Retroviral RNA packaging: sequence requirements and implications.
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Animals↗
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Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences.
The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor-joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates. Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species.
Animals↗
Spatial transcriptomics-aided localization for single-cell transcriptomics with STALocator.
Single-cell RNA-sequencing (scRNA-seq) techniques can measure gene expression at single-cell resolution but lack spatial information. Spatial transcriptomics (ST) techniques simultaneously provide gene expression data and spatial information. However, the data quality of the spatial resolution or gene coverage is still much lower than the quality of the single-cell transcriptomics data. To this end, we develop a ST-Aided Locator for single-cell transcriptomics (STALocator) to localize single cells to corresponding ST data. Applications on simulated data showed that STALocator performed better than other localization methods. When applied to the human brain and squamous cell carcinoma data, STALocator could robustly reconstruct the relative spatial organization of critical cell populations. Moreover, STALocator could enhance gene expression patterns for Slide-seqV2 data and predict genome-wide gene expression data for fluorescence in situ hybridization (FISH) and Xenium data, leading to the identification of more spatially variable genes and more biologically relevant Gene Ontology (GO) terms compared with the raw data. A record of this paper's transparent peer review process is included in the supplemental information.
Single-Cell Analysis↗
Haemonchus contortus SL2 trans-spliced RNA leader sequence.
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Animals↗
Cancer-testis antigen ACRBP: Cytotoxic response to its HLA-A2 restricted peptide and immune features in ovarian cancer.
While our prior study identified the HLA-A *0201-restricted ACRBP epitope peptide and demonstrated its capacity to generate cytotoxic T lymphocytes (CTLs) in vitro, the clinical relevance of the peptide-induced T cell reactivity in ovarian cancer (OC) patients and the in vivo anti-tumor efficacy of these CTLs remain unexplored. In this study, dendritic cells were sensitized with ACRBP peptide (ALLVLCYSI) and co-cultured with autologous CD8+T cells to induce the production of specific cytotoxic T lymphocytes (Pep-CTLs). The anti-tumor effects of Pep-CTLs were evaluated in SCID mice bearing human ovarian cancer (OC) OVCAR-3 cells. Concurrently, we co-cultured ALLVLCYSI peptide with peripheral blood mononuclear cells (PBMCs) from OC patients (HLA-A2+, ACRBP+) and assessed the number of specific T cells using ELISPOT assays. The immunological impact of the ACRBP peptide against human OC was validated through both in vitro and in vivo experiments. These findings establish a preclinical foundationfor developing ACRBP peptide-based vaccines in OC immunotherapy. To further elucidate ACRBP's role in OC treatment, the study analyzed single-cell RNA sequencing data from 8 OC patients and bulk RNA sequencing data from the Cancer Genome Atlas Project (TCGA) comprising 308 ovarian cancer cases. This analysis aimed to explore the heterogeneity among ACRBP-expressing tumor cell populations and to investigate the correlation between ACRBP expression and immune molecule expression (including MHC and chemokines) alongside chemotherapy response. These insights furnish a theoretical framework supporting the future application of ACRBP in tumor immunotherapy and strategies to prevent immune escape.
Humans↗
scPlantLLM: A Foundation Model for Exploring Single-cell Expression Atlases in Plants.
Single-cell RNA sequencing (scRNA-seq) provides unprecedented insights into plant cellular diversity by enabling high-resolution analyses of gene expression at the single-cell level. However, the complexity of scRNA-seq data, including challenges in batch integration, cell type annotation, and gene regulatory network (GRN) inference, demands advanced computational approaches. To address these challenges, we developed scPlantLLM, a Transformer model trained on millions of plant single-cell data points. Using a sequential pretraining strategy incorporating masked language modeling and cell type annotation tasks, scPlantLLM generates robust and interpretable single-cell data embeddings. When applied to Arabidopsis thaliana datasets, scPlantLLM excels in clustering, cell type annotation, and batch integration, achieving an accuracy of up to 0.91 in zero-shot learning scenarios. Furthermore, the model demonstrates an ability to identify biologically meaningful GRNs and subtle cellular subtypes, showcasing its potential to advance plant biology research. Compared to traditional methods, scPlantLLM outperforms in key metrics such as adjusted rand index (ARI), normalized mutual information (NMI), and silhouette score (SIL), highlighting its superior clustering accuracy and biological relevance. scPlantLLM represents a foundation model for exploring plant single-cell expression atlases, offering unprecedented capabilities to resolve cellular heterogeneity and regulatory dynamics across diverse plant systems. The code used in this study is available at https://github.com/compbioNJU/scPlantLLM.
Single-Cell Analysis↗
FastR: fast database search tool for non-coding RNA.
The discovery of novel non-coding RNAs has been among the most exciting recent developments in Biology. Yet, many more remain undiscovered. It has been hypothesized that there is in fact an abundance of functional non-coding RNA (ncRNA) with various catalytic and regulatory functions. Computational methods tailored specifically for ncRNA are being actively developed. As the inherent signal for ncRNA is weaker than that for protein coding genes, comparative methods offer the most promising approach, and are the subject of our research. We consider the following problem: Given an RNA sequence with a known secondary structure, efficiently compute all structural homologs (computed as a function of sequence and structural similarity) in a genomic database. Our approach, based on structural filters that eliminate a large portion of the database, while retaining the true homologs allows us to search a typical bacterial database in minutes on a standard PC, with high sensitivity and specificity. This is two orders of magnitude better than current available software for the problem.
Algorithms↗
Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing.
PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.
Bacterial Typing Techniques↗
The phylogeny of the cactophilic yeasts based on the 18S ribosomal RNA gene sequences: the proposals of Phaffomyces antillensis and Starmera caribaea, new combinations.
The complete sequences of the 18S rRNA gene fragments of the type strains of the cactophilic yeast species, Pichia antillensis, Pichia caribaea, Phaffomyces opuntiae, Phaffomyces thermotolerans, Starmera amethionina var. amethionina, and Starmera amethionina var. pachycereana were determined and compared. The type strain of Phaffomyces opuntiae had two kinds of the 18S rRNA gene sequences of which base differences were counted to be 15 and of which the percent similarity was calculated to be 99.1. The type strains of P. antillensis, P. caribaea, and Starmera amethionina var. pachycereana had the Q-7 system. The phylogenetic analyses showed that the genera Phaffomyces and Starmera were monophyletic and distant from each other and from the other species examined of the ascogenous teleomorphic genera, and that P. antillensis and P. caribaea were included within the clusters of the genera Phaffomyces and Starmera, respectively. The two Pichia species were transferred to the genera Phaffomyces and Starmera as the new combinations, Phaffomyces antillensis and Starmera caribaea. The new family Phaffomycetaceae was proposed as the type genus Phaffomyces.
Base Sequence↗
Complement expression profiles in human glomerular mesangial cells, endothelial cells, podocytes and proximal tubular epithelial cells.
BACKGROUND: Local expression of complement components in the kidney has been reported sporadically in both diseased and normal kidneys. This study aimed to comprehensively characterize the expression of complement components in human glomerular mesangial cells (GMCs), glomerular endothelial cells (GECs), podocytes, and proximal tubular epithelial cells (PTECs) in non-diseased renal tissue. METHODS: Complement expression in cultured human renal intrinsic cells was initially evaluated using reverse transcription polymerase chain reaction and immunofluorescence staining. These findings were further examined using publicly available single-cell RNA-sequencing datasets and 10×Genomics single-cell RNA sequencing of non-diseased human kidney tissue. The analyses focused on complement components involved in the initiation of the classical, lectin, and alternative pathways, as well as components shared among these activation pathways, terminal pathway components, complement regulators, and complement receptors. RESULTS: Complement components unique to the initial phase for classical pathway (C1S, C1R, C2, C4), lectin pathway (MBL2, FCN1, MASP1), alternative pathway (CFB, CFD), and the C3 component shared by the three activation pathways were detected in these cells. The components shared by the terminal pathways including C5, C6, C7, C8 and C9 exhibited lower expression, while complement regulators (CFH, CFI, CD55/DAF, CD46/MCP, CD59, C4BPB, PROS1/Protein S) or receptors (CD93/C1QR1, CR1), particularly membrane-bound proteins, such as DAF, MCP and CD59, which inhibit complement activation and the formation of the membrane attack complex, showed relatively high expression. CONCLUSION: These results showed that all four types of intrinsic renal cells expressed multiple complement components associated with the classical, lectin, and alternative pathways. In non-diseased kidney tissue, complement regulatory molecules involved in the control of complement activation showed relatively higher expression, whereas components of the terminal complement pathway were expressed at relatively lower levels, suggesting that renal intrinsic cells maintain a locally poised but tightly regulated complement system.
Humans↗
[Antigenic and genetic characterizations of Victoria like strain of influenza B viruses isolated in China in 2001].
BACKGROUND: To understand the antigenic and genetic characteristics of Victoria like strain of influenza B virus isolated recently and to provide a scientific evidence for influenza surveillance and monitoring of influenza epidemic in future. METHODS: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign. RESULTS: B/Sichuan/63/2001 and B/Zhejiang/2/2001 viruses were antigenically different from B/Shandong/7/97 strain. The substitution of nucleotide sequences of HA1genes of them compared with those of B/Shandong/7/97strain resulted in the change of amino acid sequences in antigenic determinants on HA1 protein domain. The phylogenetic analysis also indicated that strains isolated recently were genetically different from B/Shandong/7/97/strain. However, there was neither differences on the antigenicity nor genetic partern between these two isotates. CONCLUSIONS: The antigenic drift of Victoria-like strain of influenza B virus isolated recently in China has further occurred.
Amino Acid Sequence↗
Proceedings: Bacteriophage ms2 rna: nucleotide sequence determination of the A protein gene.
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Base Sequence↗
Comparison of RNA terminal sequences of phages f2 and Q-beta: chemical and sedimentation equilibrium studies.
The terminal fragment obtained by hydrolysis with ribonuclease T(1) of the ribonucleic acid from the bacteriophage Qbeta has been isolated and purified. The results of chemical and enzymatic hydrolysis of this fragment and of the intact RNA itself indicate that the fragment has the composition (10 Cp, 4 Up), and that the RNA has the terminal sequence -GP(9 Cp, 4 Up) CpA. These conclusions are supported by the results of an application of the sedimentation-equilibrium method in which the molecular weight of the Qbeta fragment was compared with that of the corresponding fragment from f2 phage RNA for which the terminal sequence, -GpUpUpApCpCpApCpCpCpA had previously been determined.
Coliphages↗
Mitochondrial DNA transfer RNA gene sequence variations in patients with mitochondrial disorders.
Many different pathogenic mutations in the mitochondrial (mt) transfer RNA (tRNA) genes have been reported for patients with mitochondrial encephalomyopathy. Although some of them are recurrent, most have only been described once and appear to be restricted to one patient or to one family. The incidence of mt tRNA gene alterations is not known, even though the frequency of some recurrent mutations has been analysed both in patients and in the general population. In this study, we describe the results of stepwise screening for sequence variations in the mt tRNA genes of 166 patients selected according to several criteria. Extensive sequence analysis of the tRNA genes was performed using denaturing gradient gel electrophoresis. A total of 31 patients (19%) were found to harbour significant levels of a pathogenic mutation, thus confirming the importance of mt tRNA mutations in human pathology. Forty-three different sequence variations were found, illustrating the great diversity of the mtDNA sequence in humans. The functional assessment of all these sequence variations represented a difficult task; it was mostly based on indirect data, such as the phylogenetic conservation of modified nucleotides and the proportions of variant species in different tissues of the index case or in blood of maternal relatives. Direct demonstration of a correlation between the proportion of heteroplasmic sequence variations and the cytochrome c oxidase defect was performed at the single muscle-fibre level. Eleven heteroplasmic sequence variations were found, six of which are new mutations. One is a known Caucasian polymorphism but the other 10 are pathogenic. Two of them are the well-known pathogenic MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (A3243G) and MERRF (myoclonic epilepsy with ragged-red fibres) (A8344G) point mutations. They were found in 23 patients. The eight other mutations were restricted to one patient. The pathogenic nature of these mutations was demonstrated directly for five of them and hypothesized from indirect arguments for the other three. Thirty-two homoplasmic sequence variations were found. Twenty-nine were considered to be polymorphisms, even though 15 of these were found for the first time in our patients and two others had been reported previously as pathogenic. The pathogenic nature of three homoplasmic variants remains questionable.
Adolescent↗
Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits.
The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes. These regions show large species-dependent variations in sequence and size. This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison. Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits. The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate. ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences. This program uses energy minimization to find the most stable secondary structure of an RNA sequence. The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism. Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.
Aldehydes↗
Differential amplification of sequence heterogeneous ribosomal RNA genes and classification of the 'Fragaria multicipita' phytoplasma.
Ribosomal (r) RNA interoperon sequence heterogeneity in the 'Fragaria multicipita' phytoplasma, a member of group 16SrVI, was initially observed in RFLP patterns of rDNA amplified in the polymerase chain reaction (PCR), and was confirmed through sequence analysis of cloned rDNA. Sequences from operons rrnA and rrnB were amplified in PCR primed by primer pair P1/P7 but from only rrnA in PCR primed by primer pair R16mF2/R16mR1. Preferential amplification of DNA from operon rrnA was explained by base mismatches between the R16mF2/R16mR1 primers and primer annealing sites in rrnB. The results revealed potential for classification of a phytoplasma into two different subgroups within a 16S rRNA group, if the phytoplasma's 16S rRNA gene sequences are independently characterized. It is suggested that the rRNA operon containing species-specific signature sequence(s) should be specified, and where possible sequences from both 16S rRNA genes should be included, in descriptions of new 'Candidatus Phytoplasma species'.
Acholeplasmataceae↗
The sequence of the 6S RNA gene of Pseudomonas aeruginosa.
From the gram-negative eubacterium Pseudomonas aeruginosa we have isolated a stable 6S RNA, approximately 180 nucleotides in length. The RNA was partially sequenced and identified by comparison with the known Escherichia coli 6S RNA sequence. Southern hybridizations revealed a single copy gene coding for the 6S RNA. DNA from other prokaryotes, i.e. E. coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermophilus and Halobacterium maris mortui, did not give detectable hybridization signals. The 6S RNA gene was cloned in E. coli and its complete primary structure was determined. Although the 6S RNA sequences from P. aeruginosa and E. coli share only a 60.4% homology, we are able to propose a common secondary structural model.
Base Sequence↗
Identity of the 5'-terminal RNA nucleotide sequence of the satellite tobacco necrosis virus and its helper virus: possible role of the 5'-terminus in the recognition by virus-specific RNA replicase.
A pancreatic ribonuclease digest of (14)C-labeled tobacco necrosis virus RNA was fractionated according to charge by column chromatography. Individual fractions were dephosphorylated with alkaline phosphatase and rechromatographed. The fraction, originally containing oligonucleotides with seven negative charges, separated into two components corresponding to five (-5) and two negative charges (-2). The -5 fraction was derived from the internal oligonucleotides while the -2 fraction must have originated from a 5'-pyrophosphorylated terminal trinucleotide. The sequence of this terminal trinucleotide was determined by column chromatography on DEAE-cellulose in a triethyl ammonium carbonate gradient, using the appropriate markers. The radioactivity chromatographed with a (ApGp)U marker. The order of the Ap and Gp was determined after ribonuclease T(1) and alkaline phosphatase digestion. The radioactivity in the product chromatographed with an ApG marker. The 5'-terminus of tobacco necrosis virus RNA was therefore determined as ppApGpUp..., which is identical to the terminus of the RNA of its satellite virus as previously determined (J. Mol. Biol., 38, 59 (1968); Science, 160, 1452 (1968)). The 5' pyrophosphate in both viruses was probably formed by an in vivo enzymatic removal of a gamma-phosphate from a triphosphate, and its presence in both viruses suggested a common site of synthesis. The identity of the 5'-terminal sequences is considered not to be fortuitous and is discussed from the standpoint of their role as a recognition site for the virus-specific RNA replicase.
Carbon Isotopes↗