A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes)

A goldfish (Carassius auratus auratus) bacterial artificial chromosome genomic library (BAC library) was constructed from one aquarium-bred male specimen (tetraploid, 4n=100, genome size=3.52 pg/cell). The library consists of 128,352 positive clones with an average insert size of 150.4 kb, covering the genome 11-fold. All clones were spotted onto nylon filters and thus are available for screening of genomic regions of interest, such as candidate genes, gene families, or large-sized syntenic DNA regions of cyprinid species. Preliminary screens with two genes were conducted with hybridizing probes to the genes RAG1 and lgi1. RAG1 is a single-copy gene in zebrafish and is duplicated in C. a. auratus. We found a very close correlation between the number of positive BAC clones and the expected library coverage. Two copies of lgi1 were found in zebrafish. We have detected four different copies in C. a. auratus, not in the expected abundance, which indicates some variation in the coverage of the BAC library. The preliminary screens indicate that many duplicated genes that resulted from the ancient fish-specific genome duplication persist in the tetraploid goldfish genome. Hence, the BAC library will provide a useful resource for the future work on comparative genomics, polyploidy, diploidization, and evolutionary genomics in fishes.     

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.     

Development of single-cell PCR methods for the Raphidophyceae

Many Raphidophytes are important algal bloom-forming species. Morphology-based identification of these species is often ambiguous, however, as many species are very similar in shape and size. To accurately detect the presence of these species in pre-bloom conditions, single-cell PCR is probably the most rapid and convenient method. However, direct single-cell PCRs with conserved primers are apparently not effective, probably due to the impermeability of the cell wall. We report here an effective detergent-based pre-PCR cell lysis method, which turned out to be a critical step for effective single-cell PCR of the Raphidophytes. Two PCR-based methods, nested SC-PCR and SC-RAPD, were evaluated. The nested SC-PCR involves two consecutive PCRs, the first of which is performed with the D1 and D2 primers (external primers) resulting in an amplification of a partial LSU rRNA gene. The second amplification is performed with primers targeting the LSU domain and specifically annealing to Chattonella ovata and Chattonella marina only. The SC-RAPD performed with the established random primers, RP1–RP4, produced unique haplotypes that could be exploited to differentiate the two Chattonella species. The assay was demonstrated to be sensitive, with the lowest detection limit of a single Raphidophyceae cell. The method developed is a valuable tool for the study of intra-specific variations of the Raphidophytes and represents a platform for further development of species-specific SC-RAPD for all members of the Raphidophyceae.     

Bridging Proteomics and Medical Science - the 5th HUPO Brain Proteome Workshop in Dublin, Ireland

More than 70 interested colleagues attended the 5th Workshop of the HUPO Brain Proteome Project (HUPO BPP) at the UCD Conway Institute, Dublin, Ireland. An overview of the outcome of the pilot study was presented and the new subprojects "Clinical Neuroproteomics of Human Body Fluids" as well as "Cerebellum 2D-mapping" were announced. In addition the election of the HUPO BPP committees and the future directions of this project were discussed and decided. The meeting was enhanced by several talks highlighting the application of proteomics in biomedical research.     

Automated reprocessing pipeline for searching heterogeneous mass spectrometric data of the HUPO Brain Proteome Project pilot phase

The newly available techniques for sensitive proteome analysis and the resulting amount of data require a new bioinformatics focus on automatic methods for spectrum reprocessing and peptide/protein validation. Manual validation of results in such studies is not feasible and objective enough for quality relevant interpretation. The necessity for tools enabling an automatic quality control is, therefore, important to produce reliable and comparable data in such big consortia as the Human Proteome Organization Brain Proteome Project. Standards and well-defined processing pipelines are important for these consortia. We show a way for choosing the right database model, through collecting data, processing these with a decoy database and end up with a quality controlled protein list merged from several search engines, including a known false-positive rate.     

Influence of modulated structural dynamics on the kinetics of alpha-chymotrypsin catalysis. Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Although the chemical nature of the catalytic mechanism of the serine protease alpha-chymotrypsin (alpha-CT) is largely understood, the influence of the enzyme's structural dynamics on its catalysis remains uncertain. Here we investigate whether alpha-CT's structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation [Solá RJ & Griebenow K (2006) FEBS Lett 580, 1685-1690] was used to generate a series of glycosylated alpha-CT conjugates with reduced structural dynamics, as determined from amide hydrogen/deuterium exchange kinetics (k(HX)). Determination of their catalytic behavior (K(S), k(2), and k(3)) for the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) revealed decreased kinetics for the catalytic steps (k(2) and k(3)) without affecting substrate binding (K(S)) at increasing glycosylation levels. Statistical correlation analysis between the catalytic (DeltaG( not equal)k(i)) and structurally dynamic (DeltaG(HX)) parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the alpha-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing the Gaussian network model revealed structural insights into the relation between the protein's surface glycosylation, the resulting structural dynamic changes, and the influence of these on the enzyme's collective dynamics and catalytic residues. The experimental and theoretical results presented here not only provide fundamental insights concerning the influence of glycosylation on the protein biophysical properties but also support the hypothesis that for alpha-CT the global structural dynamics directly influence the kinetics of enzyme catalysis via mechanochemical coupling between domain motions and active site chemical groups.