A New Internal Ribosomal Entry Site 5′ Boundary Is Required for Poliovirus Translation Initiation in a Mouse System

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1–8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.The single-stranded genome of poliovirus has mRNA polarity, is approximately 7,500 nucleotides (nt) in length, is polyadenylylated (45), and is linked covalently at its 5′ end to a small protein called VPg (30, 41). The RNA itself is infectious; cells transfected with the RNA produce progeny virions that are infectious. Poliovirus RNA harbors a long 5′ noncoding region of approximately 740 nt that is important for viral RNA and protein syntheses. A possible cloverleaf-like structure formed by the 5′-proximal end of the RNA (approximately 90 nt) is a probable cis element that regulates the synthesis of the plus-strand RNA (1). nt 120 to 590 of the poliovirus RNA make up the internal ribosomal entry site (IRES) (32), which directs the viral translation initiation step in a 5′-end- and cap-independent manner (17, 25, 29, 44). The IRES is assumed to carry a number of secondary structures (10, 40), and multiple host cellular factors are required for its functions.Translation of poliovirus does not occur in a cell-free wheat germ translation system, and it occurs only inefficiently and usually incorrectly in rabbit reticulocyte lysates (RRL) (9). The poor translation in RRL, however, is markedly improved by the addition of factors from HeLa cells (5, 9, 33). Other IRESs, such as the IRESs of encephalomyocarditis virus RNA (18) and hepatitis C virus RNA (43), are highly functional in the RRL system. These observations indicate that individual IRESs with different structures may require quantitatively and/or qualitatively different sets of host factors for their activities.Determinants for strain-specific neurovirulence (replication ability) of poliovirus type 1 in the central nervous system (CNS) have been mapped in the IRES region, particularly at nt 480 of the genome, by using monkey neurovirulence tests on recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains (19, 24). Similar results were obtained when the recombinants were tested for their relative neurovirulence levels by using transgenic (Tg) mice carrying the human gene for the poliovirus receptor (15, 20, 34). Thus, the IRES seems to be an important regulatory element for strain-specific expression of poliovirus neurovirulence. These two animal models show no difference in the development of the disease, even though replication of the virus in vivo must involve a number of biological interactions between viral and host factors. These results suggest that host factors of monkeys and mice, including IRES-related factors, support the expression of poliovirus neurovirulence (replication) in much the same way. However, it is possible that species differences between the IRES-related host factors of monkeys and mice exist.Several mutants with alterations in the stem-loop II (SLII) region were constructed from an infectious cDNA clone of the virulent Mahoney strain of poliovirus type 1 (39). The mutants replicated well in primate cells and in the CNS of monkeys but did poorly in mouse cells expressing human poliovirus receptor and in the CNS of the Tg mice carrying the human PVR gene (39). The replication of the mutant strains in mouse cells was blocked at the IRES-dependent translation initiation step, indicating that the function of the SLII as a part of the IRES is deficient in mouse cells but still active in primate cells. These differences in how the SLII mutants acted in the two animal models point to an interaction between the SLII and SLII-related host factors that could be a determinant for host-specific replication of poliovirus.To gain a deeper insight into the molecular basis of the function of the SLII region within the IRES, revertants that acquired the ability to replicate in mouse cells were isolated from the SLII mutants. Genetic analysis of mutation sites in the revertant genomes revealed that nt 107 within the 5′ noncoding region of poliovirus RNA influenced the efficiency of the IRES-dependent translation initiation process and that the remaining mutation sites (nt 120 to 161), in addition to nt 107, were required for the expression of the full revertant phenotype.     

Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on the activity of the enhancer in transiently transfected Drosophila S2R⁺ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.     

A Role for auxin during actinorhizal symbioses formation?

The symbiotic interaction between the soil bacteria Frankia and actinorhizal plants leads to the formation of nitrogen-fixing nodules resembling modified lateral roots. Little is known about the signals exchanged between the two partners during the establishment of these endosymbioses. However, a role for plant hormones has been suggested.Recently, we studied the role of auxin influx activity during actinorhizal symbioses. An inhibitor of auxin influx was shown to perturb nodule formation. Moreover we identified a functional auxin influx carrier that is produced specifically in Frankia-infected cells. These results together with previous data showing auxin production by Frankia lead us to propose a model of auxin action during the symbiotic infection process.Key words: lateral roots, nitrogen fixation, Frankia, AUX1, actinorhizal symbioses, phenylacetic acid, auxin influxActinorhizal symbioses result from the interaction between the soil actinomycete Frankia and plants belonging to eight angiosperm families collectively called actinorhizal plants.¹ This symbiotic interaction leads to the formation of a new organ on the root system, the actinorhizal nodule, where the bacteria are hosted and fix nitrogen.² Unlike legume nodules, actinorhizal nodules are structurally and developmentally related to lateral roots.³ Little is known about the signals exchanged between the two partners during the establishment of the symbiosis.² Diffusible signals are emitted by Frankia at early stages of the interaction resulting in root hair deformation.² The chemical nature of these signals remains unknown, however, detailed studies revealed that they are different from rhizobial Nod factors.⁴ Phytohormones are chemicals that control many developmental processes⁵ and have been linked to many plant-microbe interactions. Recently, we studied the role of auxin influx in actinorhizal nodule formation in the tropical tree Casuarina glauca.⁶     

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.     

Changes of Con A Receptor Sites on Mammalian Sperms during Capacitation and Acrosome Reaction

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A continuous spectrophotometric assay for pyrimidine-5′-nucleotidase

A simple method is presented for the determination of pyrimidine-5′-nucleotidase activity using a continuous spectrophotometric assay system. Activity is determined by measuring inorganic phosphate generation using a linked indicator system that produces uric acid in the presence of inosine, purine nucleoside phosphorylase, and xanthine oxidase. This method has several advantages over any of the methods currently in use.     

Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3′ untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3′ UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3′ UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5′ end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5′ UTR, since a hairpin structure abolishes expression of a fused reporter gene.     

Simple radioassay for uridine phosphorylase and 5′-nucleotidase

A simple micromethod was developed for the accurate measurement of the activity of uridine phosphorylase in the direction of both ribosylation and phosphorolysis. The technique was applicable also to the measurement of cytoplasmic 5′-nucleotidase. In this micromethod the radioactive products were separated from the substrates by electrophoresis on cellulose acetate membrane in 0.1 m borate buffer, pH 9.0, at 150 V for 40 min. These micromethods are highly sensitive and capable of utilizing small samples (4–30 μg protein); they are valid in crude tissue extracts of rat liver and hepatoma.     

Role of Calcium and Adenosine Cyclic 3′-5′ Phosphate in Action of Adrenocorticotropin

ALTHOUGH adenosine cyclic monophosphate (cyclic AMP) has been proposed as a mediator through which many hormones exert their physiological effects¹, it is also well established that calcium plays a crucial role in hormone release². Both calcium^3,4 and cyclic AMP^1,5 have been implicated in the action of adrenocorticotropin (ACTH) on the adrenal cortex and although various hypotheses have been advanced concerning their roles in steroid production and release, elucidation of their functions in the adrenal gland is hindered because most studies have been carried out on in vitro systems where the physiological release response cannot be studied. The isolated cat adrenal gland perfused in situ ⁶ approximates the situation in vivo, yet eliminates the influence of several factors, including the anterior pituitary. In the intact adrenal preparation one can also measure both steroid synthesis and release and can better evaluate the respective effects of cyclic AMP and calcium on these processes.     

A 5′UTR SNP of GHRHR locus is associated with body weight and average daily gain in Chinese cattle

Growth hormone-releasing hormone receptor (GHRHR) has important functions in the regulation of the growth hormone axis and the development and proliferation of pituitary somatotropes. Moreover, some mutations in mouse GHRHR can induce the dwarfism. The objective of this paper is to reveal the association of GHRHR with growth traits in three Chinese cattle breeds, including Nanyang cattle (NY, 220), Qinchuan cattle (QC, 114), and Jiaxian cattle (JX, 142). A novel single nucleotide polymorphism (NM_181020:c.102C>T) in 5′UTR of GHRHR was identified using PCR–SSCP and DNA sequencing. The frequency of NM_181020:c.102C allele ranged from 0.926 to 0.956. We found that the locus was significantly associated with NY cattle’s body weight (BW) of 6?months, with average daily gain (ADG) of 0–6?months, and as well as with ADG of 6–12?months (p?<?0.05). The data suggested that the polymorphism (NM_181020:c.102C>T) of the GHRHR could be a molecular marker candidate for breeding of NY cattle in favor of BW.     

DAP5 associates with eIF2β and eIF4AI to promote Internal Ribosome Entry Site driven translation

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5′UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2β and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.     

A Simple Multi-Gram Synthesis of 5′-Hydrogenphosphonates and 5′-Phosphorofluoridates of Sugar Modified Nucleosides

Abstract

Treatment of 3′-fluoro-3′-deoxythymidine (FLT), 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxyadenosine (ddA) with tris(1,1,1,3,3,3-hexafluoro-2-propyl)phosphite or phosphorous acid and N,N'-dicyclohexylcarbodiimide produced the corresponding nucleoside 5′-hydrogenphosphonates. Reaction of FLT, AZT and 3′-deoxythymidine (ddT) with fluorophosphoric acid and 2,4,6-triisopropylbenzenesulfonyl chloride lead to the corresponding nucleoside 5′-phosphorofluoridates also on a multi-gram scale. All the compounds were isolated in high pure state by chromatographic technique.     

Comparison of 3′ and 5′ biotin labelled oligonucleotides for in situ hybridisation

Oligonucleotide probes enzymatically labelled at the 3-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5-end and compared it to conventional 3-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5-end and 12 biotin residues were almost as effective as 3-enzymatic tailing. The sensitivity could be increased above that of either 3- or 5-labelling by the addition of residues at both ends of the probe. The 5-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3-enzymatic labelling.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.