Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

Production of Recombinant Hinnavin II/MSH Hybrid in Insect Cells

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. For the purpose of this study, a cDNA encoding hinnavin II‐α‐melanocyte stimulating hormone (hin/MSH) hybrid was chemically synthesized, annealed, and then cloned into transfer vector pBacPAK 9 for expression in Sf21 insect cells. Recombinant hin/MSH (rhin/MSH) hybrid was efficiently produced in baculovirus expression vector system (BEVS) as a hybrid peptide. The antibacterial activity of the rhin/MSH hybrid was compared to that of the recombinant hinnavin II (rhin), using inhibition zone and overlay assay. This new recombinant hybrid peptide may serve as an attractive candidate for powerful novel class of antimicrobial pharmaceuticals.     

Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales

Background and Aims

Detailed knowledge of variations in ploidy levels and their geographic distributions is one of the key tasks faced in polyploid research in natural systems. Flow cytometry has greatly facilitated the field of cytogeography by allowing characterization of ploidy levels at both the regional and population scale, and at multiple stages of the life cycle. In the present study, flow cytometry was employed to investigate the patterns and dynamics of ploidy variation in the taxonomically challenging complex Knautia arvensis (Dipsacaceae) and some of its allies (K. dipsacifolia, K. slovaca) in Central Europe.

Methods

DNA ploidy levels were estimated by DAPI flow cytometry in 5205 adult plants, 228 seedlings and 400 seeds collected from 292 Knautia populations in seven European countries. The flow cytometric data were supplemented with conventional chromosome counts. A subset of 79 accessions was subjected to estimation of the absolute genome size using propidium iodide flow cytometry.

Key Results and Conclusions

Five different ploidy levels (from 2x to 6x) were found, with triploids of K. arvensis being recorded for the first time. The species also exhibited variation in the monoploid genome size, corresponding to the types of habitats occupied (grassland diploid populations had larger genome sizes than relict and subalpine diploid populations). Disregarding relict populations, the distribution of 2x and 4x cytotypes was largely parapatric, with a diffuse secondary contact zone running along the north-west margin of the Pannonian basin. Spatial segregation of the cytotypes was also observed on regional and microgeographic scales. The newly detected sympatric growth of diploids and tetraploids in isolated relict habitats most likely represents the primary zone of cytotype contact. Ploidy level was found to be a major determinant of the strength of inter-cytotype reproductive barriers. While mixed 2x + 4x populations virtually lacked the intermediate ploidy level at any ontogenetic stage, pentaploid hybrids were common in 4x +6x populations, despite the cytotypes representing different taxonomic entities.Key words: Contact zone, cytogeography, flow cytometry, genome size, hybridization, Knautia arvensis, ploidy mixture, polyploidy, relict, reproductive isolation, serpentine     

The paradox high zooplankton biomass-low vegetal particulate organic matter in high turbidity zones: What way for energy transfer?

Estuarine ecosystems are characterized by high zooplanktonic biomasses, essentially constituted by copepods and mysids whose nutritional requirements are mainly provided by phytoplankton, an easily available carbon form. The Gironde estuary is characterized by high turbidities which limit light penetration in the water column and therefore primary production. Consequently, primary production is low and its availability for higher trophic level is very limited. The main goal of this study was to characterize the total vegetal particulate organic matter (POM) in high turbidity zones of the Gironde estuary during summer (a critical period characterized by high heterotrophic bacterial degradation and high zooplanktonic biomasses) and to analyse its utilization by zooplankton, using prey/predator experiments and trophic biomarkers (fatty acids). The specific goals were to define (i) how vegetal POM was exploited by the different zooplanktonic groups (protozoa, copepods and mysids) and (ii) which alternative preys could be used when vegetal POM was not sufficient to ensure their nutritional requirements.Chlorophyll biomass was very low in the MTZ during summer 2002 (0.48 ± 0.03 mg m^− 3). Total zooplankton grazing was low (19% d^− 1) probably due to a large contribution of detritus originating from terrestrial plants in vegetal POM compared to phytoplankton. The highest grazing pressure was exercised by the mysid Mesopodopsis slabberi due to its high abundances and by its almost entirely herbivorous diet (phytoplankton and small terrestrial detritus). Grazing rates (19.7 ± 4.2 and 9.6 μgC cop^− 1 d^− 1 for juveniles and adults, respectively) seemed to be sufficient to satisfy their daily carbon requirement. Grazing rate of the copepod Eurytemora affinis (139 ngC cop^− 1 d^− 1) seemed to be insufficient to cover its nutritional requirements and the copepods probably needed to complete a great part of their diet from protozoa. Grazing rates of the mysid Neomysis integer (24.7 ± 0.01 and 20.89 ± 8.45 μgC cop^− 1 d^− 1 for juveniles and adults, respectively) were higher than those of M. slabberi when feeding only on phytoplankton. However, when other preys were introduced in its environment, N. integer only fed on the copepod E. affinis with a preference for nauplii. The study revealed the great importance of protozoa and bacteria in the trophic transfers between vegetal POM and zooplankton in the MTZ during summer, despite the low protozoa grazing pressure on vegetal POM (3.1%). The detritic food chain probably implies various trophic transfers with little direct relationships between vegetal POM and zooplankton.     

Diversity of human U2AF splicing factors

U2 snRNP auxiliary factor (U2AF) is an essential heterodimeric splicing factor composed of two subunits, U2AF(65) and U2AF(35). During the past few years, a number of proteins related to both U2AF(65) and U2AF(35) have been discovered. Here, we review the conserved structural features that characterize the U2AF protein families and their evolutionary emergence. We perform a comprehensive database search designed to identify U2AF protein isoforms produced by alternative splicing, and we discuss the potential implications of U2AF protein diversity for splicing regulation.     

Electrophoretic behavior of the H+-ATPase proteolipid from bovine heart mitochondria

The proteolipid subunit of H⁺-ATPase was labeled by [¹⁴C]N,N-dicyclohexylcarbodiimide in bovine heart mitochondria. The radioactive labeling was followed using various systems of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). When using discontinuous SDS-PAGE (Laemmli, U.K., 1970,Nature (London)227, 680–685) a monomeric (Mr 7600±1500) and a dimeric form (Mr 17,800±1200) of the proteolipid were detected, while only the monomeric form was found on urea (8 M) containing gels (SDS-PAGE according to Laemmli; or Swank, R. T., and Munkers, K. D., 1971,Anal. Biochem. 39, 462–477). When using SDS-PAGE with Na-P_i buffer (Weber, K., and Osborn, M., 1969,J. Biol. Chem. 244, 4406–4442), only a dimeric form of the proteolipid (Mr 15,000±1000) was detected. Experimental data indicate that the different patterns of proteolipid separation are related to the presence of the two distinct proteolipid conformations in the SDS solution.     

Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A·A base pairing and a putative structure of the coralyne-induced homo-adenine duplex

Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson–Crick DNA. Hud’s laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine–adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson–Crick Hoogsteen (transWH) or Watson–Crick Watson–Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA→7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A·A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA→7 mutations, these results are consistent with the N1–N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.     

Isolation of polysaccharide-free DNA from plants

A quick procedure for the isolation of polysaccharide-free DNA from different plant species and cell suspension or callus cultures is described. The originality of the method lies in the use of a mixture of glycoside hydrolases that leads, after phenol and chloroform extraction, to the isolation of pure DNA without any polysaccharide contamination. The highly purified DNA can be used for nucleotide analysis by HPLC, RFLP analysis and PCR amplification.     

Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.