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At least 1,009 records · Page 56Linked to original sources
PubMed1994

From RNA to sequenced clones within three days: a complete protocol.

Detection of specific mRNA transcripts by the reverse transcription/polymerase chain reaction (RT/PCR) technique has become increasingly important. The technique is fast and has a very high resolution. Cloning of these PCR fragments into vectors is sometimes necessary for identification of alternative splicing products, for bacterial expression or for generation of a DNA probe. Here we present a complete protocol for RT/PCR, cloning and sequencing of PCR, cloning and sequencing of PCR products beginning with the total RNA and ending with the DNA sequence within three days. To illustrate the procedure as an example, a fragment of the human glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified from total RNA, cloned and partially sequenced. The protocol has been optimized for small scale to facilitate handling and to reduce costs.

Base Sequence↗
PubMed1981

Genotypic varieties of La Crosse virus isolated from different geographic regions of the continental United States and evidence for a naturally occurring intertypic recombinant La Crosse virus.

The tripartite ribonucleic acid (RNA) genomes of 23 alternate isolates of La Crosse virus have been analyzed by the procedure of oligonucleotide fingerprinting. By comparison with the fingerprints of the three viral RNA species (large, medium and small) of prototype La Crosse virus, the viruses have been categorized in terms of the degree of their RNA sequence relatedness. The A type La Crosse viruses, which have been recovered from Wisconsin, Minnesota, Indiana and Ohio, have viral RNA sequences that are closely related to those of prototype La Crosse virus. The B type La Crosse viruses, which have been recovered from Minnesota, Wisconsin and Illinois, have RNA sequences which, although related, are easily distinguished from those of type A viruses. A La Crosse virus isolate obtained from Rochester, Minnesota, appears to be an intertypic type A/B recombinant, it has a small size RNA segment like those of the B type La Crosse virus isolates, but medium- and large-sized RNA species like those of the A type La Crosse virus isolates. The C type La Crosse viruses have viral RNA sequences that neither closely resemble the A or B type La Crosse viruses. They have been recovered from eastern Ohio, New York State, Texas, Georgia and North Carolina.

Bunyaviridae↗
PubMed2026

scRNA-seq and bulk RNA-seq reveal the characteristics of macrophage copper metabolism and establish a risk signature in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a prevalent malignancy with an urgent need for improved prognostic stratification and treatment-response prediction. This study aimed to explore a macrophage copper metabolism-associated prognostic model and to investigate the relationship between this risk model and the tumor immune microenvironment. METHODS: The FindClusters function was used to analyze cell clusters, and CellChat and CellPhoneDB/LIANA were employed for cell-cell communication analysis. Copper metabolism-related genes were sourced from the MSigDB database. A prognostic risk model was established using least absolute shrinkage and selection operator (LASSO) analysis and multivariate Cox regression analysis, and a nomogram was constructed by integrating the prognostic model with clinicopathological factors. Additional analyses were performed to map the seven model genes in single-cell data, assess model uncertainty and robustness, evaluate macrophage/copper/cuproptosis-related transcriptional programs, and examine the correlations between risk score, immune infiltration and predicted drug sensitivity. RESULTS: Using single-cell RNA sequencing (scRNA-seq) data, we identified four macrophage subpopulations. Macrophages with high SPP1 expression showed close interaction with T cell populations and were associated with copper ion metabolism. By incorporating 141 copper metabolism-related genes and using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort, we constructed a seven-gene risk prediction model. Additional single-cell mapping showed that the model genes were detectable in the HCC single-cell dataset and showed a macrophage-associated expression pattern. The model showed moderate prognostic discrimination in TCGA-LIHC, whereas its external performance was heterogeneous and remained evaluable across external cohorts, with performance varying among datasets. Immune and mechanism-related analyses suggested that the risk signature was associated with macrophage-related infiltration, copper metabolism and cuproptosis-related transcriptional programs. Drug sensitivity analysis nominated Daporinad as a computationally predicted candidate compound, supporting Daporinad as a pharmacogenomic candidate for follow-up investigation. CONCLUSIONS: By integrating scRNA-seq and bulk RNA sequencing (RNA-seq) data, we constructed a macrophage copper metabolism-associated prognostic signature for HCC. The risk score was associated with survival, immune microenvironment features and predicted drug response, providing a transcriptomic framework for risk stratification and therapeutic hypothesis generation.

Hepatocellular carcinoma (HCC)↗
PubMed1975

Host induced alteration of avian sarcoma virus B-77 genome.

The genome of an avian oncornavirus was altered after infection of a heterologous host. This was studied with avian sarcoma virus B-77 in duck embryonic fibroblasts (DEF) and chicken embryonic fibroblasts (CEF). To detect alteration of the viral genome, we hybridized 35S B-77 RNA with normal duck DNA by either one of two techniques:when viral RNA was in excess and when DNA was in excess. The RNA of B-77 passaged only in gs minus chf minus CEF does not have homology with duck DNA. However, after several passages of B-77 through DEF the viral genome acquired duck specific RNA sequences. After 4 and 10 passages, B-77 RNA acquired 2.2 and 6.6%, respectively, complementarity to normal duck DNA. The duck specific RNA sequences were found to be covalently linked to the B-77 RNA genome. Also, the host specific sequences acquired by the virus appear to be from a region of the duck DNA which is repeated four to six times per cell. After 5 back passages in CEF some of the duck specific RNA sequences in the viral genome were lost.

Animals↗
PubMed1974

Nucleotide sequences of RNA transcribed in infected cells and by Escherichia coli RNA polymerase from a segment of simian virus 40 DNA.

The nucleotide sequence of 180 residues of an RNA transcript of DNA of simian virus 40 has been deduced. This sequence adjoins a preferred initiation site for E. coli RNA polymerase. Comparison of this sequence with that of complementary RNA of simian virus 40 from infected cells has shown that this site also adjoins the apparent 3' terminus of some of the cytoplasmic complementary RNA of simian virus 40.

Adenoviridae↗
PubMed1992

[Importance of PCR in the diagnosis of hepatitis C].

The identification of hepatitis C virus, based on DNA amplification, gives a precise estimation of the prevalence of the most frequent agent of NANB hepatitis. The first ELISA allowing the detection of anti-HCV antibodies, had too many false positive results and required the development of more sensitive and specific assays to confirm its results. PCR, allowing the hepatitis C virus diagnosis by showing directly HCV RNA sequences, offers a complementary approach to immunoserological tests. In blood donors with anti-HCV antibodies and with indeterminate or negative confirmatory tests, the finding of HCV RNA sequences reveals serum infectivity. During acute hepatitis, the delay in the appearance of anti HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on-going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness and a third one distinct from the previous two. However, its limits and constraints imply that PCR must not be considered as a routine assay. This emphasizes the need for more simple and rapid diagnostic tests, allowing the detection of HCV antigens and, as in hepatitis B, the progressive unravelling of the life cycle of HCV.

Animals↗
PubMed1985

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata.

We have determined the complete nucleotide sequence of the small-subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.

Animals↗
PubMed2025

AncestryGeni: a novel genetic ancestry classification pipeline for small and noisy sequence data.

MOTIVATION: Efforts to address health disparities are often limited by the lack of robust computational tools for inferring genetic ancestry by calculating an individual's genetic similarity to continental groups. We have already shown that a preferred alternative to self-described race is using ancestry-informative markers (AIMs) that can be classified into ancestral components and used to estimate their similarity to those of known populations to identify continental groups. However, real-world genomic data can present challenges, including limited availability of germline DNA, a small number of AIMs for each sample, and the use of different variant calling software, limiting the application of existing solutions. RESULTS: Here, we describe a novel supervised machine-learning tool AncestryGeni, which infers genetic ancestry for samples with even a hundred markers and is applicable to any genomic data, including whole exome sequencing (WES) and RNA sequencing (RNA-Seq) data. Applying AncestryGeni to a real-world genomic dataset obtained from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study, we show that it is more accurate than the commonly used FastNGSadmix when using nonstandard genomic material. We also demonstrate that when using AncestryGeni, the tumor-derived sequence obtained from WES and RNA-Seq can be a robust data source to accurately estimate an individual's genetic similarity to a continental group. AVAILABILITY AND IMPLEMENTATION: AncestryGeni pipeline is available at https://github.com/eelhaik/AncestryGeni/tree/main.

Humans↗
PubMed1993

Movement of the guide sequence during RNA catalysis by a group I ribozyme.

Ribozymes derived from the self-splicing pre-ribosomal RNA of Tetrahymena act as sequence-specific endonucleases. The reaction involves binding an RNA or DNA substrate by base pairing to the internal guide sequence (IGS) to form helix P1. Site-specific photo-crosslinking localized the 5' end of the IGS in helix P1 to the vicinity of conserved bases between helices P4 and P5, supporting a major feature of the Michel-Westhof three-dimensional structure model. The crosslinked ribozyme retained catalytic activity. When not base-paired, the IGS was still specifically crosslinked, but the major site was 37 A distant from the reactive site in the experimentally supported three-dimensional model. The data indicate that a substantial induced-fit conformational change accompanies P1 formation, and they provide a physical basis for understanding the transport of oligonucleotides to the catalytic core of the ribozyme. The ability of RNA to orchestrate large-scale conformational changes may help explain why the ribosome and the spliceosome are RNA-based machines.

Animals↗
PubMed2000

Replication-competent foot-and-mouth disease virus RNAs lacking capsid coding sequences.

RNA transcripts were prepared from plasmids encoding an infectious cDNA of foot-and-mouth disease virus (FMDV) or derivatives in which the leader (Lab and Lb) and capsid protein coding sequences were deleted or replaced by sequences encoding chloramphenicol acetyltransferase (CAT). The transcripts were electroporated into BHK cells and the expression of CAT and the FMDV 3C protease was monitored. Detection of CAT and 3C was dependent on the ability of the transcript to replicate. All of the Lb coding sequence and 94% of P1 (the capsid protein precursor) coding sequence could be deleted without any apparent effect on the ability of the RNA to replicate. Thus, no cis-acting replication element is present within this region of the FMDV genome. TRANS:-encapsidation of these FMDV replicons was very inefficient, which may explain the lack of production of defective-interfering particles in FMDV-infected cells.

Aphthovirus↗
PubMed1996

Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence.

The phylogenetic position of the phylum Haplosporidia was investigated with the complete small subunit rRNA gene sequences from 5 species in the phylum: Haplosporidium nelsoni and Haplosporidium costale, parasites of the eastern oyster Crassostrea virginica; Haplosporidium louisiana, a parasite of the mudcrab Panopeus herbstii; Minchinia teredinis, a parasite of shipworms (Teredo spp.) and Urosporidium crescens, a hyperparasite found in metacercariae of the trematode Megalophallus sp. in the blue crab, Callinectes sapidus. Multiple alignments of small subunit rRNA gene sequences included the 5 haplosporidian taxa and 14 taxa in the alveolate phyla Ciliophora, Dinoflagellida, and Apicomplexa. Maximum parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the alveolate clade, as a taxon of equal rank with the other 3 alveolate phyla, and as a sister taxon to the clade composed of the phyla Dinoflagellida and Apicomplexa. Transversionally weighted parsimony placed the haplosporidians as a sister taxon to the ciliates. A separate analysis focused on the relationships of species in the genus Haplosporidium. Analyses were conducted with the haplosporidians as a functional ingroup, using each of the alveolate phyla individually as functional outgroups. The results indicated that species in the genus Haplosporidium do not form a monophyletic assemblage. As such, the present morphological criteria for distinguishing the genera Haplosporidium and Minchinia are insufficient.

Animals↗
PubMed2002

Transcriptional regulation by antitermination. Interaction of RNA with NusB protein and NusB/NusE protein complex of Escherichia coli.

A recombinant heterodimeric NusB/NusE protein complex of Escherichia coli was expressed under the control of a synthetic mini operon. Surface plasmon resonance measurements showed that the heterodimer complex has substantially higher affinity for the boxA RNA sequence motif of the ribosomal RNA (rrn) operons of E.coli as compared to monomeric NusB protein. Single base exchanges in boxA RNA reduced the affinity of the protein complex up to 15-fold. The impact of base exchanges in the boxA RNA on the interaction with NusB protein was studied by (1)H,(15)N heterocorrelation NMR spectroscopy. Spectra obtained with modified RNA sequences were analysed by a novel generic algorithm. Replacement of bases in the terminal segments of the boxA RNA motif caused minor chemical shift changes as compared to base exchanges in the central part of the dodecameric boxA motif.

Algorithms↗
PubMedDate not supplied

Simultaneous sequencing of RNA by solid-phase chemical degradation using DE 81 anion-exchange paper.

A solid-phase chemical degradation method for simultaneous sequencing of RNA and RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves the following operations: (1) immobilization of the 3'-end labeled RNAs or RNA fragments on DE 81 paper; (2) washing; (3) modification reactions; (4) washing; (5) sorting of the paper segments; (6) aniline reaction; (7) lyophilization; (8) desorption of the RNA by TEAB; and (9) lyophilization.

Animals↗
PubMed1982

Nucleotide sequence analyses and predicted coding of bunyavirus genome RNA species.

We performed 3' RNA sequence analyses of [(32)P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3'-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3'-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3'-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3'-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.

Base Sequence↗
PubMed1995

The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.

The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex-specific alternative splicing of doublesex (dsx) pre-mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre-mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre-mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female-specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site.

Alternative Splicing↗