isoacceptor B upfield to ca. -15 ppm in that of isoacceptor A. Because the sequences of the two isoacceptors of each tRNA differ only at one position in the D loop, that normally occupied by a dihydrouridine residue, we assign the 4.5 ppm peak in the spectrum of fluorine-labeled tRNA(1Val) to FUra17 and the resonance at 4.6 ppm in the spectrum of fluorouracil-substituted tRNA(mMet) to FUra20. A reciprocal 19F[19F] nuclear Overhauser effect is observed between the downfield peaks A and B in the 19F NMR spectrum of 19F-labeled tRNA(1Val). Assuming that fluorine-labeled tRNA(1Val) has a structure similar to that of yeast tRNA(Phe), only FUra54 and -55 are close enough (4-5 A) to give an appreciable 19F homonuclear Overhauser effect. Peaks A and B have therefore been assigned to FUra54 and -55. As the temperature is raised from 30 to 45 degrees C, the intensity of peak B (6.6 ppm) in the spectrum of 19F-labeled tRNA(1Val) gradually shifts upfield to 6.4 ppm (Tm = 36 degrees C), indicating a temperature-dependent slow exchange of the corresponding 5-fluorouracil residue between two magnetically distinct environments.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1987

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs.

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.

Animals↗
PubMed1987

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA. A 19F nuclear magnetic resonance relaxation study.

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).

Chemical Phenomena↗
PubMed1987

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA.

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

Anticodon↗
PubMed1987

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies.

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1987

Cytoplasmic Z-RNA.

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

Animals↗
PubMed1986

Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA.

19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have pKa's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1986

Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.

Anticodon↗
PubMed1983

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA.

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized. This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNAMetm and tRNAVal1, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with 19F spectrum of the model compound 5'-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (delta FUra = -31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd). At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at - 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at - 15 ppm. Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz). Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at -33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.

Escherichia coli↗
PubMedDate not supplied

Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids.

The dissociation and assembly of quadruplex DNA structures (and a few quadruplex RNAs) have been characterized at several levels of rigor, ranging from gross descriptions of factors that govern each process, to semiquantitative comparisons of the relative abilities of these factors to induce stabilization or destabilization, to quantitative studies of binding energies (thermodynamics), transformational rates (kinetics), and analysis of their transition-state energies and mechanisms. This survey classifies these factors, describes the trends and focuses on their interdependencies. Quadruplex assembly is induced most efficiently by added K(+) and elevating the strand concentration; however, Na(+), NH(4)(+), Sr(2+), and Pb(2+) are also very effective stabilizers. Quadruplex dissociation is typically accomplished by thermal denaturation, "melting"; however, when the quadruplex and monovalent cation concentrations are low enough, or the temperature is sufficiently high, several divalent cations, e.g., Ca(2+), Co(2+), Mn(2+), Zn(2+), Ni(2+) and Mg(2+) can induce dissociation. Stabilization also depends on the type of structure adopted by the strand (or strands) in question. Variants include intramolecular, two- and four-stranded quadruplexes. Other important variables include strand sequence, the size of intervening loops and pH, especially when cytosines are present, base methylation, and the replacement of backbone phosphates with phosphorothioates. Competitive equilibria can also modulate the formation of quadruplex DNAs. For example, reactions leading to Watson-Crick (WC) duplex and hairpin DNAs, triplex DNAs, and even other types of quadruplexes can compete with quadruplex association reactions for strands. Others include nonprotein catalysts, small molecules such as aromatic dyes, metalloporphyrins, and carbohydrates (osmolytes). Other nucleic acid strands have been found to drive quadruplex formation. To help reinforce the implications of each piece of information, each functional conclusion drawn from each cited piece of thermodynamic or kinetic data has been summarized briefly in a standardized table entry.

Base Pairing↗