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At least 1,531 records · Page 85Linked to original sources
PubMed1982

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.

Animals↗
PubMed1988

Structure of an unusual sea urchin U1 RNA gene cluster.

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.

Animals↗
PubMed2025

IGF1R deficiency mitigates acute lung injury by promoting anti-inflammatory transcriptional profiles.

BACKGROUND: Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and COVID-19 are characterized by hyperinflammation, commonly referred to as "cytokine storm". The insulin-like growth factor (IGF) pathway, particularly the type 1 receptor (IGF1R), plays a critical role in lung homeostasis and has been implicated in the pathogenesis of pulmonary inflammatory diseases. In mice, widespread Igf1r deficiency attenuates lung inflammation and alveolar damage in bleomycin (BLM)-induced ALI. METHODS: We analyzed single-cell RNA sequencing datasets from lung tissue of COVID-19 cases and control donors as well as mouse lungs to determine Igf1r and IGF family expression across pulmonary cell types. Furthermore, we conducted bulk RNA sequencing on lungs from Igf1r-deficient mice three days after BLM or saline instillation, followed by differential expression and functional enrichment analyses. Findings were further tested through protein detection, assessment of DNA damage and methylation in lung tissues, and functional assays using Igf1r-deficient primary mouse embryonic fibroblasts (MEFs). RESULTS: IGF1R was broadly expressed across multiple cell types in both human and mouse lungs under normal and pathological conditions. Other IGF family members showed cell-type-specific expression, which was modulated by lung injury. Transcriptomic profiling revealed differentially expressed genes between BLM-challenged and control mouse lungs, detecting biological processes and signaling pathways involved in ALI pathobiology. Igf1r deficiency in BLM-challenged mice reversed a large fraction of the transcriptional changes triggered by BLM, including "cytokine storm"-related gene expression. Functional enrichment analysis additionally revealed significant modulation of pathways related to DNA damage, metabolic reprogramming, mitochondrial homeostasis, and epigenetic regulation. In vitro, Igf1r-deficient MEFs exhibited decreased mitochondrial respiration and glycolysis, protection against BLM-induced nuclear damage and mitochondrial accumulation, and decreased histone H3 acetylation. Moreover, Igf1r-deficient mouse lungs displayed increased global DNA methylation following BLM challenge. CONCLUSIONS: IGF1R is a key modulator of the inflammatory and molecular response to ALI pathogenesis. IGF1R deficiency dampens the "cytokine storm", modifies transcriptional and epigenetic profiles and promotes protective cellular responses. These findings highlight IGF1R signaling as a potential therapeutic target in ARDS and related lung injuries.

Animals↗
PubMed1997

Sequence-selective RNA scission by oligoamine--DNA conjugates (1).

Ethylenediamine and diethylenetriamine, which are active for RNA hydrolysis, are attached to the 5'-ends of synthetic oligonucleotides by urethane linkages. The resultant artificial ribonucleases selectively hydrolyze the substrate RNA at the target phosphodiester linkage. Acid/base cooperation in the oligoamines for the catalysis is confirmed in terms of kinetic evidences.

Base Sequence↗
PubMed2002

Microbial identification by immunohybridization assay of artificial RNA labels.

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Bacteria↗
PubMed1983

Search for Sendai 6/94 viral RNA in the antigen-free cell line Cl-C-2 isolated from human multiple sclerosis brain tissue.

The viral antigen-free cell line Cl-C-2, obtained from multiple sclerosis brain tissue by cell fusion with CV-1 cells, was examined for the presence of intracellular virus-specific RNA sequences of the persistent Sendai 6/94 virus by nucleic acid hybridization. As a specific probe for this assay, an in vitro synthesized cDNA was used. Oligodeoxyguanidylic acid served as a primer for the initiation of cDNA synthesis. The 6/94 RNA was detectable as expected in the viral antigen-expressing cell lines Cl-E-8 and Cl-F-2, which were used as a reference of the same source. In the viral antigen-free cell line Cl-C-2, however, no viral RNA sequences have been found by hybridization experiments. Corresponding superinfection studies confirmed the conclusion that in cell line Cl-C-2 no viral components are present. The lack of expression of viral proteins and of protection against superinfection seems to be correlated with the lack of viral RNA in Cl-C-2 cells, which may eliminate the persistent virus by a cellular defense mechanism.

Animals↗
PubMed2004

Dissecting the requirement for subgenomic promoter sequences by RNA recombination of brome mosaic virus in vivo: evidence for functional separation of transcription and recombination.

Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3. In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.

Base Sequence↗
PubMed1982

Separation and sequence of the 3' termini of M double-stranded RNA from killer yeast.

Four subspecies of M double-stranded RNA from a killer strain of Saccharomyces cerevisiae were isolated. Each subspecies were susceptible to heat cleavage, presumably at an internal 190 base pair A,U-rich region, generating two discrete fragments corresponding to each side of the A, U-rich region. Enzymatic and chemical RNA sequence analysis defined the 3'-terminal 175 bases for the larger fragment (M-1) and 231 bases for the smaller fragment (M-2). All four subspecies of M have identical size and 3'-terminal sequences. Potential translation initiation codons are present on the corresponding 5' termini of both fragments, and a possible 18S ribosomal RNA binding site is also present on the 5' terminus of M-1. Stem and loop structures for the 5' and 3' termini of M-1 may function as recognition sites for replication, transcription, and translation.

Amino Acid Sequence↗
PubMed1998

Analysis of protein complexes assembled on apolipoprotein B mRNA for mooring sequence-dependent RNA editing.

Apolipoprotein B (apoB) mRNA editing involves a cytidine-to-uridine transition at a select site catalyzed by a cytidine deaminase known as APOBEC-1. This enzyme cannot edit RNA alone but acquires site-specific editing capacity in the context of additional protein factors (auxiliary proteins). These proteins are currently hypothesized to assemble with APBEC-1 as a holoenzyme complex or editosome. Auxiliary proteins are the focus of ongoing research as they presumably serve important structural and regulatory roles in the editosome. The abilities of auxiliary proteins to interact with APOBEC-1 and apoB RNA and to promote RNA editing activity are important endpoints used as proof that proteins are functionally involved in apoB RNA editing. This article reviews the discovery of the editosome and provides detailed protocols for its isolation and subfractionation.

APOBEC-1 Deaminase↗
PubMedDate not supplied

In vitro selection of a ligase ribozyme carrying alkylamino groups in the side chains.

A novel ligase ribozyme was in vitro selected from a random-sequence RNA library including N(6)-aminohexyl-modified adenine residues in place of natural adenine residues. This ribozyme mediated the formation of a phosphodiester bond with a DNA oligonucleotide through condensation with a 5'-triphosphate moiety on the ribozyme. Among the clones isolated from this selection, one was shown to accelerate ligation about 250-fold compared to the original random-sequence RNA library. Almost no rate acceleration was observed when the N(6)-aminohexyl-groups on adenine residues were omitted. Furthermore, ligation was dependent on the presence of a template DNA oligonucleotide that bridged the two strands.

Base Sequence↗
PubMed2026

Single-cell transcriptomic atlas of Alzheimer's disease middle temporal gyrus reveals region, cell type, and sex specificity of gene expression with novel genetic risk for MERTK in female.

BackgroundAlzheimer's disease (AD), the most common age-related neurodegenerative disease, is closely associated with both amyloid-β plaque and neuroinflammation. Two thirds of AD patients are female, and they have a higher disease risk; women with AD have more extensive brain histological changes than men along with more severe cognitive symptoms and neurodegeneration.ObjectiveThis study aimed to determine how sex difference induces structural brain changes and molecular cell vulnerabilities in AD, with a focus on identifying sex-specific transcriptional alterations and genetic risk factors.MethodsWe performed single nucleus RNA sequencing on postmortem brains from individuals with AD and age- and sex-matched controls, focusing on the middle temporal gyrus, a cortical brain region strongly affected by the disease, and integrated single nucleus RNA sequencing results with genome-wide association study (GWAS) data using cell type-specific enrichment and generalized gene-set analysis approaches. The analysis pipeline is provided with threshold information.ResultsWe identified a selectively vulnerable subpopulation of layer 2/3 excitatory neurons that were RORB-negative and CDH9-expressing in both males and females. Disease-associated, but sex-independent, reactive astrocyte signatures were also present. In clear contrast, the microglia signatures of AD brains differed between males and females. Integrating single cell transcriptomic data with results from GWAS, we identified MERTK genetic variation as a candidate novel risk factor for AD selectively in females.ConclusionsTaken together, our single cell atlas of middle temporal gyrus revealed a unique cellular-level view of sex-specific transcriptional changes in AD, illuminating GWAS identification of sex-specific AD genes. These data serve as a rich resource for interrogation of the molecular and cellular basis of AD.

Alzheimer's disease↗
PubMed1996

Enterovirus persistence and myocardial damage detected by 111In-monoclonal antimyosin antibodies in patients with dilated cardiomyopathy.

BACKGROUND: Patients with dilated cardiomyopathy in whom enteroviruses in the myocardium are detected are more likely to die than those in whom no viruses have been demonstrated. The presence of enterovirus RNA in the myocardium at endomyocardial biopsy has been shown to be the strongest predictor of reduced survival. These results raise the question as to whether persistent virus might be responsible for continuing myocardial damage. Detection of myocardial cell damage is assessed using 111Indium-labelled monoclonal antimyosin antibodies. The present study was undertaken to address the question of whether the presence of myocardial cell damage by such antibodies in patients with dilated cardiomyopathy can be correlated with enterovirus persistence. PATIENTS AND METHODS: A series of 19 consecutive patients diagnosed as having chronic dilated cardiomyopathy who were referred for evaluation for heart transplantation were studied with 111Indium labelled monoclonal antimyosin antibodies. These patients and 10 controls were screened for enterovirus RNA sequences in endomyocardial biopsy tissue by hybridization with an enterovirus group-specific cDNA probe. RESULTS: Antimyosin uptake, indicative of myocardial cell damage, was observed in 16 of 19 patients (84%), with dilated cardiomyopathy, and enterovirus RNA sequences were detected in endomyocardial biopsies from four of these 16 patients (25%), but not in myocardium from the remaining three patients with a negative antimyosin scan, nor from any of 10 controls. CONCLUSIONS: Although these data do not establish a causal relationship between virus persistence in the myocardium and myocardial damage, the results obtained in the preliminary study support the hypothesis that enterovirus persistence is associated with continuing myocardial damage in patients with dilated cardiomyopathy.

Adult↗
PubMed1993

Serodiagnosis of chronic hepatitis C in Japan by second-generation recombinant immunoblot assay.

The seroprevalence of antibodies to hepatitis C virus (HCV) by a second-generation recombinant immunoblot assay (RIBA-2) was tested using 4 recombinant antigens. The results were correlated with those of C100-3 enzyme-linked immunosorbent assay (ELISA) and with the detection of HCV-RNA sequences by the polymerase chain reaction. Sera were obtained from 27 C100-3 ELISA-positive Japanese patients with chronic non-A, non-B liver disease and from 29 C100-3 ELISA-negative patients. All C100-3 ELISA-positive patients and 19 (66%) out of 29 C100-3 ELISA-negative patients reacted to two or more RIBA-2 antigens (reactive by RIBA-2). Of the remaining 10 C100-3 ELISA-negative patients, one patient reacted to just one antigen (indeterminate by RIBA-2), while 9 reacted to none of the 4 antigens (non-reactive by RIBA-2). Forty-three (93%) of the 46 RIBA-2-reactive patients and the single RIBA-2-indeterminate patient tested positive for HCV-RNA sequences, whereas only one (11%) of the 9 RIBA-2-non-reactive patients tested positive. These findings indicate that HCV infection is more prevalent than expected from the results of C100-3 ELISA, and that RIBA-2 is a more sensitive assay for estimating the presence of HCV infection in chronic liver disease.

Aged↗
PubMed1985

A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure.

The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

Base Sequence↗
PubMed1978

Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides.

Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.

Base Sequence↗
PubMed1974

Nucleotide sequence relationships of avian RNA tumor viruses: measurement of the deletion in a transformation-defective mutant of Rous sarcoma virus.

Stocks of cloned helper-independent Rous sarcoma virus (RSV) spontaneously segregate transformation-defective (td) mutants that appear to have an RNA genome composed of smaller subunits than those of the patent virus. Differential hybridization and competitive hybridization techniques involving reactions between viral RNA and proviral sequences in host cell DNA (under conditions of initial DNA excess) were used to measure the extent of the deletion in a td mutant of Prague strain (Pr) of RSV (Pr RSV-C). Viral 60 to 70S RNA sequences labeled to 1 to 5 x 10(7) counts per min per mug with (125)I were characterized with respect to their properties in hybridization reactions and used to reinforce data obtained with [(3)H]RNA of lower specific activity. By these techniques, about 13% +/- 3% of the sequences Pr RSV-C that formed hybrids with DNA from virus-induced sarcomas appeared to be deleted from the genome of td Pr RSV-C. Studies comparing hybridization of RNA from Pr RSV-C and td Pr RSV-C with RSV-related sequences in normal cells, and competition experiments with RNA from the endogenous chicken oncornavirus Rous-associated virus type 0 (RAV-0) provided evidence that the majority, if not all, of the RNA sequences of Pr RSV-C deleted from its transformation-defective mutant are not represented in normal chicken DNA. Competition studies with a leukosis virus, RAV-7, indicated this virus also lacks a genome segment of about the same size as the deletion in the td mutant. Finally, the genome of all three "exogenous" viruses was found to lack a small segment (about 12%) of sequences present in the endogenous provirus of RAV-O.

Animals↗
PubMed1994

Chronic liver disease and active hepatitis C virus infection in patients with antibodies to this virus.

AIMS: To assess the association between active hepatitis C virus (HCV) infection and liver damage in randomly selected patients with antibodies to the virus. METHODS: Thirty three consecutive subjects with serologically confirmed positivity for antibodies to HCV were studied for the presence of liver and circulating viral sequences by using the reverse transcription polymerase chain reaction (RT-PCR) and specific primers for the 5'-untranslated region (5'-UTR) of the HCV genome. Parallel clinical, biochemical, and histological investigations were carried out in all cases. RESULTS: A comparative virological and histological investigation showed the presence of molecular signs of active viral replication and different degrees of liver damage in all cases. Baseline values of liver and plasma samples from all the patients showed (with one exception) the presence of detectable HCV RNA sequences, despite alanine amino transferase activities being within normal values or within 1.5 times the upper limit of normal in 13 of them. Examination of percutaneous liver biopsy specimens showed the presence of confirmed liver damage (ranging from chronic persistent hepatitis to cirrhosis) in all 33 patients. CONCLUSIONS: Circulating HCV RNA sequences (a direct sign of active HCV infection) are associated with liver damage, even in the absence of clinical or biochemical signs of overt liver disease. Parallel molecular, histological, and clinical follow up of these patients is needed to understand precisely the natural history of HCV infection and for correct clinical management.

Base Sequence↗