Complexes with truncated RNAs from the large domain of Archaeoglobus fulgidus signal recognition particle

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

Evolutionary relatedness of some primate models of Plasmodium

Primate--and, specifically, monkey--malaria infections are commonly used for understanding the pathology of and immune response to the human disease because they are thought to resemble most closely the host-parasite relationship found in humans. Plasmodium cynomolgi is used extensively as a model for the human parasite, P. vivax, and P. knowlesi is used primarily as a model for the development of erythrocytic-stage vaccines. Both of these simian parasites can naturally infect man, resulting in mildly symptomatic episodes of the disease. The phylogenetic relationship between these two simian parasites and previously characterized Plasmodium species, including P. vivax, was examined by comparison of the asexually expressed small- subunit ribosomal RNA genes. Our analysis confirmed that P. vivax is most closely related to P. cynomolgi and that it remains an appropriate model of the human pathogen. Furthermore, with P. knowlesi and P. fragile, these two species form a group of closely related species, distant from other Plasmodium species. What is considered to be the most ancient of the human malaria pathogens, P. malariae, was also included in the analysis and does not group at all with other simian or human parasites.      

Evolution and phylogenetic information content of the ITS-1 region in the tiger beetle Cicindela dorsalis

Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.      

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

A new practical tool for deriving a functional signature for herbaceous vegetation

Hypothesis: For any one time and place a ‘functional signature’ can be derived for a sample of herbaceous vegetation in a way that concisely represents the balance between the different clusters of functional attributes that are present among component species. Methods: We developed a spreadsheet‐based tool for calculating functional signatures within the context of the C‐S‐R system of plant functional types. We used the tool to calculate and compare signatures for specimen British vegetation samples which differed in management regime and location in time. Conclusion: The integrative power of the ‘C‐S‐R signature’ is useful in comparative studies involving widely differing samples. Movements in the signature can be used to indicate degree of resistance, resilience, eutrophication and dereliction. Systems of plant functional types other than C‐S‐R might also be approached in this way. Availability: The tool can be downloaded free of charge from the first author's web pages or from the journal's electronic archive.     

Solution structure and backbone dynamics of the TGFbeta type II receptor extracellular domain

Isoforms of transforming growth factor beta (TGFbeta) are 25 kDa homodimeric polypeptides that signal by binding and bringing together two related, functionally distinct cell surface receptors designated as TbetaR1 and TbetaR2. Here, we report the solution structure of the 13.8 kDa extracellular domain of human TbetaR2 (ecTbetaR2) as calculated from N(N)-H(N), C(alpha)-H(alpha), and C(alpha)-C(O) residual dipolar coupling restraints in conjunction with NOE distance, dihedral angle, and scalar coupling restraints. Comparison of the free ecTbetaR2 solution structure with the TGFbeta3-bound ecTbetaR2 crystal structure reveals backbone conformations that superimpose with RMSDs of 1.0 A over the regions of regular secondary structure and 1.4 A overall. The differences in structure fall mainly in loop regions that are either poorly defined by the available NMR data or are involved in crystal contacts. The noted similarities between the NMR structure of the free form and the crystal structure of the TGFbeta-bound form are also consistent with the close correspondence, 0.16 A RMSD for regions of secondary structure and 0.51 A RMSD overall, for the crystal structure of free ecTbetaR2 as compared to the crystal structure of TGFbeta3-bound ecTbetaR2. Despite the apparent similarities between the free and the bound forms, there appears to be small but significant differences in structure involving the interfacial contact region of the receptor. Measurements of backbone (15)N relaxation times and interpretation of these by the model-free formalism with axial diffusional anisotropy further reveal significant ms to micros time scale motions centered about two of the conserved disulfide bonds and in several residues that comprise the TGFbeta binding surface. Together, these observations indicate that binding likely occurs through a mechanism with a small component of induced fit character, whereby flexibility within the receptor facilitates the transition to the TGFbeta-bound state.     

UNC5H1 induces apoptosis via its juxtamembrane region through an interaction with NRAGE

The UNC5Hs are axon guidance receptors that mediate netrin-1-dependent chemorepulsion, and dependence receptors that mediate netrin-1-independent apoptosis. Here, we report an interaction between UNC5H1 and NRAGE. Our experiments show that this interaction is responsible for apoptosis induced by UNC5H1, and this level of apoptosis is greater than the amount induced by either UNC5H2 or UNC5H3. We mapped the NRAGE binding domain of UNC5H1 to its ZU-5 domain and show that this region, in addition to an adjacent PEST sequence, is required for UNC5H1-mediated apoptosis. Chimeric UNC5H2 and UNC5H3 receptors, containing the NRAGE binding domain and PEST sequence of UNC5H1, bind NRAGE and cause increased levels of apoptosis. UNC5H1 expression does not induce apoptosis in differentiated PC12 cells, which down-regulate NRAGE, but induces apoptosis in native PC12 cells that endogenously express high levels of NRAGE and in differentiated PC12 cells when NRAGE is overexpressed. Together, these results demonstrate a mechanism for UNC5H1-mediated apoptosis that requires an interaction with the MAGE protein NRAGE.