Construction and characterization of a sheep BAC library of three genome equivalents

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96. Received: 2 November 1998 / Accepted: 29 January 1999     

Studies on potential use of tin(IV) porphyrin in a role of proteins’ label

We present an electrochemical and optical characterization of 5,10,15,20-tetraphenylporphyrin tin(IV) dichloride (Sn–tpp) in terms of its potential use as a hybrid proteins’ label. Our research comprised Sn–tpp and Sn–tpp in the presence of model proteins selected as to mimic a receptor or surface blocking agents: bovine serum albumin, ovalbumin, and immunoglobulin G. In the course of the study, we determined optimal conditions for analysis by means of differential pulse voltammetry, ultraviolet–visible spectrophotometry, and spectrofluorimetry. In electrochemical detection, the influence of the working electrode, solvent, and supporting electrolyte was examined. Displacements of the received signals along the potential axis (a shift of the potential) and changes in signal intensities due to the addition of proteins were observed and analyzed. Simultaneously, the suitability of Sn–tpp as a label in optical detection mode was assessed by using spectroscopic techniques. The obtained results prove Sn–tpp to be applicable in dual and triple detection systems. Such an approach will improve the reliability of the analysis and, at the same time, will allow for widening the range of the linear response with some overlapping ranges of concentrations.     

Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.     

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact.