Molecular dynamics studies of axis bending in d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5): effects of sequence polarity on DNA curvature

Gel retardation studies and other experiments indicate that DNA sequences containing the d(GA4T4C)n motif are curved, whereas those of identical composition but with a reverse sequence polarity, the d(GT4A4C)n motif, are straight. Hydroxyl radical cleavage experiments show that d(GA4T4C)n shows a unique signature, whereas d(GT4A4C)n behaves normally. To explain these results at a molecular level, molecular dynamics (MD) simulations were performed on the DNA duplexes d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5) to 3.0 and 2.5 ns, respectively. The MD simulations are based on the Cornell force field implemented in the AMBER 4.1 modeling package and performed in a neutral solution of anionic DNA with K+, Cl- and Mg2+ at concentrations roughly comparable to a ligase buffer. Long range interactions were treated by the particle mesh Ewald method. Analysis of the results shows that the calculated dynamical structure of d(G5-(GA4T4C)2-C5) exhibits strong gross curvature, consistent with the observed behavior. The most significant locus of curvature in the MD structure is found at the central C15-G16 step, with an average roll angle of 12.8(+/-6.40)deg. The d(G5-(GT4A4C)2-C5) MD structure exhibited significantly less gross curvature. Analysis of results indicates that the reduction in gross curvature in the d(G5-(GT4A4C)2-C5) trajectory originates from the effect of the T10-A11 and T20-A21 steps, which showed average roll angles of 12.5(+/-5)deg. These three steps, T10-A11, C15-G16 and T20-A21, are half-helix turns away from one another, and their contributions to concerted bending cancel out. The A-tracts in the MD structure are essentially straight. The dynamical structure of d(G5-(GA4T4C)2-C5) exhibited minor groove deformation comprised of expansion at the 5' end of A-tracts and progressive narrowing towards the 3' end, consistent with and elaborating the interpretation of hydroxyl radical chemical probing results.     

A simple and rapid thyroxine radioimmunoassy (T4-RIA) in unextracted human serum; a comparison of T4-RIA and T4 displacement assay, T4(D), in normal and pathologic sera

A simple, rapid and accurate thyroxine radioimmunoassay (T4-RIA) in unextracted serum or plasma has been described, and for comparison T4 determinations have also been made by a T4(D) procedure using Abbott Tetrasorb kits. T4-RIA procedure basically involved denaturation of serum to dissociate T4-protein bond, and T4 released was allowed to react with [125I]T4-labeled T4 antiserum elicited by immunizing rabbits against bovine thyroglobulin. The displaced unbound [125I]T4 was rapidly taken up by an anionic resin sponge within 15 min and this sponge [125I]T4 uptake was linearly related to T4 present in standards or serum. The denaturation of serum effected by trichloroacetic acid-sodium hydroxide permitted virtually 100% T4 extraction recovery in normal, pregnancy, hypo- and hyperthyroid sera whereas 72.9-87.6% T4 recovery from normal serum (and with large individual differences) was noted with lower alcohols in T4(D) procedure. Cumbersome and/or tedious steps such as pre-extraction, centrifugation, time consuming bound and unbound hormone separation procedures, etc. are obviated in T4-RIA and the entire assay can be completed in the same tube in approximately an hour. These attributes along with increased sensitivity and specificity and the need for only microamounts of test sera (25-50 mul) in T4-RIA offer distinct advantages over T4(D) procedures, and in simplicity excel even other T4-RIAs. T4-RIA values in physiological and pathological states were highly correlated (r = 0.97) with T4(D) measurements and no differences between these two techniques were found. The reported discrepancies between T4-RIA and T4(D) measurements in human sera and some of the reasons for attributing these inconsistencies to probable methodological errors and variations are discussed.     

Triiodothyronine (T3) and thyroxine (T4) levels in Rana curtipes during development and metamorphosis

During premetamorphosis, levels of circulating triiodothyronine (T3) and thyroxine (T4) were below the limits of detection of RIA. They became detectable in late prometamorphic stages. A gradual increase in T3 and T4 was observed during this period. A sharp rise in hormone levels was apparent at the onset of metamorphic climax. Peak levels of both hormones were found at Taylor-Kollros stage XXI. The T3 reached a peak level of 101.4 ng/dl, about 3 fold increase over the level at prometamorphosis. Thereafter the circulating hormones (particularly T4) decreased rapidly and reached levels similar to prometamorphic stages. Significant high levels of thyroid hormones (T3 and T4) in metamorphic climax suggest that like other anurans, elevation of these hormones is required for normal metamorphosis in R. curtipes tadpoles.     

Abundant (A)n.(T)n mononucleotide repeats in the pig genome: linkage mapping of the porcine APOB, FSA, ALOX12, PEPN and RLN loci

A computer analysis revealed that the mononucleotide repeat (A)n.(T)n is about five times as common as (CA)n.(GT)n repeats in the porcine genome, with frequency estimates of one every 7kb and 30kb, respectively. Seven mononucleotide repeats with n = 12-25 located close to coding sequences were analysed for polymorphism using polymerase chain reaction (PCR) amplification and polyacrylamide gel electrophoresis. All loci were variable with 3-6 alleles and heterozygosities of 0.26-0.69 based on investigation of 10 unrelated pigs (two wild boars and eight domestic sows). Repeat length correlated with degree of polymorphism. A comparison with (CA)n.(GT)n polymorphisms suggested that the number of repeat units rather than the total length of the repeat region is the common denominator that governs polymorphism at both mono- and dinucleotide repeat loci. (A)n.(T)n polymorphisms allowed linkage mapping of relaxin to chromosome 1, apolipoprotein B to chromosome 3, aminopeptidase N to chromosome 7, arachidonate 12-lipoxygenase to chromosome 12, and follistatin to chromosome 16. The rich abundance of potentially informative (A)n.(T)n repeats will increase the chances of finding a PCR-based marker near any DNA sequence of interest.     

Molecular modelling of Me(2+)-(8-hydroxy-quinolinate)2 complexes using ZINDO and ESSF methods

The Me2+ (8-hydroxy-quinolinate)1-2 system was studied using semi-empirical (ZINDO) and molecular mechanics (ESFF) methods for a range of bivalent metals comprising alkaline earth metals (up to Sr2+) and the first two rows of transition metals. The structural validation of the optimisation calculations showed that ESFF is in general an efficient predictor of the structure of the complexes. On the other hand, ZINDO offers an appropriate tool for describing the mechanisms of complex formation for the studied system. The spectral validation based on the comparison of spectral data with predicted molecular geometric parameters shows that ZINDO offers a better tool for describing the mechanisms of complex formation. Finally, the consistent relationship (in relative terms) between SCF energies and equilibrium constants, and between computed charges at the oxygen ligation atom and pH, showed that ZINDO can be used as a tool for the design of chelating agents. For the system studied the CPU times are not prohibitive, even for a metal-comprehensive investigation.     

On the Gracefulness of Graph (jC4n) U Pm

The present paper deals with the gracefulness of unconnected graph (jC4n) U Pn, and proves the following result: for positive integers n, j and m with n ≥ 1, j ≥ 2, the unconnected graph (jC4n) U Pm is a graceful graph for m ＝ j - 1or m ≥ n + j, where C4n is a cycle with 4n vertexes, -Pm is a path with m + 1vertexes, and (jC4n) U Pm denotes the disjoint union of j - C4n and Pm.