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51.
For nonspecific binding of oligopeptides and other cationic ligands, including proteins, to nucleic acid oligomers, we develop a model capable of quantifying and predicting the salt concentration dependence of the binding free energy (deltaG(o)obs) by way of an analytic treatment of the Coulombic end effect (CEE). Ligands, nucleic acids, and their complexes (species j of valence Zj) are modeled as finite lattices with absolute value(Zj) charged residues; the CEE is quantified by its characteristic length Ne (specified in charged residues) and its consequences for the free energy and ion association of the oligomer. Expressions are developed for the individual site binding constants Ki as a function of position (site number i) of a bound ligand on a nucleic acid and for the observed binding constant Kobs as an ensemble average of Ki. Analysis of deltaG(o)obs = -RT ln Kobs and Sa Kobs identical with (partial differential ln Kobs)/(partial differential ln a(+/-)) for binding of the oligopeptide KWK6 (ZL = +8) to single-stranded (ss) dT(pdT)(absolute value(ZD) oligomers (dT-mers) where ZD = {-6, -10, -11, -14, -15} in the range 0.1-0.25 M Na+ yields Ne = 9.0 +/- 0.8 residues at each end, demonstrating that both KWK6 and the above dT-mers are sufficiently short so that the CEE extends over the entire molecule. The dependences of Kobs and of Sa Kobs on absolute value(ZD) for a given ZL are determined by the difference between 2Ne and the net number of charged residues Q in the complex (Q identical with absolute value(ZD) - ZL). For Q < 2Ne, characteristic of complexes of KWK6 with this set of dT-mers, the distribution of binding free energies deltaG(o)obs = -RT ln Ki for sites along the DNA oligomer is parabolic, and Kobs and Sa Kobs are strongly dependent on absolute value(ZD). For Q > or = 2Ne, the distribution of binding free energies deltaG(o)obs is trapezoidal, and the dependence of Kobs and Sa Kobs on absolute value(ZD) is weaker. Application of the model to nonspecific binding of human DNA polymerase beta to ssDNA demonstrates the significance of the CEE in determining Kobs and Sa Kobs of binding of a cationic site on a protein to a DNA oligomer. 相似文献
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The ACHE and BCHE genes, encoding the acetylcholine hydrolysing enzymes acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE), co-amplify with several oncogenes in leukemic patients with platelet deficiency (thrombocytopenia). This and other experiments implicated ACHE and BCHE in the development of bone marrow megakaryocytes, the progenitors of platelets. Therefore, we wished to find out whether cholinesterase gene amplification would also occur in non-cancerous platelet disorders and, if so, whether oncogenes would amplify in such cases as well. The autoimmune disease systemic lupus erythematosus (SLE) presents an appropriate model system for this issue, since patients with SLE may suffer from thrombocytopenia resistant to most treatment modalities. Here, we report a 40-80-fold amplification of genomic sequences from the ACHE and BCHE genes as well as the C-raf, V-sis and C-fes/fps oncogenes in peripheral blood cells from an SLE patient with severe thrombocytopenia. PvuII restriction analysis and DNA blot hybridization of the amplified ACHE and BCHE sequences demonstrated apparent aberrations in both genes, suggesting that malfunctioning of modified, partially amplified cholinesterase genes may be involved in the etiology of thrombocytopenia associated with SLE. These observations imply that cholinergic mechanisms regulate megakaryocytopoiesis, shed new light on the diverse hematologic findings characteristic of SLE, and may become valuable as diagnostic, treatment and prognostic tools in the follow-up of patients suffering from thrombocytopenia associated with SLE. Furthermore, these findings reinforce the notion that cholinesterase gene amplifications are causally related with platelet abnormalities in multiple hemopoietic disorders. 相似文献
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Background
Quantifying the amount of standing genetic variation in fitness represents an empirical challenge. Unfortunately, the shortage of detailed studies of the genetic architecture of fitness has hampered progress in several domains of evolutionary biology. One such area is the study of sexual selection. In particular, the evolution of adaptive female choice by indirect genetic benefits relies on the presence of genetic variation for fitness. Female choice by genetic benefits fall broadly into good genes (additive) models and compatibility (non-additive) models where the strength of selection is dictated by the genetic architecture of fitness. To characterize the genetic architecture of fitness, we employed a quantitative genetic design (the diallel cross) in a population of the seed beetle Callosobruchus maculatus, which is known to exhibit post-copulatory female choice. From reciprocal crosses of inbred lines, we assayed egg production, egg-to-adult survival, and lifetime offspring production of the outbred F1 daughters (F1 productivity). 相似文献56.
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Ballin JD Prevas JP Bharill S Gryczynski I Gryczynski Z Wilson GM 《Biochemistry》2008,47(27):7043-7052
Acrylamide quenching is widely used to monitor the solvent exposure of fluorescent probes in vitro. Here, we tested the utility of this technique to discriminate local RNA secondary structures using the fluorescent adenine analogue 2-aminopurine (2-AP). Under native conditions, the solvent accessibilities of most 2-AP-labeled RNA substrates were poorly resolved by classical single-population models; rather, a two-state quencher accessibility algorithm was required to model acrylamide-dependent changes in 2-AP fluorescence in structured RNA contexts. Comparing 2-AP quenching parameters between structured and unstructured RNA substrates permitted the effects of local RNA structure on 2-AP solvent exposure to be distinguished from nearest neighbor effects or environmental influences on intrinsic 2-AP photophysics. Using this strategy, the fractional accessibility of 2-AP for acrylamide ( f a) was found to be highly sensitive to local RNA structure. Base-paired 2-AP exhibited relatively poor accessibility, consistent with extensive shielding by adjacent bases. 2-AP in a single-base bulge was uniformly accessible to solvent, whereas the fractional accessibility of 2-AP in a hexanucleotide loop was indistinguishable from that of an unstructured RNA. However, these studies also provided evidence that the f a parameter reflects local conformational dynamics in base-paired RNA. Enhanced base pair dynamics at elevated temperatures were accompanied by increased f a values, while restricting local RNA breathing by adding a C-G base pair clamp or positioning 2-AP within extended RNA duplexes significantly decreased this parameter. Together, these studies show that 2-AP quenching studies can reveal local RNA structural and dynamic features beyond those that can be measured by conventional spectroscopic approaches. 相似文献
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