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Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2′OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.  相似文献   
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Dopaminergic abnormalities are implicated in the pathogenesis of Tourette syndrome (TS) and chronic multiple tics. We used the transmission-disequilibrium test (TDT) method to test for linkage disequilibrium between a specific allele (the seven-repeat allele (DRD4*7R) of the exon 3 VNTR polymorphic site) at the D4 dopamine receptor locus (DRD4) and expression of chronic multiple tics and TS. This particular allele had been shown in functional studies to have different binding properties compared with the other common alleles in this DRD4 polymorphic system. We studied 64 family trios (consisting of an affected person and two parents, at least one heterozygous for DRD4*7R), including 12 nuclear family trios and 52 trios from four large TS kindreds. The DRD4*7R allele was transmitted significantly more frequently than expected (chi 2 TDT ranging from 8.47 [P < .004] to 10.80 [P = .001], depending on breadth of disease definition and inclusion or exclusion of inferred genotypes). Confirmation of this finding will depend on either replication in other samples or the identification of a transmitted functional mutation within this sample.  相似文献   
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Motivation: Sequence alignment is the problem of finding theoptimal character-by-character correspondence between two sequences.It can be readily solved in O(n2) time and O(n2) space on aserial machine, or in O(n) time with O(n) space per O(n) processingelements on a parallel machine. Hirschberg's divide-and-conquerapproach for finding the single best path reduces space useby a factor of n while inducing only a small constant slowdownto the serial version. Results: This paper presents a family of methods for computingsequence alignments with reduced memory that are well suitedto serial or parallel implementation. Unlike the divide-and-conquerapproach, they can be used in the forward-backward (Baum-Welch)training of linear hidden Markov models, and they avoid data-dependentrepartitioning, making them easier to parallelize. The algorithmsfeature, for an arbitrary integer L, a factor proportional toL slowdown in exchange for reducing space requirement from O(n2)to O(n). A single best path member of this algorithm familymatches the quadratic time and linear space of the divide-and-conqueralgorithm. Experimentally, the O(n1.5)-space member of the familyis 15–40% faster than the O(n)-space divide-and-conqueralgorithm. Availability: The methods will soon be incorporated in the SAMhidden Markov modeling package http: //www.cse.ucs-c.edu/research/compbio/sam.html. Contact: wzrph{at}cse.ucsc.edu  相似文献   
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