On the NMR structure determination of a 44n RNA pseudoknot: Assignment strategies and derivation of torsion angle restraints

The complete T- and pseudoknotted acceptor arm of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA has been studied by NMR spectroscopy. Resonance assignment and the gathering of restraints of the 44-mer are impeded by spectral complexity as well as by line broadening. The latter is caused by local dynamical effects in the pseudoknot domain in the molecule. These specific problems could be solved by using different field strengths and selectively ¹³C/¹⁵ labeled samples. Experiments for assigning the sugar spin systems were adjusted to satisfy the requirements of this system. Furthermore, the quality of the structure could be improved by determining the backbone torsion angles , and , using new approaches that were tailored for use in large RNA molecules.     

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a ¹⁵N, ¹H-HMQC spectrum of uniformly ¹⁵N/²H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different ¹⁵N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-¹⁵N-HMQC and 3D¹⁵ N-HMQC-NOESY-¹⁵N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly ¹⁵N/¹³C/²H-labelled S. aureus DNA gyrase B.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 20 (S 1197). Compound 20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure–activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the -amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of 20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

Refined radiation hybrid map of mouse Chromosome 17

We have made a radiation hybrid map of mouse Chromosome (Chr) 17 with 75 microsatellite markers, including those from McCarthy et al. (Genome Res 7, 1153–1161, 1997). Seventy-four of the markers are linked at LOD > 9, and all link at LOD > 5. A LOD 3 framework of 18 markers was used to construct a placement map. The order obtained is in good agreement with genetic maps, and distance estimates give an idea of how recombination rates vary across the chromosome. Recombination is remarkably low with respect to RH break frequency in the region from the centromere to the end of H2. This is similar in interspecific and intersubspecific crosses despite the inversion of a substantial part of this region in Mus spretus with respect to Mus musculus. Received: 10 April 1998 / Accepted: 18 June 1998     

The use of forest inventory data for a National Protected Area Strategy in Guyana

Forest inventories are largely neglected in the debate of national parks selection in Guyana (and probably elsewhere). Because taxonomic data are often scant and biased towards are as of high collecting effort, large scale forest inventory data can be a useful tool adding to a knowledge database for forests. In this paper the use of forest inventories to select national parks in Guyana is assessed. With the data of a large scale inventory five forest regions could be distinguished and two were added on the base of existing other information. Forest composition in Guyana is largely determined by geology at a national level and soil type at regional level. Species diversity is higher in the south of Guyana, possibly due to higher disturbance and is also higher on the better soils. It is concluded that a selection of national parks in Guyana should include a sample of all seven regions, including as much soil variation as possible. Because of land use conflicts in central Guyana, this area is in need of quick attention of Guyana's policy makers.     

Use of the IGF BAC library for physical mapping of the Arabidopsis thaliana genome

In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis –IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double-end clone-limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.     

High-resolution histographical mapping of glucose concentrations in developing cotyledons of Vicia faba in relation to mitotic activity and storage processes: glucose as a possible developmental trigger

Previous studies provided evidence that the carbohydrate status triggers developmental processes in the growing cotyledons of Vicia faba . We describe here the high-resolution mapping of glucose concentrations in tissue sections of developing faba bean cotyledons by quantitative bioluminescence and single-photon imaging. Patterns of local glucose distributions are compared with tissue cell type, mitotic index and the distribution pattern of starch. During cotyledon differentiation, gradients in the glucose concentration emerge which are related to the particular cell type. Higher concentrations are found in non-differentiated premature regions of the cotyledon whereas mature starch-accumulating regions contain particularly low concentrations of glucose. In addition, glucose concentration is correlated to mitotic activity. The glucose distribution pattern is therefore related to the developmental gradient. Our data provide for the first time evidence for steep glucose gradients across developing plant embryos and favour the idea that sugar gradients may have morphogenic functions in developing cotyledons.