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At least 1,387 records · Page 77Linked to original sources
PubMed1997

Sequence similarities between large subunit ribosomal RNA gene intervening sequences from different Helicobacter species.

When the 23S rRNA genes from several Helicobacter species were amplified by PCR and compared with similar amplicons derived from H. pylori, they were seen to be enlarged in size. Sequencing of these enlarged genes from H. mustelae, H. canis (two strains) and H. muridarum identified insertions of novel sequence (intervening sequences, IVSs) sized between 93 and 377 bp located at nt 545, in place of an 8-nt sequence in the conventionally sized H. pylori gene. These IVSs were not present elsewhere in the genome. All strains with such IVSs lacked intact 23S rRNA which was replaced by two fragment whose sizes were consistent with cleavage at either side of the particular IVS. The predicted secondary structures of the four IVSs were characterised by base pairing at the 5' and 3' ends to form a stem. The four IVSs exhibited significant sequence inter-relationships. Further relationships were also observed between them and similar elements in both small and large subunit rRNA genes of other Helicobacter and Campylobacter species. Alignment of each IVS with the other such elements identified blocks of related sequence consistent with insertion/deletion events, indicating possible evolutionary relationships.

Base Sequence↗
PubMed2026

Translation of scRNA-seq to a clinical blood test for infection diagnostics.

INTRODUCTION: Early and accurate triage of patients with febrile illness is crucial for appropriate treatment. While standard inflammatory biomarkers are often nonspecific, transcriptome analysis of peripheral blood has diagnostic potential. However, bulk gene expression data is often confounded by changes in cell count proportions, a more robust quantification of gene expression in specific single-cell types, such as monocytes, is required to serve as a reliable clinical biomarker. AREAS COVERED: Various methods to obtain single-cell-type gene expression results, including the gold standard of gene expression analysis after cell sorting and single-cell RNA sequencing, which are difficult to implement in the routine settings are discussed. Other method to interrogate gene expression of a single cell-type is needed. Finally, monocyte cell-type specific ratio-based biomarker (RBB, called Direct Leukocyte Single cell-type Transcript Abundance, or DIRECT LS-TA) which can estimate single cell-type (monocyte) specific gene expression without cell sorting is introduced. EXPERT OPINION: Traditional diagnostic test for differentiating infection has several limitations requiring breakthrough including turn-around time and cost. DIRECT LS-TA provides a reliable way to quantify monocyte-specific gene expression that strongly correlates with gold-standard methods. It is more affordable than single-cell RNA sequencing and can be readily implemented in clinical laboratories using widely available quantitative PCR or digital PCR machines.

Humans↗
PubMed1995

Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

Amino Acid Sequence↗
PubMed1996

Phylogeny of the Chlamydomonadales (Chlorophyceae): a comparison of ribosomal RNA gene sequences from the nucleus and the chloroplast.

Phylogenetic analyses of nuclear-encoded small-subunit rRNA sequences and chloroplast-encoded large-subunit rRNA sequences from flagellate green algae representing the order Chlamydomonadales were found to show considerable congruence. In general, the chloroplast data set exhibited more robust support for comparable lineages than the nuclear data set. The phylogenetic inferences derived from the independent data sets support some, but also challenge many, traditional taxonomic and phylogenetic concepts regarding the green flagellates. Results from phylogenetic analyses of both molecular data sets support six distinct lineages that include taxa from the biflagellate genus, Chlamydomonas, and a basal lineage that comprises taxa from the quadriflagellate genus, Carteria. Both data sets support the conclusion that Chlamydomonas is not monophyletic. Although the chloroplast data are ambiguous regarding the question of Carteria monophyly, the nuclear data fail to support Carteria monophyly. The chlorococcalean genus Chlorococcum was found to have affinities with the Chlamydomonadales, indicating that the traditional concepts of both Chlorococcales and Chlamydomonadales may need revision. The genus Dunaliella is allied within the Chlamydomonadales, supporting the contention that it has lost a typical glycoprotein cell wall.

Animals↗
PubMed1998

Differential regulation of A gamma and G gamma fetal hemoglobin mRNA levels by hydroxyurea and butyrate.

In clinical studies, both hydroxyurea and butyrate increase fetal hemoglobin expression and ameliorate the symptoms of sickle cell anemia. However, comparative studies of the effects of hydroxyurea and butyrate on the expression of the individual fetal hemoglobin genes, A gamma and G gamma, have not been performed. The present study reports the effects of hydroxyurea and butyrate on steady-state A gamma and G gamma mRNA levels in K562 cells. Because the high degree of homology between the A gamma and G gamma cDNA sequences precludes the use of large cDNA probes for detection of individual fetal hemoglobin gene products, we investigated the specificity of two 20-base oligonucleotide probes synthesized from the region of greatest sequence diversity between these genes. Hybridization experiments demonstrated that the A gamma oligonucleotide probe was specific for A gamma DNA and RNA sequences and the G gamma oligonucleotide probe was specific for G gamma DNA and RNA sequences. These oligonucleotide probes detected both A gamma and G gamma mRNAs in K562 cells. In K562 cells treated with 2 mM sodium butyrate for 168 hours, the G gamma mRNA level increased 3.6-fold, whereas the A gamma mRNA level was not significantly different from untreated cells. Similar results were obtained when K562 cells were treated with 80 microM hydroxyurea. The G gamma mRNA level increased 2.3-fold at 168 hours, whereas the A gamma mRNA level did not change. The above results demonstrate that both butyrate and hydroxyurea selectively increase G gamma expression. Selective regulation of individual fetal hemoglobin genes is also seen in human development, where approximately 70% of the total fetal hemoglobin in the fetus is G gamma. Therefore, understanding the mechanisms by which butyrate and hydroxyurea differentially regulate fetal hemoglobin gene expression may provide insights into the developmental regulation of hemoglobin expression as well as the mechanisms of action of pharmacological agents currently being used to treat sickle cell disease.

Butyrates↗
PubMed1992

Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro.

A conserved positive-acting RNA sequence was found to be required for the neuron-specific splicing of the mouse c-src N1 exon. The sequence lies in the intron between exons N1 and 4, close to the N1 donor site. Normally, only the neural-specific splicing of exon N1 required this sequence. When the intron downstream of N1 was shortened, splicing at the constitutive exon 4 acceptor also became dependent on the activating sequence. The neuronal and nonneuronal patterns of src splicing were reconstituted in vitro. HeLa cell extracts spliced exon 4 to exon 3, skipping exon N1. Weri-1 retinoblastoma cell extracts spliced exon 4 to exon N1 as well as to exon 3. Both patterns of splicing were dependent on the activating sequence. A 123 nt RNA containing just the activating sequence specifically inhibited both patterns of src splicing, indicating that factors bound to the activator were required for its effects.

Animals↗
PubMed1995

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.

Base Composition↗
PubMed2003

Bistable secondary structures of small RNAs and their structural probing by comparative imino proton NMR spectroscopy.

We investigate 25-34 nucleotide RNA sequences, that have been rationally designed to adopt two different secondary structures that are in thermodynamic equilibrium. Experimental evidence for the co-existence of the two conformers results from the NH...N 1H NMR spectra. When compared to the NH...N 1H NMR spectra of appropriate reference sequences the equilibrium position is easily quantifiable even without the assignment of the individual NH resonances. The reference sequences represent several Watson-Crick base-paired double helical segments, each encountered in either of the two conformers of the bistable target sequence. In addition, we rationalize the influence of nucleotide mutations on the equilibrium position of one of the bistable RNA sequences. The approach further allows a detailed thermodynamic analysis and the evaluation of secondary structure predictions for multistable RNAs obtained by computational methods.

Base Composition↗
PubMedDate not supplied

The small subunit ribosomal RNA gene sequence of Pleistophora anguillarum and the use of PCR primers for diagnostic detection of the parasite.

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels (Anguilla japonica) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum, enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post infection. No PCR product was obtained with templates prepared from clinically healthy control animals.

Anguilla↗
PubMed2026

Transcriptomic Changes Associated with Electroacupuncture in a DMCAO Model of Delayed Cognitive Impairment.

INTRODUCTION: Delayed Cognitive Impairment (DCIS) occurs in approximately 31% to 77% of individuals following stroke. Clinical findings have indicated that electroacupuncture may alleviate post-stroke DCIS. However, insights derived from animal models remain limited. The present study utilized a Distal Middle Cerebral Artery Occlusion (DMCAO) mouse model to investigate the potential mechanisms of electroacupuncture through hippocampal transcriptomic analysis. MATERIALS AND METHODS: Adult male BALB/c mice were subjected to DMCAO and received electroacupuncture treatment. High-throughput RNA sequencing of hippocampal tissue was performed to identify Differentially Expressed Genes (DEGs). Enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, hierarchical clustering, and Protein-Protein Interaction (PPI) network analysis, were performed to elucidate potential biological mechanisms. RESULTS: The DMCAO model exhibited features consistent with DCIS. Electroacupuncture treatment was associated with improved cognitive performance and enhanced hippocampal neuroplasticity. A total of 116 DEGs were identified in the DMCAO group compared with the sham group, while 69 DEGs were identified in the DMCAO + electroacupuncture group compared with the untreated DMCAO group. DISCUSSION: GO enrichment analysis indicated that electroacupuncture modulated biological processes related to nerve fibers, axonal development, neuronal regulation, cellular processes, and cardiovascular protection. KEGG pathway analysis indicated involvement in pathways associated with neuronal recovery and axonal function. The PPI network comprised 28 nodes and 33 interactions, with hub genes such as Gna13, Hipk2, and Stambp playing key roles. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) results were consistent with RNA sequencing findings. CONCLUSION: Electroacupuncture improved DCIS in the DMCAO mouse model. Transcriptomic analysis of the hippocampus provided preliminary evidence of the potential mechanisms underlying the therapeutic effects of electroacupuncture treatment following ischemic stroke.

Animals↗
PubMed1998

Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins.

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of beta-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

Cell Nucleus↗
PubMed1995

Cell proteins bind to a linear polypyrimidine-rich sequence within the 5'-untranslated region of rhinovirus 14 RNA.

Members of the picornavirus family initiate translation of their RNA genomes by a cap-independent mechanism in which ribosomes bind to an internal site in the 5' untranslated region (5'-UTR). This unique process requires an internal ribosome entry site (IRES), a highly structured RNA whose function is mediated in part by interactions with cell proteins. The IRES element of human rhinovirus 2 (HRV-2) extends from nucleotide (nt) 10 to between nt 544 and 568 and has been shown to interact with two cell proteins, pyrimidine tract-binding protein (pPTB) and p97. To map the specific regions of HRV-14 RNA that bind cell proteins, mobility shift, UV cross-linking and Western immunoblot analyses were performed. The results indicate that an RNA sequence from nt 538 to 591 interacts with pPTB and La, two proteins previously shown to functionally interact with the IRES elements of several picornaviruses. Two additional proteins, p97 and p68, were also cross-linked to nt 538 to 591 RNA. These four proteins interact with a putatively unstructured portion of the 5'-UTR that contains a polypyrimidine tract and has been shown to be present at the 3' border of sequences that are essential for IRES function of HRV-2. These protein-RNA interactions are likely to play a role in internal initiation of translation.

Base Sequence↗
PubMed1998

In vitro selection of RNAs aminoacylated by Escherichia coli leucyl-tRNA synthetase.

To investigate systematically the RNA sequences necessary for aminoacylation by Escherichia coli leucyl-tRNA synthetase, RNAs with leucylation activity were isolated by in vitro selection from a library of tRNALeu variants possessing randomized sequences in the D-loop, the variable arm, and the T-loop. After two rounds of selection, most of the selected variants showed the following features: (1) the tertiary interaction between nucleotides at positions 15 and 48 was A15-U48; (2) the continuous G18G19 sequence, which is invariant in canonical tRNAs, appeared at the fixed position in the D-loop; and (3) the nucleotide at position 20a in the D-loop was A. These selected nucleotides and their positions, concentrating on the hinge region of tRNA, were identical to those of native tRNALeu. In contrast, although the long variable arm is the most characteristic of the tRNALeu structure, the primary and secondary structures were not correlated with the leucylation activity. These findings indicate that A15-U48, A20a, and G18G19 located at specific positions are involved in the tertiary folding of leucine-accepting tRNA molecules. With increases in the selection cycle, the D-loop sequence and the secondary structure of the variable arm became similar to those of tRNALeu, suggesting that tRNALeu represents an optimized RNA sequence for leucylation.

Acetylation↗
PubMed1982

Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena.

In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.

Animals↗
PubMedDate not supplied

The mouse Cd83 gene: structure, domain organization, and chromosome localization.

Human CD83 (hCD83) is a 45 000 Mr cell-surface protein expressed predominantly by dendritic lineage cells. In this report, the genomic locus encoding mouse CD83 (Cd83) was isolated and the gene structure determined. The Cd83 gene spans approximately 19 kilobases (kb) and is composed of five exons, with two exons encoding a single extracellular immunoglobulin (Ig)-like domain. Mouse CD83 (mCD83) cDNAs were isolated by reverse transcriptase polymerase chain reaction of mouse RNA. Sequence determination revealed substantial conservation, with mCD83 and hCD83 sharing 63% amino acid identity. The transmembrane and cytoplasmic regions of CD83 were most highly conserved. Mouse CD83 mRNA of 2.4 kb was abundantly expressed in spleen and brain, but could also be detected in most tissues analyzed. These results suggest that in the mouse, as in humans, widely distributed dendritic cells may express mCD83. Chromosome localization revealed that the Cd83 gene is present on mouse chromosome 13 band A5, while the locus for the human gene (CD83) is located within a homologous region of human chromosome 6p23. Thus, the CD83 protein and gene appear to be well conserved during recent mammalian evolution. The isolation and characterization of the mCD83 cDNA and gene provides important information and tools that will facilitate the study of CD83 and dendritic cell function in a mouse model system.

Amino Acid Sequence↗
PubMed1997

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes.

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Aedes↗