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At least 415 records · Page 23Linked to original sources
PubMed2005

Isothermal RNA sequence amplification method for rapid antituberculosis drug susceptibility testing of Mycobacterium tuberculosis.

RNA transcript quantification by an isothermal sequence amplification reaction was evaluated for susceptibility testing of 15 Mycobacterium tuberculosis strains. Agreement with the proportion method on Ogawa egg medium and the BACTEC MGIT 960 system was 100 and 87% for rifampin, 93 and 100% for isoniazid, 60 and 53% for ethambutol, and 80 and 80% for streptomycin, respectively.

Anti-Bacterial Agents↗
PubMed1978

Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing.

Three synthetic oligonucleotides were prepared to be complementary to known regions of the mouse immunoglublin light chain mRNA, and their ability to prime the transcription of complementary DNA (cDNA) was studied. The sequence of the cDNA was determined by adapting for mRNA the DNA sequencing method of Sanger, Nicklen and Coulson (1977) which uses 2'3' dideoxy ribonucleotides. A continuous sequence of 532 nucleotides was obtained, 321 corresponding to the whole of the constant region of the mRNA and the remaining 211 being the complete 3' noncoding region of the mRNA. The termination codon U-A-G occurs at the expected position in the mRNA corresponding to the triplet following the C terminal cystine. The nucleotide sequence is partially corroborated by the sequence of fragments obtained previously from 32P-mRNA fingerprints and endonuclease IV digests of 32P-cDNA, and is in agreement with the amino acid sequence of the constant region, except for a rearrangement of four amino acids (between amino acid positions 163 and 166). A revision of the amino acid sequence confirms the nucleic acid sequence.

Amino Acid Sequence↗
PubMed1999

The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1 specifically interact with an exon 3-derived mouse vascular smooth muscle alpha-actin messenger RNA sequence.

Amino acids 44-53 of mouse vascular smooth muscle alpha-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5' promoter of the gene. The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5'-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Puralpha, Purbeta, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purbeta with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle alpha-actin gene in fibroblasts.

Actins↗
PubMed2006

Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III.

Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, muR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of muR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db 'set' is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate.

Base Pairing↗
PubMed1997

Human coronavirus polyadenylated RNA sequences in cerebrospinal fluid from multiple sclerosis patients.

Human coronaviruses, represented by the two prototype strains HCV-OC43 and HCV-229E, are important human respiratory pathogens, also associated with necrotizing enterocolitis. Two previous studies, one describing the electron microscopic observation of doughnut-shaped particles, resembling coronaviruses, in a perivascular inflammatory lesion of brain tissue taken at autopsy from a multiple sclerosis patient, and the other one reporting the isolation of coronaviruses from the brains of two multiple sclerosis patients, suggested the possible association between coronaviruses and human demyelinating diseases. We analysed polyadenylated RNAs extracted from cerebrospinal fluid of twenty randomly selected multiple sclerosis patients and ten patients with other neurological diseases (medullary atrophy, Parkinson's disease, polyneuropathy, senile dementia, headache and toxic polyneuropathy) by reverse transcription and polymerase chain reaction searching for HCV-OC43 and HCV-229E sequences. By hybridization analysis of amplification products, we detected HCV-OC43 polyadenylated RNAs in ten specimens of patients with multiple sclerosis. Furthermore, we found positive hybridization signals for HCV-OC43 in the other neurological diseases, except for the toxic polyneuropathy specimen. Positivity for HCV-229E was observed in seven specimens of multiple sclerosis cerebrospinal fluid; one headache cerebrospinal fluid and the medullary atrophy specimen also resulted positive for HCV-229E. Moreover, using a solid phase technique, we report for the first time the sequence of a cDNA fragment derived from RNA extracted from cerebrospinal fluid of multiple sclerosis patient, belonging to the open reading frame which codes for the HCV-OC43 nucleoprotein N. Furthermore, cDNA sequences revealed the presence of a mixed viral population.

Coronavirus↗
PubMed1999

Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd.

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

Base Sequence↗
PubMed1979

Androgenic regulation of messenger RNA sequence complexity in accessory sexual tissues of the male rat studied with fractionated complementary DNA.

Effects of androgens on mRNA sequence complexity in the rat seminal vesicle have been investigated using complementary DNA fractionated on the basis of sequence abundance. Total cDNA complementary to poly(A)-rich RNA from normal rats was hybridised with an excess of the same RNA to controlled rot values and then the free cDNA was separated from cDNA . RNA hybrids by hydroxyapatite chromatography. Three cDNA fractions were obtained with very different hybridisation characteristics. Abundant cDNA hybridised to an excess of its parental RNA with an rot 1/2 of 2.46 x 10(-3) mol 1(-1) s and is complementary to about six or seven average-sized sequences. Use of hybrid-arrested translation in a cell-free protein-synthesising system has shown that this class of mRNA includes mRNAs coding for major androgen-dependent secretory proteins. Moderate and scarce cDNA fractions each showed more complex hybridization kinetics; computer analysis suggested each is complementary to two groups of average-sized sequences. Each cDNA fraction was hybridised to excess poly(A)-rich RNA from normal or castrated rats and the kinetics compared. Castration had no effect on the total number of sequences present in any class and did not alter the relative concentration of the scarce sequences. A small (threefold) decrease was seen in the concentration of abundant sequences with a larger (tenfold) decrease in the moderate class. Both de-reases were reversed by testosterone in vivo. The results are consistent with earlier studies where the effects of testosterone on seminal vesicle mRNA were followed using a translation assay and confirm that no gross differential effects are exerted on abundant mRNA coding for major secretory proteins. The cDNA fractions were also used to investigate the overlap in genetic expression between seminal vesicle and ventral prostate. Both tissues share all the scarce sequences in the same relative abundance. Less than 0.0015% and 0.004% of prostatic mRNA is complementary to seminal vesicle abundant and moderate sequences respectively. Similarly prostatic abundant sequences account for less than 0.004% of seminal vesicle mRNA.

Animals↗
PubMed2007

Guidelines for the selection of effective short-interfering RNA sequences for functional genomics.

To avoid long-double-stranded-RNA-dependent interferon response, short-interfering RNAs (siRNAs) are widely used for RNA interference (RNAi) in mammalian cells. siRNA-based RNAi, however, may not be readily available for the large-scale gene silencing essential for systematic functional genomics, because only a limited fraction of siRNAs is capable of inducing effective mammalian RNAi. siRNAs correctly designed for the knockdown of a particular gene may also destroy the functions of unrelated genes. Here, we describe algorithms by which these serious setbacks can be eliminated in mammalian functional genomics using RNAi and a Web-based online software system for computing highly functional siRNA sequences with maximal target-specificity in mammalian RNAi.

Algorithms↗
PubMed2024

SIMS: A deep-learning label transfer tool for single-cell RNA sequencing analysis.

Cell atlases serve as vital references for automating cell labeling in new samples, yet existing classification algorithms struggle with accuracy. Here we introduce SIMS (scalable, interpretable machine learning for single cell), a low-code data-efficient pipeline for single-cell RNA classification. We benchmark SIMS against datasets from different tissues and species. We demonstrate SIMS's efficacy in classifying cells in the brain, achieving high accuracy even with small training sets (<3,500 cells) and across different samples. SIMS accurately predicts neuronal subtypes in the developing brain, shedding light on genetic changes during neuronal differentiation and postmitotic fate refinement. Finally, we apply SIMS to single-cell RNA datasets of cortical organoids to predict cell identities and uncover genetic variations between cell lines. SIMS identifies cell-line differences and misannotated cell lineages in human cortical organoids derived from different pluripotent stem cell lines. Altogether, we show that SIMS is a versatile and robust tool for cell-type classification from single-cell datasets.

Single-Cell Analysis↗
PubMed1992

An analysis of partial 28S ribosomal RNA sequences suggests early radiations of sponges.

Sequences from the 5' end terminal part of 28S ribosomal RNA were obtained and compared for 22 animals belonging to all diploblastic phyla and for a large number of representatives of triploblastic Metazoa and protists. Phylogenetic analyses undertaken using different methods showed deep radiations of phyla such as Ctenophora, Cnidaria and Placozoa but also for groups of Porifera of low taxonomic rank. Short internodes between these radiations suggested an early rapid diversification of diploblasts. A long internal branch preceding the diversification of all triploblasts analyzed could be explained either by a long period with a single ancestor or by the extinction of the earliest triploblastic radiations. Finally some unexpected relationships were revealed among Porifera.

Animals↗
PubMed1988

Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus.

Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.

Animals↗
PubMed1999

The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner.

modulo belongs to the modifier of Position Effect Variegation class of Drosophila genes, suggesting a role for its product in regulating chromatin structure. Genetics assigned a second function to the gene, in protein synthesis capacity. Bifunctionality is consistent with protein localization in two distinct subnuclear compartments, chromatin and nucleolus, and with its organization in modules potentially involved in DNA and RNA binding. In this study, we examine nucleic acid interactions established by Modulo at nucleolus and chromatin and the mechanism that controls the distribution and balances the function of the protein in the two compartments. Structure/function analysis and oligomer selection/amplification experiments indicate that, in vitro, two basic terminal domains independently contact DNA without sequence specificity, whereas a central RNA Recognition Motif (RRM)-containing domain allows recognition of a novel sequence-/motif-specific RNA class. Phosphorylation moreover is shown to down-regulate DNA binding. Evidence is provided that in vivo nucleolar Modulo is highly phosphorylated and belongs to a ribonucleoprotein particle, whereas chromatin-associated protein is not modified. A functional scheme is finally proposed in which modification by phosphorylation modulates Mod subnuclear distribution and balances its function at the nucleolus and chromatin.

Animals↗
PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗
PubMed1976

A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: some modifications in the Sanger method for RNA sequencing.

Two modifications in the Sanger two dimensional electrophoretic procedure for RNA analysis are reported. One increases resolution on the primary fingerprint to the point that digests of large RNAs, of the size 1500-3000 nucleotides yield well resolved fingerprint patterns. The other is a novel endonucleolytic procedure that proves useful in determining sequences of the large oligonucleotides produced by T1 ribonuclease. These modifications have been used in determining the catalogs of oligomers produced by T1 ribonuclease digestion of 16S rRNAs from three related organisms, Bacillus subtilis, B.pumilus and B.stearothermophilus. The possible effects of adaptation to a thermophilic niche on ribosomal RNA primary structure and the phylogenetic relatedness of the two mesophilic Bacilli are discussed.

Animals↗
PubMed1983

Regulation of protein synthesis in Tetrahymena. RNA sequence sets of growing and starved cells.

The complexity of messenger RNA in growing or starved Tetrahymena thermophila is similar and unusually high (approximately 4.5 X 10(7) nucleotides). The complexity of nuclear RNA in growing cells (approximately 7.8 X 10(7) nucleotides) is only about 1.7 times that of mRNA. The concentration of complex class (rare) messages (approximately 53 copies/growing cell and approximately 11 copies/starved cell) is low in comparison to the size of the cell. The concentration of complex nuclear transcripts is also very low (approximately 0.7 copies/growing cell nucleus and approximately 2.6 copies/starved cell nucleus) considering that the macronucleus contains 45 to 90 copies of each single copy sequence. The complex sequence sets found on polysomes of growing and starved cells overlap about 80% and about 60% of the complex nuclear transcripts appear to be held in common. About 60% of macronuclear single copy DNA is transcribed in one or both physiological states. Although growing and starved cells have extremely different fractions of their messages loaded onto polysomes, within each cell type the complex messages in polysomal and nonpolysomal cytoplasmic fractions are indistinguishable, suggesting that exchange may occur between loaded and unloaded messages. Although T. thermophila DNA has an unusually low G + C content (23%), sequences coding for complex RNAs have base ratios similar to those of total DNA. Therefore, codon usage in Tetrahymena must be extremely biased towards adenine- and uridine-rich codons.

Animals↗