Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

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.     

The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.     

Unusual conformation of a 3′-thioformacetal linkage in a DNA duplex

Summary The DNA·DNA duplex ·d(GCGCAAAACGCG) (designated duplex III) containing a 3-thioformacetal (3-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH₂O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3-TFMA-modified T6 sugar adopts an O4-endo form (an intermediate structure between the usual C2-endo and C3-endo forms). This change is accompanied by a change in the (C4–C3–S3–CH₂) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3-TFMA linkages is merely in the substitution of the T6(O3) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.Supplementary material available from the authors: One table containing J₁₂, J₁₂ and J₃₄ of duplexes I, II and III.     

Is the effect of priming plants,and a functional JAR1, negligible on the foraging behaviour and development of a generalist lepidopteran,Helicoverpa armigera?

Theory and recent literature suggest strong effects of induced plant defences in some plant herbivore systems. Few have studied behavioural effects on intact plants. Differences in foraging behaviour as well as weight gain were determined for first instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on Arabidopsis thaliana (L.) Heynh. (Brassicaceae) mutant and wild type plants, non‐primed, or primed by herbivore feeding or methyl jasmonate. The differences in feeding were primarily in the length of feeding time as opposed to the area fed on, feeding location, or frequency. More larvae dispersed from plants after priming by mite feeding than dispersed after caterpillar feeding. Other behavioural activities such as resting were not significantly affected. Early instars gained less weight feeding on ein2 (ethylene insensitive) mutant, but there was no difference in weight gain between larvae feeding on induced and non‐induced plants of the same type. We concluded that there are fitness consequences for neonates of the generalist H. armigera after feeding on induced plant tissues in some cases, and that distinct changes in behaviour are recognisable both at the fine scale and at grosser levels (dispersal). However, these changes are more subtle than might be expected.     

Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp

Aims: The aim of this study was to develop a real‐time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). Methods and Results: A duplex real‐time PCR specific for the genes encoding for β‐glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety‐six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. Conclusions: The duplex real‐time PCR assay was found to be simple, rapid, reliable and specific. Significance and Impact of the Study: If possible at all, delineation of so‐called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno‐ and serotypes. This assay clearly simplifies the differentiation of both.     

Patterns of richness and endemism of the Mexican herpetofauna,a matter of spatial scale?

The goal of this study was to compare the richness and endemism patterns of Mexican species of amphibians and reptiles at different spatial scales. We used the best available dataset of distributional ranges generated from ecological niche models and employed geographically weighted regressions (GWRs) to test whether richness and endemism were related. Patterns were found to vary with the scale used for richness and endemism, and these patterns were not coincident. The results showed that: (1) only relatively coarse spatial scales can address latitudinal patterns in amphibians and reptiles, and, in fine scales, they are related to topographic formations; (2) areas of greatest endemism for amphibians and reptiles are located in the highlands of the central or southern part of the country, although not necessarily in the same specific highlands for both groups; (3) there is a strong average correlation between richness and endemism for both groups, indicating that the same factors contribute to both patterns, but these factors act differentially in terms of regions among amphibians and reptiles; and (4) the scale at which the analysis is conducted is important, and we believe that careful consideration of spatial scale must be undertaken to avoid false conclusions. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 305–316.     

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site

Recognition of the 5′ splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem–loop of domain 1 and a complementary sequence at the 3′ end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem–loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5′ splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1–IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.     

Analysis of the conformational features of Watson–Crick duplex fragments by molecular mechanics and quantum mechanics methods

It is generally accepted that important features of the Watson–Crick duplex (WCD) originate from the molecular structure of its subunits. However, it is still unclear what properties of each subunit are responsible for significant features of the WCD structure. Computations of deoxydinucleoside monophosphate (dDMP) complexes with Na ions on the basis of the density functional theory (DFT) have shown that conformational properties of minimal single-stranded fragments of DNA play a pivotal role in the origin of the unique features of the WCD. The directionality of the sugar-phosphate backbone (SPB) and preferable ranges of its torsion angles combined with the difference in geometry between purines and pyrimidines have been found to define the nucleotide sequence dependence of the WCD three-dimensional structure. In this work, density functional theory computations were extended to minimal duplex fragments, that is, complementary dDMP (cdDMP) complexes with Na ions. Using several computational methods and various functionals, energy minima were searched for the BI conformation of cdDMP complexes with different nucleotide sequences. Two sequences were optimized using an ab initio method at the MP2/6-31++G** level of theory. An analysis of the SPB torsion angles, sugar-ring puckering, and mutual base positions in the optimized structures showed that the conformational features of cdDMP complexes with Na ions remained within the BI ranges and become more similar to the corresponding features that WCDs display in a crystal. Qualitatively, the main features of each cdDMP complex were invariant with different computational methods, although the values of certain conformational parameters could vary, but still within the limits that are typical of the corresponding family. Common functionals that are employed in DFT calculations were observed to overestimate the distance between base pairs, while MP2 computations and new complex functionals yielded structures with atom–atom contacts that are too close. Several energy minima that correspond to the BI conformation have been proven to exist for certain cdDMP complexes with Na ions, indicating that the topography of the potential energy surface is complex. This circumstance accounts for the variation of conformational parameters among duplex fragments with the same nucleotide sequence. The common AMBER and CHARMM molecular mechanics force fields reproduce many conformational characteristics of dDMPs and their complementary complexes with Na ions, but fail to reproduce certain details of the nucleotide sequence dependence of the WCD conformation.     

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Using the Capsuleen (Csul) methyltransferase as bait in the yeast two-hybrid system, we have identified a novel Drosophila protein containing multiple WD repeats and encoded by the valois (vsl) gene, which acts in pole plasm function. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase--the human homologue of Csul. We found that Vls localizes to the nuage in the nurse cells and to the pole plasm in the oocyte. Moreover vls is required for the synthesis and/or stability of Oskar and the localization of Tudor (Tud) in both the nuage and at the posterior pole of the oocyte. Furthermore, we show that Vls and a fragment of Tud interact directly in binding assay. As the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, we hypothesize that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte.     

NMR study of hydrogen exchange during the B-Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα_E3L) bound to Z-DNA revealed that the overall structure of yabZα_E3L and its interaction with Z-DNA are very similar to those of hZα_ADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα_E3L and d(CGCGCG)₂ with a variety of protein-to-DNA molar ratios. This study revealed that yabZα_E3L could efficiently change the B-form helix of the d(CGCGCG)₂ to left-handed Z-DNA via the active-mono B-Z transition pathway like hZα_ADAR1.     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

Sequence-dependent conformational heterogeneity of a hybrid DNA·RNA dodecamer duplex

Summary Two- and three-dimensional homonuclear NMR studies of a hybrid duplex RI, formed by annealing r(GCGCAAAACGCG) and d(CGCGTTTTGCGC) strands are described. NMR parameters, such as intra-and interresidue proton-proton NOEs and sugar proton coupling constants were analyzed with reference to those of the corresponding DNA·DNA duplex. Furthermore, spectral analyses were conducted on the basis of model structures of nucleic acid duplexes. Distinctive spectral patterns of the hybrid duplex reveal unique heterogeneous conformations which co-exist throughtout the sequence and are significantly different from those of model structures of either canonical A- or B-forms. Features of an intermediate conformation were observed in the DNA and RNA strands in duplex RI, the former being more B-like and the latter more A-like. Three-dimensional NOESY-NOESY spectra were analyzed and their use was demonstrated for resolving superimposed resonances and cross peaks, especially those originating from the RNA strand. The application of a useful strategy that combines the use of 2D NMR data and the known structural information for efficient 3D spectral analyses is demonstrated.     

Effects of rapeseed‐press cake glucosinolates and iodine on the performance,the thyroid gland and the liver vitamin a status of pigs

Rapeseed press cake (per kg DM 181 g EE, 341 g CP and 23.3 mmol glucosinolates) was tested in a long‐term experiment with a total of sixty pigs (live weight range 24 to 104 kg). The 3×2 factorial design consisted of three rapeseed press cake levels (no rapeseed press cake ‐ control, 75g or 150g rapeseed press cake per kg diet) each with two iodine dosages (125 or 250 μg supplementary iodine per kg diet). Reduced feed intake and depressed weight gain were found in groups receiving 150 g rapeseed press cake per kg diet, which correspond to 3.2mmol glucosinolates per kg diet. At an inclusion level of 75 g rapeseed‐press cake per kg diet no differences in feed intake and growth intensity were recorded in comparison to the rape feed free control. The rapeseed‐press cake diet increased the weight of thyroid gland and liver and decreased the serum thyroxine (T₄) concentration. Higher iodine dosage increased the serum T₄ concentration of pigs receiving 75 g rapeseed press cake per kg diet ( = 1.6 mmol glucosinolates per kg diet) to the level of the control group and retarded the enlargement of the thyroid gland. Intake of rapeseed products lowered the iodine content of the thyroid gland, however, there was no significant difference between groups given 1.6 and 3.2 mmol glucosinolates per kg diet. The vitamin A content of the whole liver and the vitamin A serum concentration were not influenced by the diets tested. However, rapeseed press cake and the glucosinolates, respectively, decreased the vitamin A concentration per gram liver due to the organ enlargement and the resulting dilution effect.     

Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.     

Chronicle of a summer and the editing of cinema‐verite

Jean Rouch and Edgar Morin's Chronicle of a Summer is held up as an innovative, seminal “work in the formation of cinéma‐vérité, and has a central position in the development of documentary and ethnographic filmmaking. Although much has been written about fthis film, little attention has been paid to the film's construction. This paper examines ‘elements of the editing structure of Chronicle of a Summer both to consider how this “work was shaped given the constraints of its production and intended reception and to ¦ analyze a relatively neglected aspect of documentary film practice.     

Canopy transpiration and water fluxes in the xylem of the trunk of Larix and Picea trees — a comparison of xylem flow,porometer and cuvette measurements

Summary Leaf gas exchange, transpiration, water potential and xylem water flow measurements were used in order to investigate the daily water balance of intact, naturally growing, adult Larix and Picea trees without major injury. The total daily water use of the tree was very similar when measured as xylem water flow at breast height or at the trunk top below the shade branches, or as canopy transpiration by a porometer or gas exchange chamber at different crown positions. The average canopy transpiration is about 12% lower than the transpiration of a single twig in the sun crown of Larix and Picea. Despite the similarity in daily total water flows there are larger differences in the actual daily course. Transpiration started 2 to 3 h earlier than the xylem water flow and decreased at noon before the maximum xylem water flow was reached, and stopped in the evening 2 to 3 h earlier than the water flow though the stem. The daily course of the xylem water flow was very similar at the trunk base and top below the lowest branches with shade needles. The difference in water efflux from the crown via transpiration and the water influx from the trunk is caused by the use of stored water. The specific capacitance of the crown wood was estimated to be 4.7 x 10^-8 and 6.3 x 10^-8 kg kg^-1 Pa^-1 and the total amount of available water storage was 17.8 and 8.7 kg, which is 24% and 14% of the total daily transpiration in Larix and Picea respectively. Very little water was used from the main tree trunk. With increasing transpiration and use of stored water from wood in the crown, the water potential in the foliage decreases. Plant water status recovers with the decrease of transpiration and the refilling of the water storage sites. The liquid flow conductance in the trunk was 0.45 x 10^-9 and 0.36 x 10^-9 mol m^-2s^-1 Pa^-1 in Larix and Picea respectively. The role of stomata and their control by environmental and internal plant factors is discussed.