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At least 1,747 records · Page 97Linked to original sources
PubMed2004

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Adenosine↗
PubMed1996

Nucleocapsid protein 10 activates dimerization of the RNA of Moloney murine leukaemia virus in vitro.

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.

Base Sequence↗
PubMed1999

Mean free energy topology for nucleotide sequences of varying composition based on secondary structure calculations.

The mean free energy generated from the secondary structure of RNA sequences of varying length and composition has been studied by way of probability theory. The expected boundaries or maximal and minimal values of a given distribution are explored and a method for estimating error as a function of the number of shuffled sequences is also examined. For typical nucleotide sequences found in biologically active organisms, the mean free energy, free energy distributions and errors appear to be scalable in terms of a fixed set of algorithm-dependent parameters and the nucleotide composition of the particular sequence under evaluation. In addition, a general semi-analytical formula for predicting the mean free energy is proposed which, at least to first-order approximation, can be used to rapidly predict the mean free energy of any sequence length and composition of RNA. The general methodology appears to be algorithm independent. The results are expected to provide a reference point for certain types of analysis related to structure of RNA or DNA sequences and to assist in measuring the somewhat related matter of complexity in algorithm development. Some related applications are discussed.

Algorithms↗
PubMed1979

The use of R-looping for structural gene identification and mRNA purification.

A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.

Base Sequence↗
PubMed1979

A study of barley stripe mosaic virus (BSMV) genome. I. Determination of sequence homology between BSMV RNA species.

The sequence homology between individual RNA species of three-component Norwich strain of BSMV (BSMV-N) has been studied by kinetic hybridization analysis using complementary DNAs obtained for each of the three BSMV-N RNA species by the method of Taylor et al. (1976). No significant sequence homology could be detected between RNA 1, on the one hand, and RNA2 and RNA3, on the other, whereas RNA2 and RNA3 were found to be highly homologous.

Base Sequence↗
PubMed2000

Bacillus sp. WW3-SN6, a novel facultatively alkaliphilic bacterium isolated from the washwaters of edible olives.

A novel Gram-positive facultatively alkaliphilic, sporulating, rod-shaped bacterium, designated as WW3-SN6, has been isolated from the alkaline washwaters derived from the preparation of edible olives. The bacterium is nonmotile, and flagella are not observed. It is oxidase positive and catalase negative. The facultative alkaliphile grows from pH 7.0 to 10.5, with a broad optimum from pH 8.0 to 9.0. It could grow in up to 15% (w/v) NaCl, and over the temperature range from 4 degrees to 37 degrees C, with an optimum between 27 degrees and 32 degrees C: therefore, it is both halotolerant and psychrotolerant. The bacterium is sensitive to a range of beta-lactam, sulfonamide, and aminoglycoside antibiotics, but resistant to trimethoprim. The range of amino acids, sugars, and polyols utilized as growth substrates indicates that this alkaliphile is a heterotrophic bacterium. D(+)-glucose, D(+)-glucose-6-phosphate, D(+)-cellobiose, starch, or sucrose are the substrates best utilized. The major membrane lipids are phosphatidylglycerol and diphosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and an unknown phospholipid. During growth at high pH, the proportion of phosphatidylglycerol is increased relative to phosphatidylethanolamine. The fatty acyl components in the membrane phospholipids are mainly branched chain, with 13-methyl tetradecanoic and 12-methyl tetradecanoic acids as the predominant components. The G + C content of the genomic DNA is 41.1 +/- 1.0 mol%. The results of 16S ribosomal RNA sequence analysis place this alkaliphilic bacterium in a cluster, together with an unnamed alkaliphilic Bacillus species (98.2% similarity).

Alkalies↗
PubMed2000

Molecular evolution of the Paramyxoviridae and Rhabdoviridae multiple-protein-encoding P gene.

Presented here is an analysis of the molecular evolutionary dynamics of the P gene among 76 representative sequences of the Paramyxoviridae and Rhabdoviridae RNA virus families. In a number of Paramyxoviridae taxa, as well as in vesicular stomatitis viruses of the Rhabdoviridae, the P gene encodes multiple proteins from a single genomic RNA sequence. These products include the phosphoprotein (P), as well as the C and V proteins. The complexity of the P gene makes it an intriguing locus to study from an evolutionary perspective. Amino acid sequence alignments of the proteins encoded at the P and N loci were used in independent phylogenetic reconstructions of the Paramyxoviridae and Rhabdoviridae families. P-gene-coding capacities were mapped onto the Paramyxoviridae phylogeny, and the most parsimonious path of multiple-coding-capacity evolution was determined. Levels of amino acid variation for Paramyxoviridae and Rhabdoviridae P-gene-encoded products were also analyzed. Proteins encoded in overlapping reading frames from the same nucleotides have different levels of amino acid variation. The nucleotide architecture that underlies the amino acid variation was determined in order to evaluate the role of selection in the evolution of the P gene overlapping reading frames. In every case, the evolution of one of the proteins encoded in the overlapping reading frames has been constrained by negative selection while the other has evolved more rapidly. The integrity of the overlapping reading frame that represents a derived state is generally maintained at the expense of the ancestral reading frame encoded by the same nucleotides. The evolution of such multicoding sequences is likely a response by RNA viruses to selective pressure to maximize genomic information content while maintaining small genome size. The ability to evolve such a complex genomic strategy is intimately related to the dynamics of the viral quasispecies, which allow enhanced exploration of the adaptive landscape.

Evolution, Molecular↗
PubMed2001

Inhibition of the B. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT.

An anti-TRAP (AT) protein, a factor of previously unknown function, conveys the metabolic signal that the cellular transfer RNA for tryptophan (tRNATrp) is predominantly uncharged. Expression of the operon encoding AT is induced by uncharged tRNATrp. AT associates with TRAP, the trp operon attenuation protein, and inhibits its binding to its target RNA sequences. This relieves TRAP-mediated transcription termination and translation inhibition, increasing the rate of tryptophan biosynthesis. AT binds to TRAP primarily when it is in the tryptophan-activated state. The 53-residue AT polypeptide is homologous to the zinc-binding domain of DnaJ. The mechanisms regulating tryptophan biosynthesis in Bacillus subtilis differ from those used by Escherichia coli.

5' Untranslated Regions↗
PubMed1992

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.

Base Sequence↗
PubMed2026

Single-cell transcriptomics reveals distinct microglial state remodeling associated with the (R)-nicotine/diosmetin combination and galantamine in LPS-challenged BV2 cells.

BACKGROUND: Microglial neuroinflammation contributes to the progression of neurodegenerative diseases, yet it remains challenging to attenuate inflammatory responses while preserving cellular function. The effects of (R)-nicotine, diosmetin, their combined administration, and galantamine on heterogeneous BV2 transcriptional states have not been compared at single-cell resolution. METHODS: LPS-stimulated BV2 microglial-like cells were treated with (R)-nicotine, diosmetin, their combination (DR), or galantamine. Single-cell RNA sequencing was performed with three biological replicates per group and integrated with RNA velocity and SCENIC regulon inference to characterize treatment-associated state redistribution, inferred local transcriptional directionality and regulon-activity patterns. Functional validation included CCK-8 metabolic activity assays, multiplex cytokine ELISA, BDNF/GDNF quantification, qPCR, and high-content immunofluorescence analysis of iNOS and Arg1 at single-cell resolution. RESULTS: LPS decreased the relative abundance of the Itgae+/Plk4+ cluster while increasing the Nmur1+/Limk2+ cluster and inflammatory effector programs. DR treatment suppressed pro-inflammatory cytokine release without significantly reducing CCK-8-assessed metabolic activity, increased the Itgae+/Plk4+ cluster proportion, and increased BDNF/GDNF relative to LPS. RNA velocity and SCENIC analyses suggested that DR and galantamine showed distinct transcriptional and regulatory patterns: DR attenuated Batf-associated inflammatory regulons and was associated with increased Atf3-linked stress-response activity, whereas galantamine preferentially engaged DNA repair and genome-maintenance programs. CONCLUSION: These findings indicate that the DR condition was associated with remodeling of LPS-challenged BV2 microglial-like states, attenuation of inflammatory programs, and increased neurotrophic outputs relative to LPS, without significantly reducing CCK-8-assessed metabolic activity. This study provides a single-cell characterization of distinct treatment-associated responses to (R)-nicotine, diosmetin, their combined administration, and galantamine.

Microglia↗
PubMed1983

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells.

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.

Adenoviruses, Human↗
PubMed1988

Unusual sequences, homologous to 5S RNA, in ribosomal DNA repeats of the nematode Meloidogyne arenaria.

There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain "lower eukaryotes" (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes. Evidence is presented for rearrangements and deletions within Meloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences in Meloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.

Animals↗
PubMed2026

A scalable, low-cost, sample hashing workflow for multiomic single-cell analysis using the Seq-Well S3 platform.

In-depth analyses of clinical samples have the potential to provide unparalleled insights into the cellular mechanisms that underlie both health and disease, as well as therapeutic and prophylactic responses. However, these specimens are often paucicellular, necessitating the use of workflows that maximize the amount of information that can be learned. Here we provide a detailed protocol for generating and analyzing single-cell multiomic data from low-input samples with the Seq-Well S3 platform. We further describe a matched pipeline for sample hashing that reduces costs and sources of technical variation in the resulting data while also enhancing throughput. In brief, our streamlined and efficient methodology involves: (1) optionally staining single-cell suspensions with antibody-oligonucleotide conjugates for cell surface protein quantification and/or sample multiplexing; (2) generating Seq-Well S3 sequencing libraries; (3) optionally producing bulk-RNA sequencing libraries via SMART-seq2 to support genetic demultiplexing; and (4) computationally analyzing the resulting data. Each step herein has been designed to leverage readily available reagents and standard laboratory equipment, substantially lowering barriers to entry for researchers. The overall Protocol can yield high-quality multiomic insights from samples in under a week.

Single-Cell Analysis↗
PubMed1991

Comparison and modification of rRNA sequencing methods.

In this study, modification of two methods of RNA sequencing resulted in more definitive sequencing bands. In one method of sequencing, the bands of lane A and lane G sometimes were not clear. Modifications of this method by changing the concentrations of ddATP and ddGTP resulted in the bands of lane A and lane G becoming more readable. Although a second sequencing method was found to have clearer bands than the first method, and the bases immediately downstream from the primer binding site could be read by using r-32P-labeled primer, the bands on the top of lane A still were not clear. Modifications of this second method by changing the ddATP/dATP ratio resulted in the bands of lane A becoming much clearer.

Base Sequence↗
PubMed2025

Droplet-Based Single-Cell 3' mRNA Sequencing of Marburg Virus-Infected Samples.

Single-cell technologies are continually evolving with emerging methods that are gradually uncovering the central DNA-RNA-protein dogma. Single-cell RNA sequencing is one arm of a multi-omic approach that achieves an astounding level of granularity to reveal the complexity of virus-host interactions at the transcriptomic level. Cell tropism, virus replication, pathogenesis, and gene expression changes mediated by the virus and the host's immune response to infection are just some areas of study that are gaining better clarity due to the high-resolution analysis afforded by the technology.We describe a single-cell sequencing protocol for Marburg virus infection in vivo using nonhuman primate blood and the 10× Chromium Next GEM single-cell genomics methodology. Working with pathogens of high consequence is logistically complicated, requiring containment in biosafety level (BSL)-4 laboratories and harsh inactivation procedures before samples can safely be removed to lower biosafety conditions. We provide procedural insight into sample isolation and processing conducted in BSL-4 and describe the requirements for safe sample removal without jeopardizing quality for down-stream sequencing and analysis in BSL-2 conditions. Characterization of complicated biological processes mediated by high-containment pathogens, typically restricted to analogous model systems, e.g., minigenome, can be achieved using live virus.

Animals↗
PubMed2002

RNA affinity tags for purification of RNAs and ribonucleoprotein complexes.

Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.

Amino Acid Motifs↗
PubMed2003

RNA editing in hornwort chloroplasts makes more than half the genes functional.

RNA editing in chloroplasts alters the RNA sequence by converting C-to-U or U-to-C at a specific site. During the study of the complete nucleotide sequence of the chloroplast genome from the hornwort Anthoceros formosae, RNA editing events have been systematically investigated. A total of 509 C-to-U and 433 U-to-C conversions are identified in the transcripts of 68 genes and eight ORFs. No RNA editing is seen in any of the rRNA but one tRNA suffered a C-to-U conversion at an anticodon. All nonsense codons in 52 protein-coding genes and seven ORFs are removed in the transcripts by U-to-C conversions, and five initiation and three termination codons are created by C-to-U conversions. RNA editing in intron sequence suggests that editing can precede intercistronic processing. The sequence complementary to the edited site is proposed as a distant cis-recognition element.

Amino Acid Substitution↗
PubMed1974

The nucleotide sequence preceding an RNA polymerase initiation site on SV40 DNA. Part 2. The sequence of the early strand transcript.

The nucleotide sequence of the RNA transcript from the "early" (E) strand of SV40 DNA immediately preceding the preferred E. coli RNA polymerase start site is G-(A-A-A-C, -A-U-)-A-A-A-A-U-G-A-A-U-G-C-A-A-U-U-G-U-U-G-U-U-G-U-U-A-A-C-U-U-G-U-U-U-A-U-U-G-C-A-G-C-U-U-A-U-A-A-U-G-G-U-U-A-C-Ap. The last nucleotide of the sequence is the first nucleotide transcribed by E. coli RNA polymerase from the "E" strand. The DNA template contains a palindrome of 17 residues that includes the Hemophilus influenza restriction endonuclease cleavage site G-T-T-A-A-Cp. The DNA which gives this transcript lies very close to one end of SV40 DNA segment in the Adeno-SV40 hybrid virus Ad2+ND3 and appears to contain sufficient untranscribed information to specify the E. coli RNA polymerase start.

Base Sequence↗