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S Hanlon

Publications and source records attributed to S Hanlon.

At least 19 recordsLinked to original sources
PubMed1992

A semi-micromethod for the determination of the extinction coefficients of duplex and single-stranded DNA.

We have developed a rapid and convenient procedure for the determination of concentrations and extinction coefficients of oligo- and polynucleotides. It offers significant advantages over other methods in terms of precision and the ability to detect artifactual or erroneous results. Samples are first completely digested with appropriate enzymes to mononucleotides and nucleosides. Using the multicomponent linear regression capabilities of commonly available spreadsheet programs, the absorbance spectrum of the digest can be analyzed as a linear combination of the contribution of the possible constituent monomers. If all the spectral components present have been included, the analysis yields the concentration of each of the monomer species whose sum is the concentration (in monomer units) of the original undigested sample. When combined with the predigest absorbance spectrum, the extinction coefficients of the intact sample can then be calculated. The analysis also yields the fractional base composition of the oligomer or polymer. The extensive spectral data provided by digital read-outs of modern spectrophotometers permit the application of sensitive tests of the goodness of fit, thus facilitating the detection of artifacts and sample inhomogeneity. Both single-strand and duplex structures can be analyzed comfortably in sample sizes of 25 to 35 nmol (total) of mononucleotides with a precision of 1%. The concentrations obtained by this method agree, on the average, within 0.2% with those determined by phosphate analysis of the same sample. The method also yields the base composition with an accuracy of ca. 5% for high-molecular-weight polymers and 2% for short oligomers (15-20 bp) when compared to the predicted values.

Base Composition↗
PubMed1990

Structure and dynamics of M13mp19 circular single-strand DNA: effects of ionic strength.

Dynamic and static light scattering, CD, and optical melting experiments have been conducted on M13mp19 viral circular single-strand DNA as a function of NaCl concentration. Over the 10,000-fold range in concentration from 100 microM to 1.0 M NaCl, the melting curves and CD spectra indicate an increase in base stacking and stability of stacked regions with increased salt concentration. Analysis of dynamic light scattering measurements of the single-strand DNA solutions as a function of K2 from 1.56 to 20 X 10(10) cm-2 indicates the collected autocorrelation functions are biexponential, thus revealing the presence of two decaying dynamic components. These components are taken to correspond to (1) global translational motions of the molecular center of mass and (2) motions of the internal molecular subunits. From the evaluated relaxation rates of these components, diffusion coefficients D0 and Dplat are determined. The center of mass translational diffusion coefficient D0, varies in a nonmonotonic manner, by 10%, from 3.75 X 10(-8) to 3.39 X 10(-8) cm2/s over the NaCl concentration range from 100 microM to 1.0 M. Likewise, the radius of gyration RG, obtained from static light scattering experiments, varies by 15% from 699 to 830 A over the same NaCl range Dplat, the diffusion coefficient of the internal subunits, displays a different dependence on the NaCl concentration and decreases, by nearly 22% in a titratable fashion, from 12.46 X 10(-8) to 10.26 X 10(-8) cm2/s, when the salt is increased from 100 microM to 1.0 M. A semiquantitative interpretation of these results is provided by analysis of the light scattering data in terms of the circular Rouse-Zimm chain. Rouse-Zimm model parameters are estimated from the experimental results, assuming the circular chains are composed of a fixed number of Gaussian segments, N + 1 = 15. The rms displacement of the internal segments, b, is estimated to be the smallest (442 A) in 100 mM NaCl. Increases of b to 467 A in 100 microM and 524 A in 1.0 M NaCl are observed. Meanwhile, the hypothetical friction factor of the internal subunits, f, progressively increases as the NaCl concentration is raised. It is inferred from the evaluated Rouse-Zimm model parameters that both the static flexibility of the circular chain and diffusive displacements of the internal subunits decrease with increases in NaCl concentration from 100 mM to 1.0 M.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Phenomena↗
PubMed1990

High sodium chloride diets injure arteries and raise mortality without changing blood pressure.

High NaCl diets often increase blood pressure and thereby accelerate lesions in arterial walls. Could high NaCl diets increase arterial lesions without raising blood pressure? To test this, 100 uninephrectomized Dahl salt-resistant (DR) rats (highly resistant to NaCl hypertension) were administered deoxycorticosterone acetate (DOCA) (250 mg/kg) in silicone implants and drinking water containing 1% NaCl for 6 weeks. Then the DOCA and saline were removed, and the rats were allowed to recover for 4 weeks. Intra-arterial mean blood pressures on all rats allowed division of the rats into two matched groups, each group with an average blood pressure of 160 mm Hg. One group continued on a 0.3% NaCl diet, whereas the other group began an 8% NaCl diet for 8 weeks. After 5 weeks on these two diets, the intra-arterial blood pressure averaged 158 mm Hg in both groups. Thus, the 8% NaCl diet produced no further increase in blood pressure in the DR rats. Nevertheless, after 8 weeks on the 8% NaCl diet, 53% of the rats (26 of 49) had died; whereas in the group on the 0.3% NaCl diet, not one rat (0 of 51) had died (p less than 0.000001). After 7 more weeks on the 8% NaCl diet, all the rats in this group had died.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Conformation and dynamics of a left-handed Z-DNA hairpin: studies of d(CGCGCGTTTTCGCGCG) in solution.

The physical properties of the DNA oligomer d(CGCGCGTTTTCGCGCG) in solvents containing 4 M NaClO4 and 0.1 M NaCl were investigated by proton NMR, optical melting, and circular dichroism spectroscopy. Results of these investigations are as follows: (i) The DNA hexadecamer exists as a unimolecular hairpin in either high or low salt. (ii) In high salt the stem region of the hairpin is in the left-handed Z conformation. (iii) In either high or low salt, the duplex stem of the hairpin is stabilized against melting by approximately 40 degrees C compared to the linear core duplex. The added stability of the hairpin is entropic in origin. (iv) In high salt, as the temperature is elevated, the equilibrium structure of the duplex stem of the hairpin shifts from the Z to the B conformation before melting. (v) In low salt, when the DNA duplex exists in the B conformation, attachment of a T4 single-strand loop to one end only slightly decreases (by 14%) the correlation time of the CH5-CH6 interproton vector. In high salt, when the DNA duplex exists in the Z conformation, the correlation time of the CH5-CH6 interproton vector decreases by 51%. Since these viscosity-corrected correlation times are taken to be indicators of duplex motions on the nanosecond time scale, this result directly suggests a larger amplitude of these motions is present in the duplex stem of the hairpin when it exists in the Z conformation.

Circular Dichroism↗
PubMed1989

Linking number anomalies in DNA under conditions close to condensation.

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗
PubMed1989

Base and conformational specificity of an amine modification of DNA.

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A-form, Z-form, or the single-strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH2O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of adduct formation under standard reaction conditions (27 degrees C, 0.63M CH2O, 0.007M n-butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI-dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG-dC) show enhanced 2'-endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA-dT) in the "X"-form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23, 1-4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B-form of polynucleotides as demonstrated by the failure of poly(dG-m5dC) in the stable Z-form to exhibit substantial reaction. The B-form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1989

A glomerular defect in prehypertensive Dahl S rats, which limits their capacity to increase GFR.

There is a great need to discover traits that predict future human hypertension. Our previous studies found abnormal glomerular filtration rates (GFRs) in isolated kidneys of Dahl S rats. Such GFR alterations were sought in intact, low NaCl, prehypertensive Dahl S rats. Such Dahl S rats are genetically susceptible to NaCl hypertension but are normotensive on low NaCl. On .3% low NaCl, BP of 12 Dahl S rats averaged 138 mm Hg v 131 in 12 Dahl R rats (NS). Glomerular filtration rates under inactin anesthesia were measured during two 20-minute periods before a 20-minute amino acid infusion and also during three 20-minute periods after the amino acid infusion. Before the amino acid infusion, GFRs averaged 3.3 mL/min in S rats v 3.25 in R rats (NS). After the amino acid infusion, R rats showed a progressive rise in GFR: 4.7, 5.1 and 5.4 in three successive 20-minute periods. In these same three periods after amino acids, the S rats had GFRs of 3.8, 3.8, and 3.8. Thus R rats had an 81% increase of GFR after amino acids, whereas S rats increased only 18%, a 78% lower increase, P less than .001. The R rats had the normal rise of GFR in response to an amino acid load. Prehypertensive S rats had virtually no increase in GFR after the amino acid infusion. Even though both S & R rats had BPs well within the normal range, the ability to increase GFR after amino acid infusion was markedly impaired in prehypertensive S rats. This defect could possibly be used to predict future hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1989

How is the NaCl signal transmitted in NaCl-induced hypertension?

Is the NaCl signal perceived as a small increase in the concentration of NaCl in extracellular fluid? We used 8 g NaCl/100 g soluble nutrients and fed only a hypertonic (1.4% NaCl) or a hypotonic (0.45% NaCl) drink to Dahl salt-sensitive (DS) rats. After 12 weeks, 11 rats receiving the hypertonic drink had a mean blood pressure of 195 mm Hg versus 195 mm Hg in 12 rats receiving the hypotonic drink. Thus, the high-NaCl signal seems unrelated to a higher NaCl concentration in extracellular fluid, thereby suggesting volume signals. Most volume controls are near the third brain ventricle (3V). As a working hypothesis, high dietary NaCl may swell the tissues surrounding 3V, which is slitlike. Such swelling would partially close the upper part of the slit and cause ependymal cells and nerve fibers on opposite walls to touch, possibly leading to hypertension in susceptible humans or rats. To test this, we stereotaxically blocked the aqueduct with inert silicone to produce hydrocephalus of 3V in DS rats and thus prevent ependymal cells and nerve fibers from touching. After blocking or sham-blocking the aqueduct, either a 6% NaCl diet or a 0.23% NaCl diet was started. Intra-arterial blood pressure was taken after 6 weeks. A group of 28 sham-blocked rats and a group of 29 blocked rats, all fed a 0.23% low NaCl diet, had equal blood pressures averaging 130 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Effects of charge modification on the helical period of duplex DNA.

Supercoiled enriched PM-2 DNA has been relaxed by treating with calf thymus topoisomerase I and used in the preparation of a family of n-butylamine adducts of varying levels of substitution. The amine is cross-linked by formaldehyde to the exocyclic amino group of G when the DNA is in duplex form. These amine adducts of covalently closed relaxed (ccr) DNA, freed of the formaldehyde and n-butylamine reactants, have circular dichroism (CD) spectral properties similar to those previously reported for the adducts of calf thymus DNA [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987-4995]. In both instances, the CD transformation effected by increasing levels of substituted cationic amine is similar to that induced by solvents of high electrolyte content. The adducts also exhibit greatly increased electrophoretic mobility compared to unreacted controls or a control treated only with formaldehyde. Mobility changes in the presence of variable amounts of ethidium bromide demonstrate that this phenomenon is attributable to the formation of negative supercoils and is not due to denaturation or unwinding of the duplex. Incremental increases in superhelicity due to the attachment of the amine have been measured by reference to a topoisomerase ladder of underivatized PM-2 DNA and converted to changes in winding angle. As the extent of substitution increases, the rotational strength of the positive band above 260 nm decreases, and the winding angle increases in the nonlinear manner observed previously for underivatized PM-2 DNA [Baase, W. A., & Johnson, W. C., Jr. (1979) Nucleic Acids Res. 6, 797-814]. In fact, the relationship between these two properties is the same for both the adducts and the underivatized ccr species. Thus, the attachment of the amine has the same conformational effects as the electrolyte content of the solvent. The effect can be rationalized in terms of the reduction of the electrostatic free energy of the duplex due to site-bound or localized cation binding in the minor groove.

Animals↗
PubMed1988

Prehypertensive Dahl S rats show no rise in glomerular filtration rate after an amino acid infusion.

When one administers a protein or amino acid load, both GFR and renal blood flow increase about 40% in normal humans as well as in dogs and rats. The protein load causes vasodilation in both the afferent and the efferent arterioles. In these prehypertensive Dahl S rats, it is likely that there is already some vasodilation of the afferent and efferent arterioles and possibly some mesangial relaxation, in order to bring the GFR to normal levels in the face of some intrinsic abnormality in glomerular filtration. Since these arterioles are already dilated, there can be little further dilation in response to the amino acid load and hence no further increase in GFR. It is possible that this limited capacity for further vasodilation could serve as a predictor of future hypertension.

Amino Acids↗
PubMed1987

Covalent attachment of an alkylamine prevents the B to Z transition in poly(dG-dC).

Covalent complexes of n-butylamine and double-stranded poly(dG-dC) were prepared by coupling the amine to exocyclic amino groups of guanine bases with CH2O. Neither the absorption spectrum above 230 nm nor the s020,w of the complexes in low to moderate ionic strength solvents, freed of excess unreacted reagents, differs significantly from that of unreacted poly(dG-dC) or a control which had been exposed only to CH2O. In contrast, the CD spectra are profoundly altered. The minimum at 252 nm becomes more negative, and the rotational strength of the positive band above 260 nm is reduced as a linear function of the extent of amine attachment. At 0.22 mol of amine per mole of nucleotide, the transformation is similar to that observed by others in poly(dG-dC) when complexed to core histones in reconstituted core particles or in concentrated LiCl solvents at temperatures below the B----Z transition. Sedimentation studies reveal that these changes in the circular dichroism (CD) spectra reflect secondary structural effects rather than the formation of aggregates or psi type structures. Raman spectra reveal, however, that these secondary structural changes must occur within the B family as the amine complex retains B backbone geometry. The conformation produced by the attachment of the amine is probably a higher winding angle (overwound) B variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1985

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine.

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E. coli DNA until 6-7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.

Acetylation↗
PubMed1983

Conformational characteristics of deoxyribonucleic acid-butylamine complexes with C-type circular dichroism spectra. 1. An X-ray fiber diffraction study.

A set of complexes of calf thymus DNA and n-butylamine, covalently cross-linked to the DNA bases with CH2O, has been examined by X-ray diffraction. The attachment of this amine to DNA has been previously shown to result in a profound reduction of the rotational strength of the positive band above 260 nm in the circular dichroism (CD) spectrum in 20 mM NaCl, pH 7, without changing any of the properties which are characteristic of a native base-stacked duplex [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987]. In 20 mM NaCl, pH 7, substitution levels of 0-0.15 mol of amine/mol of nucleotide are sufficient to produce a family of CD spectra which range from the conservative one normally seen for protein-free DNA in this solvent to the nonconservative one ascribed to the C form of DNA [Hanlon, S., Brudno, S., Wu, T. T., & Wolf, B. (1975) Biochemistry 14, 1648-1660]. Fibers of the DNA X amine complexes at 79% relative humidity (rh) and 33% rh do indeed show C-type X-ray patterns whereas the controls exhibit A forms under the same conditions. The same preparations, however, exhibit only B patterns when the fibers are examined at 98% rh and in the wet fiber form. Although we cannot rule out the possibility that molecular interactions in the fiber have imposed conformational restraints on the DNA structure that are not present in solution, these present results suggest that the conformation of DNA giving rise to the "C" CD spectrum is a variant of the B form.

Animals↗
PubMed1983

Conformational characteristics of deoxyribonucleic acid-butylamine complexes with C-type circular dichroism spectra. 2. A Raman spectroscopic study.

The derivatives of calf thymus DNA in which n-butylamine is covalently attached as described in the preceding paper in this series [Chen, C. Y., Pheiffer, B. H., Zimmerman, S. B., & Hanlon, S. (1983) Biochemistry (preceding paper in this issue)] were examined by Raman spectroscopy. As previously mentioned, these complexes exhibit profoundly decreased rotational strengths of the positive band of the circular dichroism (CD) spectrum above 260 nm, with the most heavily substituted (ca. 0.12 mol of amine/mol of nucleotide) resembling that of DNA in 11 m LiCl. Raman spectra of all complexes and their controls in the form of either fibers at 98% relative humidity or gels at 40 mg/mL in 20 mM NaCl, pH 7, show typical B-type spectra with no evidence of significant amounts of C, A, Z, or disordered forms. We have thus concluded that the assignment of the nonconservative CD spectrum of DNA typically observed in concentrated electrolyte solutions to a C form is in error. Both these Raman data and the X-ray results reported in the previous paper indicate that the structure giving rise to the C CD spectrum has B-form backbone geometry.

Animals↗
PubMed1983

Forecasting optometry grade point average.

Preoptometry grades. Optometry College Admission Test (OCAT) scores, and a number of other variables were evaluated as predictors of the grade point average earned by optometry students (N = 494) after two years at Southern California College of Optomery. The OCAT Quantitative Ability test was found to be the best single predictor (r = +0.37). This prediction was enhanced (multiple r = +0.55) with the addition of the OCAT Study Reading test score, preoptometry grade point average in college mathematics and science courses, and a rating of the preoptometry college attended. A predicted grade point average for optometry school courses was computed for each student by additively combining these variables, weighted according to their relative predictive validities. By comparing the computed values of students who actually earned a high grade point average in optometry school with those whose average was low, we were able to establish a predicted grade point cutoff score. It is shown that this can be useful in evaluating the potential of an applicant to achieve academic success in optometry school.

Achievement↗