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G Zon

Publications and source records attributed to G Zon.

At least 109 records · Page 6Linked to original sources
PubMed1987

The potentially Z-DNA-forming sequence d(GTGTACAC) crystallizes as A-DNA.

(GT)n/(CA)n sequences have stimulated much interest because of their frequent occurrence in eukaryotic DNA and their potential for forming the left-handed Z-DNA structure. We here report the X-ray crystal structure of a self-complementary octadeoxynucleotide, d(GTGTACAC), at 2.5 A resolution. The molecule adopts a right-handed double-helical conformation belonging to the A-DNA family. In this alternating purine-pyrimidine DNA minihelix the roll and twist angles show alternations qualitatively consistent with Calladine's rules. The average tilt angle of 9.3 degrees is between the values found in A-DNA (19 degrees) and B-DNA (-6 degrees) fibers. It is envisaged that such intermediate conformations may render diversity to genomic DNA. The base-pair tilt angles and the base-pair displacements from the helix axis are found to be correlated for the known A-DNA double-helical fragments.

Base Sequence↗
PubMed1987

Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits.

Arginine 179 of the alpha subunit of tryptophan synthase of Salmonella typhimurium was changed to leucine by site-directed mutagenesis. The mutant alpha subunit was expressed in S. typhimurium, purified and crystallized as the alpha 2 beta 2 complex, and characterized by kinetic studies under steady-state reaction conditions. The rate of cleavage of indole 3-glycerol phosphate (alpha reaction) is reduced by 60% in the mutant alpha 2 beta 2 complex, whereas the rate of L-tryptophan synthesis from indole and L-serine (beta reaction) is unchanged. Thus, arginine 179 is not obligatory for catalysis, for binding of indole 3-glycerol phosphate, or for interaction of the alpha and beta 2 subunits. However, changing arginine 179 to leucine does have striking effects on ligand-dependent properties of this multienzyme complex. Ligands of the alpha subunit (DL-alpha-glycerophosphate and indole 3-propanol phosphate) which strongly inhibit the beta reaction of the native alpha 2 beta 2 complex have a slight stimulatory effect on the beta reaction of the mutant alpha 2 beta 2 complex. Likewise, L-serine, a ligand of the beta subunit which produces a 5-fold reduction in the Km for the alpha ligand indole 3-glycerol phosphate in the native alpha 2 beta 2 complex, has no effect on the mutant alpha 2 beta 2 complex. These results suggest that arginine 179 of the alpha subunit plays a role in the reciprocal transmission of substrate-induced conformational changes which occur between native alpha and beta 2 subunits in the alpha 2 beta 2 complex.

Amino Acid Sequence↗
PubMed1987

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages.

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.

Acetyltransferases↗
PubMed1987

Oligodeoxyribonucleoside methylphosphonates. NMR and UV spectroscopic studies of Rp-Rp and Sp-Sp methylphosphonate (Me) modified duplexes of (d[GGAATTCC])2.

1H NMR chemical shift assignments for the title compounds were made for most of the 1H signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which were also used to establish the absolute configuration at the modified phosphorus. The chemical shifts were similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified, parent, B-type duplex [d(GGAATTCC)]2. Differences in chemical shifts were mostly localized to the nucleotides on the 5'- and 3'-sides of the modified phosphorus. The Rp-Rp isomers exhibited UV-derived Tm values similar to that of the parent duplex. On the other hand, the Sp-Sp isomers generally exhibited lower Tm values which correlated with P-CH3--H3' (n-1 nucleotide) cross peak intensities and 31P spectral parameters. The combined data argue for increased steric interactions with the Sp-P-Me methyl group as the modification position is moved toward the center of the oligomer. All of the Tm results can be explained in terms of three factors which result from replacement of a phosphate by a methylphosphonate group: reduction of oligomer charge; electronic and other substituent effects; steric interactions.

Deoxyribonucleosides↗
PubMed1987

Absorption mode two-dimensional NOE spectroscopy of exchangeable protons in oligonucleotides.

A new NMR method is described for the generation of absorption mode two-dimensional NOE spectra of oligonucleotides in H2O solution. The method yields spectra that are free of baseline distortions with excellent suppression of the intense H2O resonance. The method is demonstrated for a sample of the dodecamer d(CGCGAATTCGCG)2. All exchangeable base protons are identified and a number of new types of NOE connectivities are observed.

Amines↗
PubMed1987

Synthesis and characterization of oligodeoxyribonucleotides containing terminal phosphates. NMR, UV spectroscopic and thermodynamic analysis of duplex formation of [d(pGGAATTCC)]2, [d(GGAATTCCp)]2 and [d(pGGAATTCCp)]2.

Derivatives of the oligomer [d(GGAATTCC)]2 with 5' (5'-P), 3' (3'-P) and both 5' and 3' (5',3'-P2) terminal phosphate groups have been synthesized and studied by temperature dependent UV and NMR spectroscopic methods. Thermodynamic studies of the helix to strand transition indicate that addition of 3' phosphate groups has very little effect on the delta G degree for helix formation at 37 degrees C while addition of 5' phosphate groups adds approximately -0.5 kcal/mole to the delta G degree for duplex formation. The helix stabilization by 5' phosphate groups occurs at salt concentrations of 0.1 M and above, and is primarily enthalpic in origin. Tm studies as a function of ionic strength also indicate that the oligomers fall into two groups with the parent and 3'-P derivatives being similar but less stable than the 5'-P and 5',3'-P2 derivatives. Imino proton and 31P NMR studies also divide the oligomers into these same two groups based on spectral comparisons and temperature induced chemical shift and linewidth changes. 31P NMR analysis suggests that addition of 5' phosphate groups results in a small change in phosphodiester torsional angles in the g,t to g,g direction, indicating improved base stacking at the 5' end of the modified oligomer. No such changes are seen at the 3' end of the oligomer on adding 3' phosphate groups.

Magnetic Resonance Spectroscopy↗
PubMed1987

NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. 2. Direct observation, characterization, and reactivity studies of iminocyclophosphamide and related species.

4-Hydroxy-5,5-dimethylcyclophosphamide (6) was synthesized as a stable (to fragmentation) analogue of 4-hydroxycyclophosphamide (1). In anhydrous Me2SO-d6 (less than or equal to 0.03 mol % water), cis- and trans-6 were observed by multinuclear NMR spectroscopy to equilibrate with alpha, alpha-dimethylaldophosphamide (7) and 5,5-dimethyliminocyclophosphamide (8). Identification of 8 was based on 1H, 13C, and 31P chemical shifts, selective INEPT and two-dimensional NMR correlation experiments, and temperature-dependent equilibria data. The interconversion of cis-/trans-6 and -7 was also observed in lutidine buffer; 8 was not detected under the aqueous conditions. In Me2SO-d6, hydroxy metabolite 1 underwent dehydration to give iminocyclophosphamide (5), as evidenced by chemical shift data and a selective INEPT experiment. Concentrations of cis-/trans-1, aldophosphamide (2), and 5 were found to be temperature-dependent with higher temperatures favoring 2 and 5 in a reversible manner, thus indicating that 1/2/5 were intercoverting. The addition of small amounts of water to Me2SO-d6 solutions of imine 5 resulted in the immediate disappearance of its NMR signals. The role of imine 5 in the conversion of 1 to C-4 substituted analogues of 1 was elucidated for the formation of 4-cyanocyclophosphamide (3a) from 1 and sodium cyanide in lutidine buffer.

Cyclophosphamide↗
PubMed1987

Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.

Nuclease-resistant phosphorothioate analogs of certain oligodeoxynucleotides have been tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Phosphorothioate analogs complementary to HIV sequences, as well as noncomplementary analogs including homooligomers, exhibited potent antiviral activity. The antiviral activity was related to the base composition of the analogs, and longer phosphorothioates were more effective than shorter ones. A 28-mer phosphorothioate oligodeoxycytidine (S-dC28) at a concentration of 1 microM exhibited potent antiviral activity and inhibited de novo viral DNA synthesis as shown by Southern blot analysis. However, S-dC28 failed to inhibit gag expression in chronically infected T cells assessed by immunofluorescent assay at concentrations up to 25 microM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanism of action is likely to be complex.

Base Sequence↗
PubMed1987

NMR studies of backbone-alkylated DNA: duplex stability, absolute stereochemistry, and chemical shift anomalies of prototypal isopropyl phosphotriester modified octanucleotides, (Rp,Rp)- and (Sp,Sp)-(d-[GGA(iPr)ATTCC])2 and -(d-[GGAA(iPr)TTCC])2.

The DNA octamer (d-[GGAATTCC])2 and four alkylated analogues, (Rp)-(d-[GGA(iPr)ATTCC])2, (Sp)-(d-[GGA(iPr)ATTCC])2, (Rp)-(d-[GGAA(iPr)TTCC])2, and (Sp)-(d-[GGAA(iPr)TTCC])2 have been examined using 1H and 31PNMR spectroscopies. Duplex stability, as monitored by both NMR and optical measurements, is shown to be a function of both site and stereochemistry of the phosphotriester moiety. Chemical shift changes relative to the native octamer indicate that there are long-range perturbations in the isopropylated molecules. 1HNMR is shown to be a general means by which stereochemistry at phosphorous can be determined.

Alkylating Agents↗
PubMed1986

Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscopic studies of the Rp-Rp, Sp-Sp, and Rp-Sp duplexes, [d(GGSAATTCC)]2, derived from diastereomeric O-ethyl phosphorothioates.

2D-NOE and 1H NMR chemical shift data obtained for the title oligonucleotides were compared with similar data previously reported [Broido et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified "parent" structure, [d(GGAATTCC)]2. The spectroscopically detectable structural perturbations caused by replacement of phosphate oxygen with sulfur were mostly localized within the GsA moiety, and were greater for the Rp configuration wherein sulfur is oriented into the major groove of the B-helix. UV-derived Tm measurements gave the following order of stability for the duplexes in 0.4 M NaCl: unmodified (33.9 +/- 0.1 degrees C) approximately Sp-Sp (34.1 degrees C) greater than Rp-Rp (31.7 degrees C). The title compounds were prepared by a new and convenient synthetic route which utilized HPLC to separate the diastereomeric O-ethyl phosphorothioate precursors, (Rp)- and (Sp)-d[GG(S,Et)AATTCC], for subsequent de-ethylation by ammonia in water.

Indicators and Reagents↗
PubMed1986

Sequence-dependent cooperative interactions in A/T-containing oligo- and polydeoxyribonucleotides.

To evaluate the length and sequence dependence of the unusual interaction properties observed for nonalternating A/T sequences in deoxyribonucleic acid (DNA) [Wilson, W. D., Wang, Y. H., Krishnamoorthy, C. R., & Smith, J. C. (1985) Biochemistry 24, 3991-3999], we have synthesized the oligomers d(A-T)6, dA10 X dT10, and d(A6-T6) and evaluated their interaction with the intercalator propidium. Propidium visible spectral shifts on adding all three oligomers are quite similar. Low-temperature spectrophotometric binding measurements indicate that d(A-T)6 has a significantly larger binding constant for propidium than dA10.dT10, as with the analogous alternating and nonalternating DNA polymers. The oligomer dA10.dT10 displays positive cooperativity in its propidium binding isotherm, and its binding constant increases with increasing temperature while d(A-T)6 does not display positive cooperativity, and its binding constant decreases with temperature, again as with the analogous polymers. van't Hoff plots indicate that the propidium binding enthalpies are approximately -9 and +6 kcal/mol for the alternating and nonalternating DNA samples, respectively. The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively. A van't Hoff plot indicates that the cooperative sites d(A-T)6 have binding constants and binding enthalpies similar to dA10.dT10. Similar rate constants are observed in the sodium dodecyl sulfate driven dissociation reaction of propidium from d(A-T)6 and d(A6-T6), but the association reaction of propidium is significantly slower with d(A6-T6) than with d(A-T)6.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy.

The use of new 1H-detected heteronuclear 1H-31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4' and C5' sugar protons. Use of the new methods enables one to make sequence-specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.

DNA↗
PubMed1986

Solution structure of [d(A-T)5]2 via complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect spectra and molecular mechanics calculations: evidence for a hydration tunnel.

Pure absorption phase proton two-dimensional nuclear Overhauser effect (2D NOE) spectra at 500 MHz have been obtained for [d(5'ATATATATAT3')]2 in deuterium oxide solution at several mixing times. The 100 nonexchangeable proton resonances have been assigned. The experimental 2D NOE spectra were compared with theoretical spectra calculated by using the complete relaxation matrix analysis method [Keepers, J. W., & James, T. L. (1984) J. Magn. Reson. 57, 404-426] and x-ray diffraction determined molecular coordinates of A, B, alternating B, left-handed B, C, D, and wrinkled D forms of DNA and of energy-minimized structures calculated from the most promising X-ray crystal structures by using the molecular mechanics program AMBER, in which all hydrogens, counterions, and hydration water molecules were included. The analysis of all features of the 2D NOE spectra played an important role in extracting the promising structures, and it was concluded that the wrinkled D form yields the best fit for the 2D NOE data of the A-T decamer. The molecular mechanics calculation indicated that this model structure, whose minor groove is comparatively deep and narrow, may be energetically more stable than the B form for alternating d(A-T) DNA. Interesting features of the structure include possible intra- and interchain sugar-phosphate attractions and a hydration tunnel inside the minor groove capable of accommodating three types of water molecules that aid in helix stabilization via hydrogen bonding. Counterions (sodium) serve to reduce interchain phosphate-phosphate repulsive effects.

Base Sequence↗
PubMed1986

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus.

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.

Amino Acid Sequence↗
PubMed1986

Alkyl phosphotriester modified oligodeoxyribonucleotides. V. Synthesis and absolute configuration of Rp and Sp diastereomers of an ethyl phosphotriester (Et) modified EcoRI recognition sequence, d[GGAA(Et)TTCC]. A synthetic approach to regio- and stereospecific ethylation-interference studies.

Protected deoxynucleoside 3'-O-ethyl-N,N-diisopropylphosphoramidite reagents were prepared for use in the automated synthesis of ethyl phosphotriester (Et) modified oligonucleotides. The title diastereomers were separated by reversed-phase HPLC, and chirality at phosphorus was assigned by an improved configurational correlation scheme that was verified by NMR spectroscopic studies (accompanying paper, Part VI). This generally applicable correlation scheme involved enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; phosphite triester sulfurization to obtain diastereomeric O-ethyl phosphorothioates, d[AS(Et)T], which were separated by HPLC for stereoretentive oxidation with H2O2 to give d[A(Et)T], and stereoretentive de-ethylation with PhSH-Et3N to give diastereomeric phosphorothioates, d[AST], whose configurations at phosphorus had been assigned previously. Neither the Rp-Rp nor Sp-Sp duplex, (d[GGAA(Et)TTCC])2, was cleaved by EcoRI endonuclease under conditions that led to cleavage of both the unmodified duplex, [d(GGAATTCC)]2, and the mixture of diastereomeric phosphorothioate-modified duplexes, [d(GGAASTTCC)]2. Cleavage of the latter substrates was Sp-selective.

Base Sequence↗
PubMed1986

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.

Base Sequence↗
PubMed1986

Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process.

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii. Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (approximately 50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.

Carbohydrates↗