Polymorphism of the 5′ Untranslated Region of NHE1 Gene Associated with Type-I Diabetes

The ubiquitous form of the sodium–hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5′-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.     

Influence of the Length of the Phosphate Chain in mRNA 5′ Cap Analogues on Their Interaction with Eukaryotic Initiation Factor 4E

Abstract

The recognition of the 5′mRNA cap structure m⁷G(5′)ppp(5′)N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Deep Sequencing Shows Multiple Oligouridylations Are Required for 3′ to 5′ Degradation of Histone mRNAs on Polyribosomes

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Synthesis of an Oligonucleotide Bearing a Phosphate Function at the 5′-Terminus Using a Novel Protecting Group in Terms of a Cellulose Acetate Derivative as a Polymer-Support

Abstract

Synthesis of the title oligonucleotide bearing a phosphate function at the 5′-terminus, i.e., pCpUpCpGpUpCpCpApCpCpA, by the use of the terminal cytidylic acid unit involving a novel protecting group of 2-[2-(monomethoxytrityloxy)ethylthio]ethyl group on its 5′-phosphoryl function in terms of a cellulose acetate polymer-support is described.     

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

Metagenomic Identification of a Novel Salt Tolerance Gene from the Human Gut Microbiome Which Encodes a Membrane Protein with Homology to a brp/blh-Family β-Carotene 15,15′-Monooxygenase

The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.     

Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.It is generally assumed that sulfonated azo dyes are not degraded under aerobic conditions (14). Nevertheless, there have been some reports which suggest a conversion of certain sulfonated azo dyes under aerobic conditions (3, 7, 8, 13, 15). Furthermore, certain carboxylated analogs of sulfonated azo compounds are utilized aerobically as the sole source of carbon and energy by specifically adapted bacteria (11, 12, 16, 17). However, unequivocal evidence for the productive mineralization of a sulfonated azo compound by bacteria is lacking. In the present article the first observation of the utilization of a sulfonated azo compound as the sole source of carbon and energy by a bacterial strain is reported.Previously, a mixed bacterial culture which mineralizes sulfanilate (4-aminobenzenesulfonate) was isolated. This coculture consisted of the strains “Hydrogenophaga palleronii” S1 and Agrobacterium radiobacter S2 (4, 5). Because sulfanilate occurs as an azoaryl structural element in many azo dyes, it was of interest whether this mixed culture could adopt the ability to reduce azo bonds and release sulfanilate as growth substrate. Therefore, the model sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (CSAB) was synthesized by nitro-amine condensation starting with sulfanilic acid and 4-nitrobenzoic acid (1). The precipitated CSAB was separated from the reaction mixture by filtration and purified by repeated dissolution in alkali and precipitation with acid. The identity and purity of the bright orange product were analyzed by UV-visible light spectroscopy, elementary analysis, and high-pressure liquid chromatography (HPLC). For the solid material obtained, molar extinction coefficients of 23.74 and 1.13 mM⁻¹ cm⁻¹ in water were determined at the wavelengths of 326 and 434 nm, respectively. The elementary analytic results were consistent with the structure of CSAB. The purity of the preparation was tested by HPLC with a reversed-phase column and a solvent gradient from 1 to 90% (vol/vol) methanol and 0.3% (vol/vol) H₃PO₄. A single band which showed absorbance at a wavelength of 326 nm was eluted. At 210 nm a minor contaminant (about 15% of the signal intensity of CSAB) was detected. This compound was clearly different from either 4-nitrobenzoate or sulfanilate.The mixed culture was grown in repeated batch cultures in a mineral medium with sulfanilate (5 mM). About every 2 weeks the culture was transferred (1:10 [vol/vol]) to fresh medium, in which the sulfanilate concentration was subsequently reduced and the CSAB concentration increased (±0.5 mM each). The color of the azo dye disappeared after 2 months. The culture was transferred to a solid mineral medium with CSAB as the sole source of carbon. From this culture was obtained strain S5, which grew aerobically with the sulfonated azo compound CSAB as its sole source of carbon and energy and with a doubling time of 9.5 h (Fig. ​(Fig.1).1). The complete disappearance of the dye was demonstrated by the loss of the orange color from the medium and by HPLC analysis, whereas CSAB was not degraded in a sterile control flask. Based on its colony morphology and the results obtained with the commercial identification system Biolog GN, this strain strongly resembled “H. palleronii” S1. Recently, it was demonstrated that, in the presence of low concentrations of biotin, cyanocobalamin, and 4-aminobenzoate, strain S1 also grows in axenic culture with sulfanilate (2). Therefore the adaptation experiment was repeated in the presence of these three substances with a pure culture of strain S1. This experiment also resulted in the isolation of a strain which grew in axenic culture with CSAB as the sole source of carbon and energy. Open in a separate windowFIG. 1Aerobic growth of strain S5 with CSAB as the sole source of carbon and energy. The growth was determined photometrically (OD₅₄₆), and the turnover of CSAB was measured by HPLC with a reversed-phase column and a solvent gradient consisting of H₂O, methanol, and 0.3% H₃PO₄ with increasing concentrations of methanol (1 to 90%). An OD₅₄₆ of 1 corresponded to 0.33 mg of protein ml⁻¹.To ensure that the genetic backgrounds of strains S5 and S1 were identical, the genes for the 16S rRNAs were amplified by PCR with different universal primers (6) and sequenced in comparison to the corresponding gene from the type strain, H. palleronii DSM 63. It was found that the sequences from strains S1 and S5 were > 99.8% identical (there were only two discrepancies between the two sequences), but they showed only 97.7 to 97.9% identity with the 16S rRNA gene from H. palleronii DSM 63. It was therefore concluded that strain S5 was derived from strain S1 and that the strains do not belong to the species H. palleronii.A reductive cleavage of the azo bond of CSAB would result in the formation of 4-aminobenzoate and sulfanilate. Like the parent strain, S1, strain S5 grew in the presence of sulfanilate, 4-aminobenzoate, and 4-sulfocatechol. The doubling times with these compounds were 6.2 to 6.4 h. We therefore investigated whether reductive cleavage of CSAB by strain S5 occurs. Strain S5 was grown aerobically with 5 mM CSAB, and cell extracts were prepared (10) in different buffers. These cell extracts were incubated aerobically in cuvettes containing 50 mM Tris-HCl buffer (pH 8.0), 0.5 mM CSAB, 1 mM NADH, or 1 mM NADPH and with various mixtures of possible cofactors. The enzyme activity was measured spectrophotometrically at the absorption maximum for CSAB (at a wavelength of 434 nm), but no significant decrease in absorbance was observed. Neither addition of a membrane fraction nor performing the enzyme assays under anaerobic conditions (9) improved the turnover of CSAB in the cell-free system. Furthermore, there was no significant increase in azo reductase activity when harvested cells were resuspended in the culture supernatant instead of Tris-HCl buffer.The maximal enzyme activities observed for cell extracts were only about 30% of the activities found for intact cells. This suggested that during the disruption of the cells some important components of the azo reductase system were destroyed or some cofactors were present in only limiting quantities.Because it was difficult to obtain reproducible enzyme activities with cell extracts, the turnover of CSAB by resting cells was investigated. Cells of strain S5 were grown with CSAB (5 mM), harvested by centrifugation, resuspended in Tris-HCl at an optical density at 546 nm (OD₅₄₆) of 5.3, and incubated in a water bath shaker (140 rpm; 30°C) with 0.5 mM CSAB (Fig. ​(Fig.2).2). Thus, the transient accumulation of two metabolites in the supernatants was observed by reversed-phase HPLC (column size, 250 by 4.6 mm) (SIL 100; Grom, Herrenberg, Germany). The solvent system consisted of a solvent gradient with increasing concentrations of methanol, starting with 1% (vol/vol) methanol, 98.9% (vol/vol) water, and 0.1% H₃PO₄. The flow rate was 0.7 ml min⁻¹. The metabolites formed were identified as sulfanilate and 4-sulfocatechol by comparison of their retention times and in situ UV-visible light-spectra with authentic standards. Surprisingly, the concentration of 4-sulfocatechol in the medium increased (and decreased) during the experiment more rapidly than the concentration of sulfanilate (Fig. ​(Fig.2).2). 4-Sulfocatechol also temporarily accumulated when resting cells of strain S1 were incubated with sulfanilate (4, 5). This suggested that in the resting-cell assay the initial activity of the sulfanilate-converting enzyme was higher than the activity of the 4-sulfocatechol-oxidizing enzyme protocatechuate-3,4-dioxygenase type II. Presumably, the activity of the sulfanilate-converting enzyme decreased during the experiment more rapidly than the activity of protocatechuate-3,4-dioxygenase type II. No accumulation of 4-aminobenzoate or protocatechuate was found by HPLC analysis during the experiment. In a control experiment with cells of strain S1 grown with 4-aminobenzenesulfonate, no turnover of CSAB was observed by HPLC analysis. Open in a separate windowFIG. 2Conversion of CSAB (•) to sulfanilate (▪) and 4-sulfocatechol (□) by resting cells of strain S5. Strain S5 was grown in a mineral medium with CSAB as the sole source of carbon and energy, and resting cells were prepared as described in the text.The detection of sulfanilate derived from CSAB suggested a reductive cleavage of CSAB, yielding sulfanilate as one of the reduction products. This reaction should also proceed in the absence of oxygen. Therefore, resting cells were incubated under anaerobic conditions with CSAB. Surprisingly, the rate of CSAB turnover under anaerobic conditions was <2% of the turnover rate under aerobic conditions.A further indication of a reductive cleavage of CSAB into sulfanilate and 4-aminobenzoate was obtained by growing strain S5 with CSAB or a complex medium (HPG medium) (4). When the cells were grown in a mineral medium with CSAB and the turnover of the substrates was analyzed by HPLC, it was found that resting cells converted CSAB, 4-aminobenzoate, or 4-aminobenzenesulfonate with specific activities of 0.012, 0.026, and 0.011 μmol min⁻¹ mg of protein⁻¹, respectively. In contrast, after growth of the cells in HPG medium, these activities were only 0.007, 0.010, and 0.003 μmol min⁻¹ mg of protein⁻¹, respectively. Incubation of resting cells with CSAB and different potential inhibitors of ring cleavage dioxygenases showed that the turnover of CSAB was almost completely inhibited by the addition of 8-hydroxyquinoline or 2,2′-bipyridyl (1 mM each). The presence of 4-nitrocatechol (0.25 mM) also resulted in a pronounced reduction of the rate of CSAB turnover (6% of the rate in the absence of the inhibitor). In this system as well the formation of 4-sulfocatechol was observed.The degradation of sulfanilate and 4-aminobenzoate by strain S1 has been previously studied (5). The proposed degradation pathway for CSAB and its reduction products is shown in Fig. ​Fig.3.3. Open in a separate windowFIG. 3Proposed pathway for the degradation of CSAB by strain S5. 4AB, 4-aminobenzoate; 4ABS, 4-aminobenzenesulfonate (sulfanilate); 3,4DHB, 3,4-dihydroxybenzoate (protocatechuate); 4SC, 4-sulfocatechol; 2H4CMSA, 2-hydroxy-4-carboxymuconic semialdehyde; 3SM, 3-sulfomuconate; 4SL, 4-carboxymethyl-4-sulfobut-2-en-4-olide (4-sulfolactone); MA, maleylacetate; 3OA, 3-oxoadipate; TCC, tricarboxylic acid cycle.To obtain some information about the substrate specificity, resting cells were incubated with CSAB, 4,4′-dicarboxyazobenzene (DCAB), 4-hydroxy-4′-sulfoazobenzene, methyl orange [4-(N,N-dimethyl)-4′-sulfoazobenzene; color index (C.I.) 13025], orange II {4-[(2-hydroxy-1-naphthalenyl)azo]-benzenesulfonic acid; C.I. 15510}, or sunset yellow FCF {6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthol-6-sulfonic acid; FD&C no. 6; C.I. 15985}. Of these compounds, only CSAB and DCAB were converted by resting cells. DCAB was also utilized by strain S5 as the sole source of carbon and energy. Furthermore, no growth of strain S5 was found with acid black 24 and 52, acid blue 113, acid red 1, amaranth, direct red 81, direct yellow 4 and 50, mordant yellow 3, and naphthol blue black.The results presented in this study suggest that bacterial cultures with the ability to aerobically degrade simple sulfonated azo dyes may be obtained after preadaptation to sulfonated aminoaromatics and/or when reductive cleavage of the azo bond gives rise to an aerobically assimilable aminoaromatic structure, like 4-aminobenzoate. This selection scheme circumvents the problems observed during attempts to adapt bacteria with the ability to degrade carboxylated azo compounds for the degradation of sulfonated azo compounds (12). The ability of strain S5 to mineralize CSAB suggests that it is possible to degrade sulfonated azo dyes under aerobic conditions if biological systems which can grow and can mineralize the reduction products are available.

Nucleotide sequence accession number.

The nucleotide sequences for the 16S rRNAs from strains S5 and S1 have been deposited in the GenBank data library under accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019037","term_id":"3004497","term_text":"AF019037"}}AF019037 and {"type":"entrez-nucleotide","attrs":{"text":"AF019073","term_id":"3004495","term_text":"AF019073"}}AF019073, respectively.     

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Abstract

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of “complementary addressed modification titration” (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylaminojbenzylamino- group (C1RCH₂NH)- at 5′-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E₁, E₂ and their derivatives E₁ ^phn, E₂ ^phn bearing N- (2-hydroxyethyl)-phenazinium residues Phn- both at 5′- and 3′- ends covalently linked via ethylenediamine linker were used as effectors. Effectors E₁ and E₂ (E₁ ^Phn and E₂ ^Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8^m) reagents were used for the target modification. The reagent X8^m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8^m and effectors E₁, E₂, E₁ ^phn, E₂ ^Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group CIRCH₂NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either CIRCH₂NH- group or TT-mismatch at the junction.     

An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.     

Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg²⁺-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5′-hydroxyl group with a 2′,3′-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2′–5′ phosphodiester bonds rather than native 3′–5′ linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2′–5′ linkages. This includes deoxyribozymes with an arrangement that favors 3′–5′ linkages for a different chemical reaction, that of a 2′,3′-diol plus 5′-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2′–5′ RNA linkages upon opening of a 2′,3′-cyclic phosphate with a 5′-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2′,3′-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.[Reviewing Editor: Niles Lehman]     

Environmental Impact of the Production of Mealworms as a Protein Source for Humans – A Life Cycle Assessment

The demand for animal protein is expected to rise by 70–80% between 2012 and 2050, while the current animal production sector already causes major environmental degradation. Edible insects are suggested as a more sustainable source of animal protein. However, few experimental data regarding environmental impact of insect production are available. Therefore, a lifecycle assessment for mealworm production was conducted, in which greenhouse gas production, energy use and land use were quantified and compared to conventional sources of animal protein. Production of one kg of edible protein from milk, chicken, pork or beef result in higher greenhouse gas emissions, require similar amounts of energy and require much more land. This study demonstrates that mealworms should be considered a more sustainable source of edible protein.     

A New Gene Trap Construct Enriching for Insertion Events Near the 5′ End of Genes

The gene trap approach is based on the integration of a gene trap vector into the genome. This can be done either by electroporation of a plasmid construct or by infection with a viral vector. Commonly used viral gene trap vectors have been shown to select for integrations near the 5 end of genes. To date, no plasmid vector with a similar tendency has been reported. In this paper we describe a new plasmid vector, pKC199geo. This vector contained a short splice acceptor fragment from the Hoxc9 gene, a full length lacZ gene, including an ATG, and a reduced activity, mutant neomycin phosphotransferase gene as a selectable marker. This vector enriched the population of trapped genes in our gene trap screen for insertion events in the 5 end of genes. In the two cases examined the -galactosidase activity pattern accurately reflected the endogenous promotor activity.     

Identification of 5′-deoxypyridoxine-3-sulfate as the major urinary metabolite of 5′-deoxypyridoxine in rats with comments on the inhibition of arylsulfatase activity and manganese dioxide oxidation by neighboring groups

The major urinary metabolite of 5′-deoxypyridoxine in rats was shown to be identical to 5′-deoxypyridoxine-3-sulfate but different from 5′-deoxypyridoxine-4′-sulfate in its ultraviolet and infrared spectra, its migration in thin-layer chromatography, and its behavior in acid and base. Previous identification of the metabolite as 5′-deoxypyridoxine-4′-sulfate by other workers was based on its failure to be hydrolyzed by arylsulfatase and to be oxidized by manganese dioxide. We have now demonstrated that ortho-methyl groups inhibit arylsulfatase and that ortho-sulfate groups inhibit oxidation by manganese dioxide. Therefore, we conclude that under our conditions the major metabolite of 5′-deoxypyridoxine in the rat was the 3-sulfate.