Searching for non-B DNA-forming motifs using nBMST (non-B DNA motif search tool).

This unit describes basic protocols on using the non-B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right-handed Watson-Crick double-helix, collectively known as non-B DNA, and on using the associated PolyBrowse, a GBrowse-based genomic browser. The nBMST is a Web-based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G-quadruplex DNA), alternating purine-pyrimidine tracts (left-handed Z-DNA), and A-phased repeats (static bending). The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to an aggregate of 20 megabasepairs (Mbp).

A clustering method for repeat analysis in DNA sequences.

BACKGROUND: A computational system for analysis of the repetitive structure of genomic sequences is described. The method uses suffix trees to organize and search the input sequences; this data structure has been used previously for efficient computation of exact and degenerate repeats. RESULTS: The resulting software tool collects all repeat classes and outputs summary statistics as well as a file containing multiple sequences (multi fasta), that can be used as the target of searches. Its use is demonstrated here on several complete microbial genomes, the entire Arabidopsis thaliana genome, and a large collection of rice bacterial artificial chromosome end sequences. CONCLUSIONS: We propose a new clustering method for analysis of the repeat data captured in suffix trees. This method has been incorporated into a system that can find repeats in individual genome sequences or sets of sequences, and that can organize those repeats into classes. It quickly and accurately creates repeat databases from small and large genomes. The associated software (RepeatFinder), should prove helpful in the analysis of repeat structure for both complete and partial genome sequences.

Geometry of dendritic spines affects calcium dynamics in hippocampal neurons: theory and experiments.

The role of dendritic spine morphology in the regulation of the spatiotemporal distribution of free intracellular calcium concentration ([Ca2+]i) was examined in a unique axial-symmetrical model that focuses on spine-dendrite interactions, and the simulations of the model were compared with the behavior of real dendritic spines in cultured hippocampal neurons. A set of nonlinear differential equations describes the behavior of a spherical dendritic spine head, linked to a dendrite via a cylindrical spine neck. Mechanisms for handling of calcium (including internal stores, buffers, and efflux pathways) are placed in both the dendrites and spines. In response to a calcium surge, the magnitude and time course of the response in both the spine and the parent dendrite vary as a function of the length of the spine neck such that a short neck increases the magnitude of the response in the dendrite and speeds up the recovery in the spine head. The generality of the model, originally constructed for a case of release of calcium from stores, was tested in simulations of fast calcium influx through membrane channels and verified the impact of spine neck on calcium dynamics. Spatiotemporal distributions of [Ca2+]i, measured in individual dendritic spines of cultured hippocampal neurons injected with Calcium Green-1, were monitored with a confocal laser scanning microscope. Line scans of spines and dendrites at a <1-ms time resolution reveal simultaneous transient rises in [Ca2+]i in spines and their parent dendrites after application of caffeine or during spontaneous calcium transients associated with synaptic or action potential discharges. The magnitude of responses in the individual compartments, spine-dendrite disparity, and the temporal distribution of [Ca2+]i were different for spines with short and long necks, with the latter being more independent of the dendrite, in agreement with prediction of the model.

Simultaneous measurement of evoked release and [Ca2+]i in a crayfish release bouton reveals high affinity of release to Ca2+.

The opener neuromuscular junction of crayfish was used to determine the affinity of the putative Ca2+ receptor(s) responsible for evoked release. Evoked, asynchronous release, and steady-state intracellular Ca2+ concentration, [Ca2+]ss, were measured concomitantly in single release boutons. It was found that, as expected, asynchronous release is highly correlated with [Ca2+]ss. Surprisingly, evoked release was also found to be highly correlated with [Ca2+]ss. The quantal content (m) and the rate of asynchronous release (S) showed sigmoidal dependence on [Ca2+]ss. The slope log m/log [Ca2+]ss varied between 1.6 and 3.3; the higher slope observed at the lower [Ca2+]o. The slope log S/log [Ca2+]ss varied between 3 and 4 and was independent of [Ca2+]o. These results are consistent with the assumption that evoked release is controlled by the sum of [Ca2+]ss and the local elevation of Ca2+ concentration near the release sites resulting from Ca2+ influx through voltage-gated Ca2+ channels (Y). On the basis of the above, we were able to estimate Y. We found Y to be significantly <10 microM even for [Ca2+]o = 13.5 mM. The dissociation constant (Kd) of the Ca2+ receptor(s) associated with evoked release was calculated to be in the range of 4-5 microM. This value of Kd is similar to that found previously for asynchronous release.