An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

MOsDB: an integrated information resource for rice genomics

The MIPS Rice (Oryza sativa) database (MOsDB; http://mips.gsf.de/proj/rice) provides a comprehensive data collection dedicated to the genome information of rice. Rice (O. sativa L.) is one of the most important food crops for over half the world's population and serves as a major model system in cereal genome research. MOsDB integrates data from two publicly available rice genomic sequences, O. sativa L. ssp. indica and O. sativa L. ssp. japonica. Besides regularly updated rice genome sequence information, MOsDB provides an integrated resource for associated analysis data, e.g. internal and external annotation information as well as a complex characterization of all annotated rice genes. The MOsDB web interface supports various search options and allows browsing the database content. MOsDB is continuously expanding to include an increasing range of data type and the growing amount of information on the rice genome.     

Comparative analysis of the composition of the volatile oils of two forms of Achillea crithmifolia W. et. K.--diploid and tetraploid

The genus Achillea L. includes more than 100 species and is a polyploid complex of di-, tetra-, hexa- and octaploids, individuals.     

Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot

Despite the suitability of various lattice geometries for coarse-grained modeling of proteins, the actual packing geometry of residues in folded structures has remained largely unexplored. A strong tendency to assume a regular packing geometry is shown here by optimally reorienting and superimposing clusters of neighboring residues from databank structures examined on a coarse-grained (single-site-per-residue) scale. The orientation function (or order parameter) of the examined coordination clusters with respect to fcc lattice directions is found to be 0.82. The observed geometry, which may be termed an incomplete distorted face-centered cubic (fcc) packing, is apparently favored by the drive to maximize packing density, in a fashion analogous to the way identical spheres pack densely and follow fcc geometry. About 2/3 of all residues obey this packing geometry, while the remainder occupy other context-dependent positions. The preferred coordination directions show relatively small variations over the various amino acid types, consistent with uniform residue viewpoint. Both the extremes of solvent-exposed and completely buried residue neighborhoods approximate the same generic packing, the only difference being in the numbers (and not the orientations) of coordination sites that are occupied (or left void for solvent occupancy). We observe the prevalence of a rather uniform (tight) residue packing density throughout the structure, including even the residues packed near solvent-exposed regions. The observed orientation distribution reveals an underlying, intrinsic orientation lattice for proteins.     

Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Comparison of rice and Arabidopsis annotation

Several versions of the rice genome were published in 2002, providing a first overview of the genome content of this model monocot. At the same time, the genome of the model dicot, Arabidopsis thaliana, reached a new level of annotation as thousands of full-length cDNA sequences were integrated with the genome sequence.     

High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic <Emphasis Type="Italic">Thermotoga maritima</Emphasis> and the determinants of its thermostability

The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.