Site-specific recombinations between direct and inverted res sites of Tn2501

The resolvase gene and the putative res site of Tn2501 are not closely related to any of the previously described resolution functions. In view of this divergence, we designed genetic experiments to confirm the localization of the res site. We analyzed the activity of the Tn2501-encoded resolvase on substrates containing either directly or invertedly repeated res sites. These experiments confirm the localization of the res site that was predicted from nucleotide sequence data and show that the Tn2501 resolvase promotes site-specific inversions in vivo.     

Role of the central dinucleotide at the crossover sites for the selection of quasi sites in DNA inversion mediated by the site-specific Cin recombinase of phage P1

Summary The crossover sites for Cin-mediated inversion consist of imperfect 12 bp inverted repeats with non-palindomic dinucleotides at the center of symmetry. Inversion is believed to occur in vivo between the homologous central 2 bp crossover sequences at the inversely repeated crossover sites through introduction of 2 bp staggered cuts and subsequent reciprocal strand exchanges. The site-specific Cin recombinase acts not only on the normal crossover sites but also, less efficiently, on quasi crossover sites which have some homology with the normal sites. We identified 15 new quasi sites including 4 sites within the cin structural gene. Homology at the 2 bp crossover sequences between recombining sites favors selection as quasi crossover sites. The Cin enzyme can occasionally mediate inversion between nonidentical crossover sequences and such recombinations often result in localized mutations including base pair substitutions and deletions within the 2 bp crossover sequences. These mutations are explained as the consequences of heteroduplex molecules formed between the staggered dinucleotides and either tubsequent resolution by DNA replication or subsequent mismatch repair. Occasional utilization of quasi crossover sites and localized mutagenesis at the crossover sequences in enzyme-mediated inversion processes would be one of the mechanisms contributing to genetic diversity.     

iDHS-DSAMS: Identifying DNase I hypersensitive sites based on the dinucleotide property matrix and ensemble bagged tree

DNase I hypersensitive site (DHS) is related to DNA regulatory elements, so the understanding of DHS sites is of great significance for biomedical research. However, traditional experiments are not very good at identifying recombinant sites of a large number of emerging DNA sequences by sequencing. Some machine learning methods have been proposed to identify DHS, but most methods ignore spatial autocorrelation of the DNA sequence. In this paper, we proposed a predictor called iDHS-DSAMS to identify DHS based on the benchmark datasets. We develop a feature extraction method called dinucleotide-based spatial autocorrelation (DSA). Then we use Min-Redundancy-Max-Relevance (mRMR) to remove irrelevant and redundant features and a 100-dimensional feature vector is selected. Finally, we utilize ensemble bagged tree as classifier, which is based on the oversampled datasets using SMOTE. Five-fold cross validation tests on two benchmark datasets indicate that the proposed method outperforms its existing counterparts on the individual accuracy (Acc), Matthews correlation coefficient (MCC), sensitivity (Sn) and specificity (Sp).     

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.     

The role of binding subsite A in reactions catalyzed by hen egg-white lysozyme

The role of binding subsite A, located at the terminal of the six binding subsites of hen egg-white lysozyme, in substrate binding and catalytic reactions was investigated by kinetic studies using a chemical modification method. Computer simulation showed that, although subsite A participates in the binding of the substrate, a decrease in the affinity of subsite A to the sugar residue does not cause a lowering of the rate of substrate consumption but changes the mode of the reaction by changing the distribution of the products formed. The binding free energies of subsites for Asp-101-modified lysozymes were estimated by data-fitting from the experimental time-courses. The contribution of Asp-101 in hen egg-white lysozyme to the substrate binding at subsite A was estimated to correspond to a binding free energy of about -3 kJ/mol, 30% of the total binding free energy of subsite A. Modification of Asp-101 affected not only the binding free energy of subsite A but also that of subsite C.     

Significance of hydrogen bonding at the S(1)' subsite of calpain I.

alpha-Ketohydroxamates were synthesized as bioisosteres of alpha-ketoamides. The alpha-ketohydroxamates were generally more potent than the corresponding alpha-ketoamides. The potency of the compounds suggests that hydrogen bonding and steric bulk of substituents on the nitrogen atom of the ketoamide moiety influence calpain inhibition.     

Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites

We have studied P1 site-specific recombination by cloning a 6·5 × 10³ base EcoRI fragment (fragment 7) of P1 DNA into a λ vector and then asking whether that fragment can promote efficient recombination for λ markers that flank the fragment. Our results indicate that fragment 7 can reassort these markers very efficiently, and that this recombination can occur in the absence of the bacterial recA and recBC functions. The fragment 7 recombination system has been dissected by an analysis of deletion mutations into two components, a site (called loxP) that must be present in both partners in the recombination in order for recombination to occur, and a P1 gene (called cre), whose product is necessary for recombination. The location of the loxP site at the end of the P1 genetic map suggests that this site-specific recombination system is responsible for the lack of linkage between terminal P1 markers and therefore for the linearity of that map.     

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.     

Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system

Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions.