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.     

Cinnamate and shikimate incorporation into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins: Are there alternative pathways?

The incorporation of shikimate-[¹⁴C] and cinnamate-[¹⁴C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida. According to the dilution values, the incorporation of shikimate-[¹⁴C] was about 3–6 times better than that of cinnamate-[¹⁴C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [¹⁴C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

3′-Deoxy-3′-C-trifluoromethyl Nucleosides: Synthesis and Antiviral Evaluation

Abstract

2′,3′-Dideoxy-3′-C-trifluoromethylthymidine 9a and -uridine 9b and 3′-C-trifluoromethyl-d4T 11 were prepared in a few steps from 3′-deoxy-3′-C-trifluoromethyl-D-ribose, which synthesis was recently reported. The biological assessment of these nucleoside analogues did not reveal interesting antiviral properties against HIV-1, HSV-1, CMV, Vaccine, and Cox B4.     

A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-[α-³²P]triphosphate ([α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-[α-³²P]triphosphate ([α-³²P]KTP) as a means to 3′-end label DNA. The [α-³²P]ddATP was found to be three to five times more efficient than [α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with [α-³²P]ddATP using terminal transferase than were the 5′-ends with [γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared [α-³²P]ddATP- and [α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.     

Identification and quantitation of adenosine-3′:5′-cyclic monophosphate in plants using gas chromatography-mass spectrometry and high-performance liquid chromatography

Direct evidence has been obtained for the presence of adenosine-3:5-cyclic monophosphate (cAMP) in tobacco (Nicotiana tabacum L.) callus tissue cultures, bean (Phaseolus vulgaris L.) seedlings and immature kernels of sweet corn (Zea mays L.) through the use of a highly specific and sensitive gas chromatography-mass spectrometric assay. Levels of endogenous cAMP ranged from 70 to 126 pmol/g fresh weight. Corresponding levels of cAMP determined for the same samples using radioimmunoassay were consistently three to four times higher. Contrary to previous reports for citrus plants, measurable levels of cAMP could not be detected in young lemon leaves within the limits of detection of the mass-spectrometric assay method. In the case of tobacco callus tissue, the coumarin glucoside, scopolin, which was present in large amounts and showed similar chromatographic behaviour to cAMP, interferred strongly with the mass-spectrometric measurements of cAMP in inadequately purified extracts. The use of high-performance liquid chromatography, in addition to standard chromatographic purification methods, produced highly purified plant extracts for quantitation of cAMP and also provided a method for the separation of cAMP from its 2:3-isomer.Abbreviations cAMP adenosine-3:5-cyclic monophosphate - 2:3-cAMP adenosine-2:3-cyclic monophosphate - GC-MS-MID combined gas chromatography-mass spectrometry with selected multiple-ion-detection - HPLC high-performance liquid chromatography - RIA radioimmunoassay - TLC thin-layer chromatography     

Novel SNPs and INDEL polymorphisms in the 3′UTR of DGAT1 gene: in silico analyses and a possible association

Diacylglycerol–O–acyltransferase (DGAT1) gene encodes the rate-limiting enzyme of triglyceride synthesis. A polymorphism in this gene, DGAT1 K232A, has been associated with milk production and composition in taurine breeds. However, this polymorphism is not a good tool for ascertaining the effects of this QTL in Bos indicus (Zebu), since the frequency of the DGAT1 232A allele is too low in these breeds. We sequenced the 3′-untranslated region of DGAT1 gene in a sample of bulls of the breeds Guzerá (Bos indicus) and Holstein (Bos taurus) and, using in silico analysis, we searched for genetic variation, evolutionary conservation, regulatory elements, and possible substitution effects. Six single nucleotide (SNPs) and one insertion-deletion (INDEL) polymorphisms were found in the Guzerá bulls. Additionally, we developed a preliminary association study, using this INDEL polymorphism as a genetic marker. A significant association was detected (P ≤ 0.05) between the INDEL (DGAT1 3′UTR INDEL) and the breeding values (BV) for protein, fat, and milk yields over a 305-day lactation period. The DGAT1 3′ UTR INDEL genotype I/I (I, for insertion) was associated with lower BVs (?38.77 kg for milk, ?1.86 kg for fat, and ?1.48 kg for protein yields), when compared to the genotype I/D (D, for deletion). I/D genotype was lower D/D genotype (?34.98 kg milk, ?1.73 kg fat, and ?1.09 kg protein yields). This study reports the first polymorphism of DGAT1 3′UTR in the Guzerá breed, as well as its association with BV for milk protein, fat, and milk yields.     

Adenosine 3′:5′-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes: Solubilization,separation, and characterization of multiple forms

Multiple protein kinase activities were found in the luminal segment of the renal proximal tubule cell plasma membrane (brush border membrane). Membranes were extracted with Lubrol, with no loss in activity, and the extract was chromatographed on diethylaminoethyl cellulose with a salt gradient. With protamine as substrate, activity eluted in two peaks, designated I and IIb, and was cyclic AMP independent. With histone VII-S, one peak, designated IIa, appeared, which eluted slightly ahead of IIb and was cyclic AMP dependent. The three activities eluted in their original patterns following rechromatography. Histone kinase activity in the combined IIa+b fraction was stimulated threefold by cyclic nucleotides (K_a = 0.013 and 0.94 μM for cyclic AMP and cyclic GMP, respectively) by increasing V. Cyclic AMP binding activity eluted with histone kinase activity. Rechromatography of IIa+b on diethylaminoethyl cellulose containing 1 μm cyclic AMP resulted in passage through the column of most of the histone kinase activity (IIa) prior to the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cyclic AMP and a specific protein inhibitor of cyclic AMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones, and phosvitin in decreasing order of activity, and was insensitive to cyclic AMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cyclic AMP-dependent histone kinase activity was relatively thermostable. These findings implicate cyclic AMP-independent protamine kinases in the cyclic AMP-independent autophosphorylation of the brush border membrane.     

Tombusvirus recruitment of host translational machinery via the 3′ UTR

RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5′-cap structure and a 3′-poly(A) tail, and instead rely on a 3′-cap-independent translational enhancer (3′CITE) located in their 3′ untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 3′CITE in tombusviruses—also present in aureusviruses and carmoviruses—using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 5′ UTR of the viral genome. The specificity of 3′CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 5′ UTR relies on complementary RNA–RNA base-pairing. We show for the first time that this tripartite 5′ UTR/3′CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 5′ end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 3′CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 3′CITE function. Moreover, the virus–host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 3′CITE activity.     

Genome-wide functional screen of 3′UTR variants uncovers causal variants for human disease and evolution

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