Automated immunomagnetic processing and separation of Legionella pneumophila with manual detection by sandwich ELISA and PCR amplification of the ompS gene.

The culture-independent and automated detection of bacteria in the environment is a scientific and technological challenge. For detection alone, a number of sensitive methods are known (e.g., PCR, enzyme-linked immunosorbent assay [ELISA], fluorescent in situ hybridization) but a major problem remaining is the enrichment and separation of the bacteria that usually occur at low concentrations. Here, we present an automated capturing and separation system, which can easily be combined with one of the sensitive detection techniques. We have developed a method for enrichment and detection of Legionella pneumophila in liquid media. Concentrated microorganisms were either detected by PCR or by sandwich ELISA. The limit of detection with the immunological assay was about 750 bacteria. Using PCR, the equivalent of about 2000 genomes could be detected. The assays were then transferred to a laboratory prototype for automated processing. It was possible to automatically enrich L. pneumophila by immunomagnetic separation (IMS), and again, the bacteria were detected by sandwich ELISA and PCR amplification of the ompS gene. As a novel aspect, ompS gene was used for the first time as a target for the detection of L. pneumophila on magnetic beads. The aim of this work was to develop an automated procedure and a device for IMS of bacteria. With Legionella as a model organism, we could show that such a novel fully automated system can be an alternative to time-consuming conventional cultivation methods for detecting bacteria or other microorganisms.

Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.

The expression of the HSP70 gene in Moneuplotes crassus is controlled by a two-step process at the transcript level.

Steady state levels of the HSP70 transcript were followed by Northern hybridization in Moneuplotes crassus in order to investigate the mechanisms of the short term and long term response to heat shock in a spirotrichous ciliate. The influence of inhibitors of transcription or translation on the transcript levels was also studied. The heat shock response could be dissected into two phases. An initial protein-dependent stabilization of the mRNA was followed by an increase of the steady state transcript level that was dependent on continued transcription. As expected, the half-life of the RNA was short. Western blot analysis then showed that the HSP70 protein accumulated only upon permanent heat shock. It is concluded that the regulation of the heat shock response is a two-step process that occurs at the transcript level.

The zinc finger-associated domain of the Drosophila transcription factor grauzone is a novel zinc-coordinating protein-protein interaction module.

About one-third of the more than 300 C2H2 zinc finger proteins of Drosophila contain a conserved sequence motif, the zinc finger-associated domain (ZAD). Genes that encode ZAD proteins are specific for and expanded in the genomes of insects. Only three ZAD-encoding gene functions are established, and the role of ZAD is unknown. Here we present the crystal structure of the ZAD of Grauzone (ZAD(Grau)), a Drosophila transcription factor that specifically controls the maternal Cdc20-like APC subunit Cortex. ZAD forms an atypical treble-clef-like zinc-coordinating fold. Head-to-tail arrangement of two ZAD(Grau) molecules in the crystals suggests dimer formation, an observation supported by crosslinking and dynamic light scattering. The results indicate that ZAD provides a novel protein-protein interaction module that characterizes a large family of insect transcription factors.

Two protein-protein interaction sites on the spliceosome-associated human cyclophilin CypH.

Cyclophilins are a family of proteins that share a common, highly conserved sequence motif. Cyclophilins bind transiently to other proteins and facilitate their folding. One member of the family, hCypH, is part of the human spliceosomal [U4/U6.U5] tri-snRNP complex; it associates specifically and stably with the U4/U6-specific protein 60K. Here, we demonstrate that recombinant hCypH exhibits peptidyl-prolyl isomerase (PPIase) activity, and describe mutagenesis studies demonstrating that it shares the catalytic pocket with other members of the cyclophilin family. However, neither the PPIase activity nor the catalytic pocket is required for binding of protein 60K. Rather, hCypH contains a small insertion in a loop of the otherwise conserved cyclophilin backbone, and this minor change creates a highly specific binding site that is responsible for the association of this cyclophilin, but not others, with protein 60K. hCypH is thus the first small cyclophilin shown to have a second protein-protein interaction site and the ability to bind stably to another protein. Since the catalytic pocket and the second binding site are located on opposite sides of the cyclophilin structure, this opens up the interesting possibility that hCypH may serve as a bridge mediating interactions between protein 60K of the U4/U6 snRNP and other as yet unknown factors.

Crystal structure of a complex between human spliceosomal cyclophilin H and a U4/U6 snRNP-60K peptide.

The spliceosomal cyclophilin H is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle, interacting with homologous sequences in the proteins U4/U6-60K and hPrp18 during pre-mRNA splicing. We determined the crystal structure of the complex comprising cyclophilin H and the cognate domain of U4/U6-60K. The 31 amino acid fragment of U4/U6-60K is bound to a region remote from the cyclophilin active site. Residues Ile118-Phe121 of U4/U6-60K expand the central beta-sheet of cyclophilin H and the side-chain of Phe121 inserts into a hydrophobic cavity. Concomitantly, in the crystal the cyclophilin H active site is occupied by the N terminus of a neighboring cyclophilin H molecule in a substrate-like manner, indicating the capacity of joint binding to a substrate and to U4/U6-60K. Free and complexed cyclophilin H have virtually identical conformations suggesting that the U4/U6-60K binding site is pre-shaped and the peptidyl-prolyl-cis/trans isomerase activity is unaffected by complex formation. The complex defines a novel protein-protein interaction mode for a cyclophilin, allowing cyclophilin H to mediate interactions between different proteins inside the spliceosome or to initiate from its binding platforms isomerization or chaperoning activities.