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At least 1,585 records · Page 88Linked to original sources
PubMed2006

Improved synthesis of oligonucleotides with an allylic backbone. Oligonucleotides containing acyclic, achiral nucleoside analogues: N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases.

An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (DeltaT(m) per modification -1 to -5 degrees C).

Amides↗
PubMed2005

PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family.

Protein arginine methylation is a common post-translational modification that has been implicated in signal transduction, RNA processing, transcriptional regulation, and DNA repair. A search of the human genome for additional members of the protein arginine N-methyltransferase (PRMT) family of enzymes has identified a gene on chromosome 12 that we have termed PRMT8. This novel enzyme is most closely related to PRMT1, although it has a distinctive N-terminal region. The unique N-terminal end harbors a myristoylation motif, and we have shown here that PRMT8 is indeed modified by the attachment of a myristate to the glycine residue after the initiator methionine. The myristoylation of PRMT8 results in its association with the plasma membrane. The second singular property of PRMT8 is its tissue-specific expression pattern; it is largely expressed in the brain. A glutathione S-transferase fusion protein of PRMT8 has type I PRMT activity, catalyzing the formation of omega-NG-monomethylated and asymmetrically omega-NG,NG-dimethylated arginine residues on a recombinant glycine- and arginine-rich substrate. PRMT8 is thus an active arginine methyltransferase that is membrane-associated and tissue-specific, two firsts for this family of enzymes.

Amino Acid Motifs↗
PubMed2001

Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers.

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.

Ethylene Glycols↗
PubMed1996

Differential expression of reg-I and reg-II genes during aging in the normal mouse.

A cDNA termed reg (for regenerating gene) has been isolated from a rat pancreatic DNA library. Reg expression has been shown to correlate with changes in beta cell mass and function. This finding has been recently challenged by studies showing a non-beta-cell-dependent regulation of reg expression. All studies to date, however, have neglected the fact that two nonallelic reg genes (reg-I and reg-II) exist in several species. In studying the regulation of each individual copy gene, we investigated reg-I and -II gene expression in a naturally occurring modification of beta-cell physiology: normal aging. RNA was isolated from individual pancreata of 1-, 3-, 9-, 20-, and 30-month-old C57BL/6J mice (n > or = 3 per group) and subjected to slot-blot analysis using homologous probes for reg-I, reg-II, insulin, and elastase-I. A progressive age-dependent decrease in total reg mRNA levels (reg-I and -II) was detected. At 30 months of age, total reg mRNA levels were approximately 45% of the level detected in 1-month-old mice (p = .01). This paralleled the decrease in insulin mRNA levels (p = .01), which fell below 50%; by contrast, mRNA levels for elastase-I increased with age (p = .05). Analysis of RNA isolated from purified islets did not reveal any mRNA for reg, suggesting that in the normal mouse, reg is primarily a product of the exocrine pancreas. Reg mRNA were detectable in RNA extracts from stomach, duodenum, and small intestine. By hybridization of total pancreatic RNA with oligonucleotide probes which specifically recognize reg-I or reg-II sequences, we show that reg-I mRNA levels declined with age (p = .001) while reg-II mRNA levels remained unchanged. These data demonstrate that in mouse pancreas the two nonallelic reg genes are differentially expressed during aging and that the decrease in reg-I mRNA levels parallels the decrease in insulin gene expression. Differential regulation of reg-I and reg-II genes may explain the presence of conflicting data in the current literature.

Aging↗
PubMed1995

Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template.

Avian myeloblastosis virus and Maloney murine leukemia virus RNase H-reverse transcriptases pause when they encounter a 2'-5' linkage or a 2'-thiophosphate in their template RNAs, but eventually read through these backbone modifications. Both reverse transcriptases pause after the 2'-5' linkage but before the 2'-thiophosphate. These results suggest that in the absence of precise information concerning the behavior of a given reverse transcriptase with respect to a particular lesion or modification, caution should be exercised in the interpretation of primer extension data that is being used to determine the existence of, or map the position of, a crosslink, site of chemical modification or non-standard linkage in an RNA template.

Avian Myeloblastosis Virus↗
PubMed1997

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.

Nucleic Acid Hybridization↗
PubMed1998

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.

Animals↗
PubMed2006

Sugar modification as a means to increase the biological performance of oligonucleotides.

Modification of the ribose unit in DNA and RNA profoundly influences the self-rcognition and the biological properties of the nucleic acids. Conformational restriction of the ribose units, as in LNA and tricyclo-DNA, has been identified as a powerful tool to increase DNA and RNA affinity as well as biological stability and antisense properties. Apart from that sugar modified DNA analogues, as homo-DNA, have shown to be orthogonal base-pairing systems which by virtue of non-crosscommunicating with the natural nucleic acids open novel applications in biotechnology.

DNA Probes↗
PubMed2004

Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid.

Transgenic techniques are used to enhance and improve crop production, and their application to the production of chemical resources in plants has been under investigation. To achieve this latter goal, multiple-gene transformation is required to improve or change plant metabolic pathways; when accomplished by plant nuclear transformation, however, this procedure is costly and time consuming. We succeeded in the metabolic engineering of the tobacco plant by introducing multiple genes within a bacteria-like operon into a plastid genome. A tobacco plastid was transformed with a polycistron consisting of the spectinomycin resistance gene and three bacterial genes for the biosynthesis of the biodegradable polyester, poly[(R)-3-hydroxybutyrate] (PHB), after modification of their ribosome binding sites. DNA and RNA analysis confirmed the insertion of the introduced genes into the plastid genome and their polycistronic expression. As the result, the transplastomic tobacco accumulated PHB in its leaves. The introduced genes and the PHB productivity were maternally inherited, avoiding genetic spread by pollen diffusion, and were maintained stably in the seed progeny. Despite the low PHB productivity, this report demonstrates the feasibility of transplastomic technology for metabolic engineering. This "phyto-fermentation" system can be applied to plant production of various chemical commodities and pharmaceuticals.

Base Sequence↗
PubMed2004

Monoallelic gene expression: a repertoire of recurrent themes.

The development of mature B and T cells in the lymphoid system involves a series of molecular decisions that culminate in the expression of a single antigen receptor on the cell surface, a phenomenon termed allelic exclusion. While feedback inhibition of the recombinase-activation gene proteins evidently plays an important role in the maintenance of allelic exclusion, the initial restriction of rearrangement to only one allele in each cell seems to be achieved through monoallelic epigenetic changes. Epigenetic mechanisms involved in the establishment of allelic exclusion also play a central role in other types of monoallelic expression, including X-chromosome inactivation in female cells, and parental imprinting. In all three systems, the inequality of the two alleles seems to be achieved mainly by differential DNA methylation, asynchronous DNA replication, differential chromatin modifications, unequal nuclear localization, and non-coding RNA. In this review, we discuss the unifying features among these monoallelically expressed systems and the unique characteristics displayed by each of them.

Alleles↗
PubMed1987

Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate.

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

Bacteroides↗
PubMed2002

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases.

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.

Bromovirus↗
PubMed2001

Cycles of transcription and translation do not comprise the gonadotropin-releasing hormone pulse generator in GT1 cells.

Neural control of reproduction is achieved through episodic GnRH secretion, but little is known about the molecular mechanisms underlying pulse generation. The ultradian time domain of GnRH release suggests mechanisms ranging from macromolecular synthesis to posttranslational modification could be involved. We tested if messenger RNA (mRNA) or protein synthesis are components of the pulse generator by determining the effects of transcription and translation inhibitors on episodic GnRH release from immortalized GT1-1 GnRH neurons. Time course and efficacy of transcription and translation blockade were assessed by determining the ability of specific inhibitors to block the robust, rapid induction of c-fos mRNA or protein accumulation by forskolin (10 microM). The transcription inhibitors actinomycin D (ACT-D, 20 microM) or 5,6-dichlorobenzimidazole riboside (DRB, 100 microM), or the translation inhibitors anisomycin (ANI, 10 microM) or puromycin (PUR, 10 microM) were applied to GT1-1 cells 30, 15, or 0 min before forskolin. Northern and Western blots revealed blockade of transcription and translation was rapid and essentially complete. GT1-1 cells were perifused for a 90- to 120-min control period then for 100-130 min with vehicle or inhibitor to examine pulsatile GnRH secretion. GnRH interpeak intervals, peak amplitude, and peak area were not different between control and experimental periods of cells treated with vehicle (n = 15), ACT-D (n = 10), DRB (n = 6), ANI (n = 8), and PUR (n = 6; P > 0.05). This study presents the first clear evidence that the series of reactions resulting in secretion of a GnRH pulse do not include cycles of transcription and translation. Although these mechanisms would be required to replenish components of the pulse generator, they are not integral components of this oscillator. We hypothesize that posttranslational events underlie episodic GnRH release in GT1-1 cells.

Animals↗
PubMed2000

[Application of radionuclides in pharmaceutical fields].

Application studies of radionuclides in pharmaceutical fields are described concerning two major categories, such as the use of gamma-emitter in nuclear medicine and beta-emitter in tracer investigation. In the former radionuclide an effort has been made to develop radioactive new diagnostic agents in the nephro-urology field. Of several agents examined two new radiopharmaceuticals, 99mTc-malic acid and 99mTc-dimercaptopropionic acid proved to be sufficient for renal imaging with adequate concentration in experimental animals and humans. Useful information was obtained for diagnosis of renal diseases using both the labeled agents. Identification and purity tests of radiopharmaceuticals also set forth for official compendia such as the Japanese pharmacopoeia and so on. In the latter one the fates of two medicines, thalidomide and norphenylephrine, were studied and the safety evaluation of some xenobiotics including food additives, plasticizers and domestic chemical products, etc. was carried out in animals using synthesized 14C- or 3H-labeled compounds. In addition, the metabolism of n-propyl-N-nitrosourea, a carcinogen, in rats, and chemical modification of high molecular substances such as DNA, RNA and proteins by 1,1'-ethylene-bis(1-nitrosourea) (EBNU), a carcinostatic agent, were studied in vitro by the use of the tracer technique.

Animals↗
PubMed2005

An end-healing enzyme from Clostridium thermocellum with 5' kinase, 2',3' phosphatase, and adenylyltransferase activities.

We identify and characterize an end-healing enzyme, CthPnkp, from Clostridium thermocellum that catalyzes the phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. CthPnkp is a homodimer of an 870-amino-acid polypeptide composed of three catalytic domains: an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyl transferase domain of polynucleotide ligases. The distinctive feature of CthPnkp vis-à-vis known RNA repair enzymes is that its 3' end modification component belongs to the calcineurin-type phosphatase superfamily. It contains putative counterparts of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. As with lambda phosphatase, the 2',3' cAMP phosphatase activity of CthPnkp is specifically dependent on nickel or manganese. We identify homologs of CthPnkp in other bacterial proteomes.

Amino Acid Sequence↗
PubMed2026

Epigenetic alterations in rheumatoid arthritis: multilayer mechanisms and translational opportunities.

Rheumatoid arthritis (RA) is a chronic inflammatory disease driven by immune dysregulation, in which genetic susceptibility and environmental exposures promote persistent synovitis, progressive joint damage, and systemic comorbidities. Recent epigenomic studies show several recurring abnormalities. Many RA susceptibility variants lie outside protein-coding sequence and map to immune-cell and synovial fibroblast regulatory elements, linking inherited risk to enhancer activity, methylation quantitative trait effects, and distal gene control. Blood-based epigenome-wide association studies identify disease-associated DNA methylation signatures, but these signals require careful control for leukocyte composition, smoking, treatment exposure, and disease stage. RA fibroblast-like synoviocytes also display stable methylome remodeling, including relative hypomethylation at loci involved in inflammation, migration, matrix degradation, and apoptosis resistance, while TET3-associated 5-hydroxymethylcytosine has emerged as a functional contributor to chemokine production and invasive stromal behavior. Histone modifications, chromatin accessibility, and 3D genome organization define pathogenic regulatory states and connect non-coding risk loci to effector genes in immune and stromal compartments. Finally, miRNAs, lncRNAs, circRNAs, snoRNAs, extracellular RNAs, and m6A-related pathways add post-transcriptional and chromatin-linked layers with potential biomarker value. We synthesize these findings and discuss translational opportunities for diagnosis, stratification, flare monitoring, and therapeutic targeting, while emphasizing incomplete replication, uneven evidence across epigenetic layers, biospecimen variability, and the need for causal, longitudinal, cell-type-resolved validation.

Humans↗
PubMed1995

Functional organization of tau proteins during neuronal differentiation and development.

Tau proteins play major regulatory roles in the organization and integrity of the cytoskeletal network. In neurons, a specific axonal compartmentalization of tau has been shown. However, recent studies demonstrate that tau displays a widespread distribution in a variety of non-neuronal cell types. These proteins have been found in human fibroblasts and in several transformed cell lines. The heterogeneous family of tau is formed by a set of molecular species that share common peptide sequences. There is a single gene that contains several exons encoding for the six different tau isoforms in mammalian brain. Alternative splicing of a common RNA transcript as well as post-translational modifications contribute to its heterogeneity. Tau isoforms generated by splicing differ from one another by having either three or four repeats in their C-terminal half, and a variable number of inserts in their N-terminal moiety. These repeats have been shown to constitute microtubule-binding motifs. In this review some relevant aspects of tau function and its regulation are analyzed. Three major topics are discussed. The first one focuses on the tau roles in regulating the interactions between microtubules with actin filaments and with intermediate filament systems. Another problem deals with the question of whether tau isoforms segregate into functionally different subsets of microtubules in axonal processes, or tau associates with these polymers in a random fashion. The third question that emerges is the involvement of tau and tau-like proteins in morphogenetic events. The regulation of the interactions of DMAP-85, a recently discovered tau-like protein, with the cytoskeleton during development of Drosophila melanogaster is analyzed.

Amino Acid Sequence↗
PubMed1998

rRNA chemical groups required for aminoglycoside binding.

Through an affinity chromatography based modification-interference assay, we have identified chemical groups within Escherichia coli 16S ribosomal RNA sequence that are required for binding the aminoglycoside antibiotic paromomycin. Paromomycin was covalently linked to solid support via a nine atom spacer from the 6"'-amine of ring IV, and chemical modifications to an A-site oligonucleotide that disrupted binding were identified. Positions in the RNA oligonucleotide that correspond to G1405(N7), G1491(N7), G1494(N7), A1408(N7), A1493(N7), A1408(N1), A1492(N1), and A1493(N1), as well as the pro-R phosphate oxygens of A1492 and A1493 in 16S rRNA are chemical groups that are essential for a high-affinity RNA-paromomycin interaction. These data are consistent with genetic, biochemical, and structural studies related to neomycin-class antibiotics and provide additional information for establishing an exact model for their interaction with the ribosome.

Adenosine↗