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At least 91 records · Page 5Linked to original sources
PubMed1996

Rice tungro spherical virus: nucleotide sequence of the 3' genomic half and studies on the two small 3' open reading frames.

Rice tungro spherical virus (RTSV) consists of a single-stranded RNA genome of about 12 kilobases that contains one large open reading frame, ORF 1 and two small ORFs 2 and 3 at its 3' end (Shen et al., 1993, Virology 193:621-630); it was suggested that ORF 2 was expressed via a frameshift. To study the genomic information of RTSV and the variation between different RTSV isolates, the 3' half of a Philippine isolate and parts of a Thai and an Indian isolate were cloned and sequenced. Significant sequence differences were found in ORF 2 and in the 3' non-translated region. Additional stop codons have been revealed in the previously described ORF 2 in several independent clones from the three different virus isolates, the most conserved stop codon in the middle of ORF 2 being confirmed by direct RNA sequencing. These results suggest that ORF 2 could only express a peptide of about 5 kDa instead of 12 kDa as proposed earlier. Polyclonal antisera were raised against ORF 2 and 3 proteins as fusions with glutathione-S-transferase. Using these antisera we failed to detect any virus-specific peptides in extracts from infected rice plants and in virus preparations. The nucleotide sequence of the 3' end of our RTSV isolates contains several small ORFs and does not contain a repeat of 256 nucleotides found in the published sequence. These results indicate that RTSV could contain an unusually long 3' non-coding region of 1240 nucleotides in length.

Amino Acid Sequence↗
PubMed1993

Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes.

The amino acid sequences at the haemagglutinin cleavage sites of 9 avian influenza A viruses of H5 subtype (5 high and 4 low pathogenicity for chickens) and 21 of H7 subtype (13 high and 8 low pathogenicity for chickens) were determined by direct RNA sequencing, PCR amplification sequencing or both. None of the viruses of low pathogenicity had multiple basic amino acids at the cleavage site. All highly pathogenic viruses had an insert of basic amino acids at the cleavage site, except A/chicken/Scotland/59 (H5N1) for which the multiple basic amino acids appeared as substitutions and not insertions. All highly pathogenic viruses examined conformed to the amino acid motif of R-X-R/K-R at the cleavage site which is considered to be essential for high pathogenicity in chickens, with the notable exception of highly pathogenic virus A/turkey/England/50-92/91 (H5N1) which had the sequence R-K-R-K-T-R adjacent to the cleavage site.

Amino Acid Sequence↗
PubMed1997

Design of the hairpin ribozyme for targeting specific RNA sequences.

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

Base Sequence↗
PubMed1990

Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S.

Four major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were constructed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutralization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130 residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites.

Amino Acid Sequence↗
PubMed1989

Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis.

Base Sequence↗
PubMed2026

Functional chimeric mRNAs encode proteins in mammalian immunity.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Journal Article↗
PubMed1989

The completed sequence of lymphocytic choriomeningitis virus reveals a unique RNA structure and a gene for a zinc finger protein.

The arenavirus, lymphocytic choriomeningitis virus (LCMV) has a single-stranded RNA genome composed of a large (L) and a small (S) RNA segment. The completed sequence of LCMV, presented here, reveals a formerly unknown gene (Z) on the L genomic segment. This gene is encoded in the positive or message-sense of the viral genomic RNA, whereas the adjacent gene (L) is in the genome-complementary, or negative sense. The ambisense polarity of the genes on the L RNA reiterates the polarity of genes on the small (S) genomic segment. The Z gene encodes a 10-kDa protein containing a single zinc-finger sequence (Cys2His2). A small RNA representing the message sense of the Z gene is found in infected cells and within virions. In contrast to the known LCMV proteins having structural or enzymatic functions, the predicted Z gene product is most likely to be an RNA-binding protein with a regulatory role. The encapsidation of a message sense Z RNA suggests a role for this gene immediately following virus penetration. The L/Z intergenic region is rich in cytidylic acid (C) and presents an unusual RNA structure. All cDNA clones of the intergenic region differ from each other within a certain poly(C) stretch and lack a 30-base region present in the direct RNA sequence. Finally, the completed sequence establishes that the L RNA 5' end is complementary to its 3' end. The L RNA termini, similar to the S RNA termini, have a small but potentially important asymmetry of sequence. LCMV is the first arenavirus to be completely sequenced.

Amino Acid Sequence↗
PubMed1993

Chimeric EWS-FLI1 transcript in a Ewing cell line with a complex t(11;22;14) translocation.

Peripheral neuroectodermal tumors include the differentiated neuroepithelioma and the undifferentiated Ewing's tumor. Despite clinical and pathological differences, both malignancies are characterized by a t(11;22)(q24;q12) translocation which is observed in > 90% of the cases. Molecularly, the translocation is underlaid by a rearrangement between the EWS and Fli-1 genes on chromosomes 22 and 11, respectively. Because of the difficulties in the differential diagnosis between various small round cell tumors of childhood, including Ewing's tumor, a molecular diagnostic assay would be desirable. A prerequisite for predicting the reliability of such a test resides in the molecular elucidation of the peripheral neuroectodermal tumor cases which do not exhibit the prototypical translocation. We have analyzed one such case of Ewing's tumor-derived cell line with a t(11;22;14)(q24;q12;q11) translocation. An EWS-Fli-1 fusion transcript was evidenced by polymerase chain reaction amplification of a reverse transcription product obtained from total RNA. Direct sequencing was performed to demonstrate that the molecular rearrangement in this particular Ewing sample resulted in a fusion transcript similar to those observed in tumors with the prototypical translocation.

Base Sequence↗
PubMed1990

Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA.

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.

Base Sequence↗
PubMed1989

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation.

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

Blotting, Northern↗
PubMed2026

Maternal immune activation perturbs the brain epitranscriptome.

Maternal immune activation (MIA) results in abnormal fetal neurodevelopment and an increased risk of neurodevelopmental disorders. Altered RNA translation has been implicated in the pathophysiology of MIA-associated neurodevelopmental deficits, but more precise mechanisms underlying disruption in RNA metabolism are lacking. Here, we characterize key components of the RNA epitranscriptomic machinery, which refers to the set of reversible chemical modifications on RNA molecules that influence RNA function, including translation, stability, splicing, and localization. Using spatial transcriptomics, we define cell type- and brain region-specific distribution of epitranscriptome regulators in the developing mouse brain. We also use direct RNA sequencing to define how MIA changes the brain epitranscriptome landscape. We identify the demethylase FTO as being notably perturbed in the context of MIA. Using pharmacological and genetic approaches, we target FTO to ameliorate behavioral phenotypes in MIA offspring. In total, this work expands upon mechanisms of translational misregulation in MIA and identifies new targets for therapeutic manipulation.

Animals↗
PubMed1979

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.

Animals↗
PubMed1984

Multicomponent RNA plant virus infection derived from cloned viral cDNA.

In vitro transcripts from mixtures of appropriate brome mosaic virus (BMV) cDNA clones are infectious when inoculated onto barley plants. Infectivity depends on in vitro transcription and on the presence of transcripts from clones of all three BMV genetic components. Infectivity is destroyed by RNase after transcription, but it is insensitive to RNase before or to DNase after transcription. Virion RNAs from plants infected with cDNA transcripts hybridize to BMV-specific probes and coelectrophorese with virion RNAs propagated from conventional inoculum. Direct RNA sequencing shows that a deletion in the noncoding region of one infectious BMV clone is preserved in viral RNA from plants systemically infected with transcript mixtures representing that clone.

Journal Article↗
PubMed1990

Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes.

By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5' end) and direct RNA sequencing of purified U3 RNA from tomato (3' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.

Base Sequence↗
PubMed1994

Structure and regulation of the chicken erythroid delta-aminolevulinate synthase gene.

Erythroid cells regulate heme biosynthesis in a manner that is distinct from all other cell types. While heme negatively regulates the synthesis of the housekeeping delta-aminolevulinate synthase (ALAS-N) in all non-erythroid cells, the expression of an erythroid-specific isozyme (ALAS-E) is developmentally regulated in red blood cells. As a first step towards understanding the molecular basis for the transcriptional regulation of ALAS-E during erythropoiesis, we cloned and characterized the chicken ALAS-E locus. This gene spans 18 kbp and is composed of eleven exons. The intron/exon structure of erythroid ALAS was found to be conserved among several vertebrate species. Direct RNA sequencing identified a 5' untranslated region that is derived from two continuous exons and is predicted to form a very stable stem-loop structure that bears resemblance to the ferritin iron-responsive element. Tissue-specific expression of the ALAS-E gene was analyzed by transient transfection assays in hematopoietic cells of both erythroid and non-erythroid origins. These experiments identified distal (-784 to -505 bp) and proximal (-155 to +21 bp) promoter elements which are required for high level, erythroid-specific transcription.

5-Aminolevulinate Synthetase↗
PubMed1997

The EMBL Nucleotide Sequence Database.

The EMBL Nucleotide Sequence Database is a comprehensive database of DNA and RNA sequences directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications. In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute (EBI) and constitutes Europe's primary nucleotide sequence resource. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases.

Academies and Institutes↗
PubMed1992

Antigenic and genetic characterization of the haemagglutinins of recent cocirculating strains of influenza B virus.

The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991, B/Hong Kong/22/89- or B/Yamagata/16/88-like viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA1 domains of representative viruses confirmed the continued existence of two main lineages among recent strains of influenza B virus and identified unique amino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.

Amino Acid Sequence↗
PubMed2025

The UTRs of Leishmania donovani vary in length and are enriched in potential regulatory structures.

Leishmania spp. regulate gene expression largely post-transcriptionally, yet untranslated regions (UTRs) remain poorly delineated. We generated high-quality genome and transcriptome datasets for Leishmania donovani strain 1S2D (Ld1S) by combining PacBio HiFi de novo assembly with Oxford Nanopore direct RNA sequencing of promastigotes and axenic amastigotes. The genome assembly consists of 65 scaffolds totaling ~33.3 Mb. Structural comparisons to LdBPK282A1 revealed numerous rearrangements, including some reshuffling genes among polycistronic transcription units and validated by polycistronic reads from RNA sequencing. Promastigote and amastigote RNA sequencing produced 469,010 and 46,729 monocistronic reads containing a spliced-leader and a polyA tail sequences, defining 8,479 transcripts and supporting 7,415 of the 7,969 annotated protein coding genes, as well as 604 putative long non-coding RNAs. We annotated UTRs for 4,921 genes and observed that putative RNA G-quadruplexes were markedly enriched in UTRs. We also noted that 31.9% and 11.5% were expressed into multiple isoforms in promastigotes and amastigotes, respectively. Collectively, these data provide a comprehensive annotation of L. donovani genes and their UTRs and reveal widespread and stage-specific UTR length polymorphisms, and, overall, points to an important role of 3' UTR in post-transcriptional regulation in L. donovani.

Journal Article↗