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G T Walker

Publications and source records attributed to G T Walker.

28 records · Page 2Linked to original sources

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↗

The TFIIIA recognition fragment d(GGATGGGAG).d(CTCCCATCC) is B-form in solution.

The deoxyoligonucleotide d(GGATGGGAG).d(CTCCCATCC) is a portion of the gene recognition sequence of transcription factor IIIA (TFIIIA). The crystal structure of this oligonucleotide was shown to be A-form (Mc Call, M., Brown, T., Hunter, W.N., and Kennard, O. 1986 Nature 322, 661-664). The present study employs NMR, optical, chemical and enzymatic techniques to investigate the solution structure of this DNA 9-mer. NMR COSY experiments indicate 16 of the 18 residues are predominantly south (C2'-endo) sugar conformation. NMR NOESY indicates glycosidic angles in the range predicted for B-form DNA as opposed to A-form. Related DNA and RNA self-complementary 18-mer sequences, d(GGATGGGAGC-TCCCATCC), with U substituted for T in RNA, were studied by circular dichroism. CD spectra support B-form structures for the DNA 9-mer and the DNA 18-mer, and A-form for the RNA 18-mer. High trifluoroethanol concentrations induce a B- to A-form transition in the DNA oligonucleotides. Enzymatic and chemical probes also illustrate significant differences between the DNA and the RNA oligonucleotides. We find no evidence to support an A-form conformation for the TFIIIA recognition sequence d(GGATGGGAG).d(CTCCCATCC) in solution.

Binding Sites↗

Deoxycytidine methylation does not affect DNA.RNA hybrid formation or B-A transitions of (dG)n.(dC)n sequences.

Optical thermal denaturation and circular dichroism (CD) experiments were performed with the following non-selfcomplementary duplex DNA, RNA and DNA.RNA hybrids: (I) dGAG3C3G3CTC.dGAGC3G3C3TC, (II) dGAG3m5C3G3m5CTC.dGAGm5C3G3m5C3TC, (III) rGAG3C3G3CUC.rGAGC3G3C3UC, (IV) dGAG3C3G3CTC.rGAGC3G3C3UC, (V) rGAG3C3G3CUC.dGAGC3G3C3TC, (VI) dGAG3m5C3G3m5CTC.rGAGC3G3C3UC, (VII) rGAG3C3G3CUC.dGAGm5C3G3m5C3TC. Duplex stabilities (delta G degrees at 60 degrees C) increase in the order: I less than IV less than II = V = VI less than VII less than III. Large enthalpic stabilization is associated with intrastrand stacking of guanosine (rG) residues. CD spectroscopy indicates B-form conformations for the unmethylated and methylated DNA (I,II), A-form geometry for the RNA (III), and DNA.RNA hybrid (IV - VII) conformations resembling but not identical to A-RNA. C5-methyldeoxycytidine does not significantly influence DNA conformation, DNA.RNA hybrid formation, or the ability of DNA to adopt an A-type conformation in trifluoroethanol solutions.

Base Composition↗

B-Z cooperativity and kinetics of poly(dG-m5dC) are controlled by an unfavorable B-Z interface energy.

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m5dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl2, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl2 in 50 mM NaCl, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 +/- 0.3 kcal/mol and is length independent. Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 +/- 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event. Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.

DNA↗

Fluorescence-detected circular dichroism of ethidium bound to poly(dG-dC) and poly(dG-m5dC) under B- and Z-form conditions.

The equilibrium binding of ethidium to poly(dG-dC) and poly(dG-m5dC) under conditions favoring B and Z forms was investigated with fluorescence-detected circular dichroism (FDCD) and optical titration methods. FDCD spectra indicate a similar geometry for the intercalated ethidium under both B- and Z-form conditions, even at low levels of bound ethidium. The magnitude of the 310-330-nm FDCD band as a function of the bound drug to base pair ratio (r) indicates ethidium binds to poly(dG-dC) in 4.4 M NaCl and to poly(dG-m5dC) in 25 mM MgCl2 by clustering. Under these conditions, circular dichroism spectra indicate the polymer is largely Z form. Thus, it appears ethidium clusters into regions it has induced into a right-handed form. For all conditions studied, the FDCD spectra provided no evidence for a left-handed binding site. Under B-form conditions, binding is random.

Chemical Phenomena↗

A temporary arteriovenous shunt (Scribner) in the management of traumatic venous injuries of the lower extremity.

The use of a temporary arteriovenous shunt distal to the repair of a traumatic venous injury of the lower extremity in eight patients is reported. Three patients sustained injury to the superficial femoral vein, three had common femoral vein injuries, one had a through-and-through injury of common iliac vein, and one had a popliteal venous injury. Seven patients had associated arterial injuries. Venous repairs included four vein patches, two end-to-end anastomoses, a single venography, and one autogenous vein interposition graft. At the end of each operation, a distal A-V shunt utilizing the posterior tibial artery and vein was constructed. In two patients the shunt clotted within hours after insertion and could not be reopened. In the remaining six patients, the temporary A-V shunt was left in place an average of 10 days (range, 3-15). Followup venograms obtained 2 to 15 days postoperatively revealed patent venous repairs in all patients whose shunt remained functional for 72 hours or more. Noninvasive Doppler studies were obtained in four patients 3 to 8 months post repair and revealed no evidence of deep venous obstruction. No patient with a functioning distal A-V shunt had significant limb edema following repair. This technique appears to improve patency rates of venous repairs and has several distinct advantages over previously described A-V anastomotic fistulas.

Adult↗

Splenic abscess caused by Blastomyces dermatitidis.

We have reported a case of fungal splenic abscess caused by Blastomyces dermatitidis. Splenic abscess is an uncommon disorder, and fungus as the causative organism is rare. The diagnosis of splenic abscess can be rapidly made with radionuclide and CT scanning and ultrasonography. Splenectomy with appropriate antifungal chemotherapy is the currently recommended therapy.

Abscess↗

Ethidium binding to left-handed (Z) DNAs results in regions of right-handed DNA at the intercalation site.

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.

Chemical Phenomena↗

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix.

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗