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At least 19 recordsLinked to original sources
PubMed1988

Steroid-induced cell proliferation in vivo is associated with increased c-myc proto-oncogene transcript abundance.

Enhanced c-myc transcript abundance has been observed in a variety of human malignancies, in normal liver tissue induced to proliferate in vivo by partial hepatectomy and in cells in culture induced to proliferate with the addition of protein hormones and growth factors. Little is known, however, about the expression of cellular proto-oncogenes in cells induced to proliferate in vivo by steroid hormones. Experiments reported here indicate that when cells of the immature chicken oviduct are induced to undergo rapid in vivo proliferation by application of the estrogen hormone 17 beta-estradiol, the onset of this proliferation is associated with a rapid, large, and transient increase in c-myc transcript abundance. When estrogen is administered to chickens in which the oviduct has already differentiated, neither massive cell proliferation nor large increases in c-myc transcript abundance are induced. We conclude that the abundance of c-myc transcripts in vivo correlates well with the degree of cell proliferation induced by steroid hormone.

Animals↗
PubMed2025

A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

RNA Editing↗
PubMed1990

An abundant transcript induced in differentiating human endothelial cells encodes a polypeptide with structural similarities to G-protein-coupled receptors.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) inhibits the growth of human endothelial cells and induces differentiation into capillary-like, tubular structures. We have isolated cDNA clones induced by PMA in the presence of cycloheximide and report the characterization of a novel immediate-early cDNA clone, termed edg-1, from human endothelial cells. The 3-kilobase edg-1 transcript is rapidly induced when endothelial cells are treated with PMA and superinduced in the presence of cycloheximide. While superinduction is due, at least in part, to the stabilization of the edg-1 transcript, nuclear run-on analysis demonstrates that the transcription of edg-1 is stimulated by PMA. Although the edg-1 transcript is very abundant in endothelial cells, transcripts related to human edg-1 are also detected at lower levels in vascular smooth muscle cells, fibroblasts, melanocytes, and cells of epithelioid origin. The deduced polypeptide sequence of edg-1 contains seven transmembrane domains with significant structural similarities to G-protein-coupled receptors (GPRs). Although the identity of the ligand for edg-1 is presently unknown, the structure of edg-1 polypeptide strongly implies that the edg-1 translation product is an inducible endothelial cell GPR. Since GPRs are involved in diverse biological processes such as signal transduction, cell proliferation, and differentiation, the characterization of human edg-1 as a highly inducible and abundant endothelial cell GPR suggest that it may be involved in the processes that regulate the differentiation of endothelial cells.

Amino Acid Sequence↗
PubMed2026

Genome-wide survey of spliceosomal snRNA transcripts across hundreds of human biosamples reveals abundant transcription but low maturation level of snRNA variants.

Small nuclear RNAs (snRNAs) are essential components of the spliceosome and are encoded by large, multicopy gene families. However, their genome-wide identification and quantification have remained challenging due to high sequence similarity among family members. To address this, we utilized RAMPAGE (Rapid Amplification of cDNA Ends) data from the ENCODE project to comprehensively profile nascent transcription of spliceosomal snRNAs across 115 human biosamples. We identified 74 expressed snRNA variants, characterized by canonical promoter features including bidirectional transcription flanking a positioned nucleosome, active histone modifications, and evolutionary conservation- features largely absent from unexpressed variants. These transcriptional events were corroborated by total RNA-seq and Bru-seq data, yet the majority of these variants showed extremely low levels in small RNA-seq, indicating post-transcriptional bottlenecks for snRNA processing and maturation. Our findings reveal new layers of regulation in snRNA variant expression and suggest that selective post-transcriptional processing plays a critical role in shaping the functional snRNA repertoire and its contribution to splicing regulation.

Journal Article↗
PubMed1992

Whole mount in situ detection of low abundance transcripts of the myogenic factor qmf1 and myosin heavy chain protein in quail embryos.

We describe a whole mount in situ hybridization procedure to detect and localize low abundance transcripts (qmf1) and myosin heavy chain proteins in quail embryos. The critical factor in transcript detection was the extent of proteinase K digestion. Optimal digestion increased probe accessibility and reduced background. The use of digoxigenin-labeled probes and immunological detection with anti-digoxigenin-alkaline phosphatase conjugated antibody allowed signal development in 6-10 h, unlike the 7 days required using 35S-labeled probes. Myosin heavy chain proteins were detected using immunofluorescence and confocal laser scanning microscopy to allow precise localization of signal within developing embryos.

Animals↗
PubMed1992

Changes in IGF-I and -II, IGF binding protein, and IGF receptor transcript abundance after uterine artery ligation.

Altered IGF activity may be one mechanism involved in the pathogenesis of intrauterine growth retardation (IUGR). We assessed the expression of IGF, IGF binding protein (IGFBP), and IGF receptor transcripts in liver, carcass, and placenta of fetal rats with IUGR resulting from unilateral uterine artery ligation. We found that uterine artery ligation on d 17 of gestation resulted in reduced body weight, liver weight, and placental weight on d 20 in the fetuses from the ligated uterine horn (UA-lig) compared with those from the opposite, nonligated uterine horn (UA-nonlig) and those from dams with no surgery or anesthesia. As assessed by solution hybridization, UA-lig fetuses exhibited significantly higher hepatic IGFBP-1, IGFBP-2, and IGF-II transcript abundance than UA-nonlig controls (increased 110, 50, and 31%, respectively). The only major difference among groups in carcass and placenta mRNA abundance was a 44% decrease in placental IGF-II expression in UA-lig pups compared with pups from dams that had had no surgery or anesthesia. Serum IGFBP, analyzed by ligand blot, showed a 2.4-fold increase in the doublet IGFBP-1/-2 band in UA-lig fetuses. Serum immunoreactive IGFBP-2 was unchanged among the groups, indicating that IGFBP-1 accounted for the increase in doublet intensity. Our results suggest that increased serum IGFBP-1 concentrations may decrease IGF activity in serum and thus inhibit IGF-stimulated cell proliferation or, by crossing the endothelial border, inhibit the activity of locally produced IGF. Decreased IGF-II expression in placenta also may contribute to decreased placental growth and, in turn, to IUGR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2026

Translating single-cell RNA sequencing into monocyte direct leukocyte subpopulation-transcript abundance assay ratio-based biomarkers (IFI27/PSAP or IFI27/CTSS) for clinical detection of viral infection.

A rapid method for triaging febrile patients by aetiology (e.g., viral or bacterial infection) using gene expression in peripheral blood (PB) is an intensively researched area. However, gene expression in blood represents a composite sum of gene expression of all the component cell types present in the sample. As a result, numerous genes are measured in most proposed signatures. Herein, we propose a simple ratio-based biomarker (RBB) called direct leukocyte subpopulation-transcript abundance assay (DIRECT LS-TA) that recapitulates gene expressions of a single cell type in PB (i.e., monocytes). Based on single-cell RNA sequencing (scRNAseq) data and bulk expression data, IFI27 and SIGLEC1 are found as interferon-stimulated genes (ISGs) predominantly expressed by monocytes. The DIRECT LS-TA method can use a simple ratio of two genes measured in PB as an RBB to represent the target gene expression in monocytes without the need for monocyte purification. Both scRNAseq and bulk RNA sequencing datasets were used to evaluate the correlation between ISG expression in monocytes and PB, with a particular focus on monocyte expression of IFI27. An iceberg plot of bulk transcriptome data was used to identify genes that were predominantly expressed by monocytes in PB. DIRECT LS-TA RBBs of the three genes (IFI27, IFI44L and SIGLEC1) were evaluated by group-wise comparison, receiver operating characteristic and meta-analysis. In addition, the conventional interferon (IFN) score was evaluated for comparison of diagnostic performance. In viral infection datasets, DIRECT LS-TA of IFI27 (IFI27/PSAP or IFI27/CTSS) was most intensely activated (p value by t test <1e-9) and had the best area under the curve (0.94) among the three potential monocyte ISGs analysed. DIRECT LS-TA SIGLEC1 was also another monocyte biomarker but showed a lower activation (p<9e-5). IFI27/PSAP showed better diagnostic performance than the conventional IFN score. On the other hand, IFI44L was not a predominant monocyte expression gene. DIRECT LS-TA of IFI27 (IFI27/PSAP or IFI27/CTSS) measured in PB was the best biomarker of viral infection and IFN activation among ISGs predominantly expressed by monocytes. It performed even better than the conventional IFN score which required quantification of eight genes. The results suggest that DIRECT LS-TA of IFI27 is a monocyte-informative biomarker which is easy to determine in PB without the need for cell sorting.

Humans↗
PubMed1987

Cell cycle regulated synthesis of an abundant transcript for human chromosomal protein HMG-17.

The abundance and cell cycle dependent expression of the mRNA for human nonhistone protein HMG-17 were studied in synchronized HeLa cells. Slot blot analysis indicates that the HMG-17 mRNA is a very abundant message, significantly more so than histone or actin mRNA. RNA prepared from tissue culture cells contains higher amounts of HMG-17 transcripts than RNA prepared from liver suggesting a correlation between the rate of cell division and HMG-17 mRNA levels. HMG-17 mRNA is present in the cells throughout the cell cycle however there is a significant increase in the mRNA levels late in S phase suggesting that the protein is deposited on chromatin after nucleosome assembly. Synthesis of the HMG-17 transcript is not coupled to DNA replication suggesting that the cell cycle related expression during late S phase is regulated in a different manner from that of the nucleosomal histones.

Cell Cycle↗
PubMed2026

Protocol to improve isoform-level quantification of low-abundance transcripts via STALARD pre-amplification.

STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 &#x3bc;g input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.

Gene Expression↗
PubMed1973

RNA synthesis in cells infected with herpes simplex virus. VII. Control of transcription and of transcript abundancies of unique and common sequences of herpes simplex virus 1 and 2.

Analysis of the kinetics of hybridization in liquid of labeled herpes simplex virus (HSV) 1 and 2 DNAs with excess unlabeled RNA extracted at 2 (early) and 8 (late) h postinfection revealed the following. (i) The RNA transcripts present in the HSV-1-infected cells at 2 and 8 h postinfection are complementary to 44 and 48% of HSV-1 DNA. The RNA transcripts present in the HSV-2-infected cells at 2 and 8 h postinfection are complementary to 21 and 50% of HSV-2 DNA. (ii) The transcripts present in 2-h HSV-1- or HSV-2-infected cells treated with cycloheximide are complementary to 44 and 45% of the respective DNAs. (iii) The RNA transcripts present in the HSV-1-infected cells at 2 h postinfection and in HSV-2-infected cells at 8 h postinfection form 2 classes, abundant and scarce, differing in molar concentrations. The RNA transcripts present in the HSV-2-infected cells at 2 h postinfection form only one abundance class. (iv) The transcripts present in the HSV-1-infected cells at 8 h postinfection are complementary to 24% of HSV-2 DNA and therefore 50% of the transcribed HSV-1 sequences are shared by the two viruses. Of the RNA sequences complementary to HSV-2 DNA, 13% arise from HSV-1 templates specifying abundant RNA and 11% arise from HSV-1 templates specifying scarce RNA. Thus, the DNA sequences shared in common by HSV-1 and HSV-2 DNAs constitute 71% of the HSV-1 templates specifying abundant RNA and 39% of sequences specifying scarce RNA.

Base Sequence↗
PubMed1990

A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts.

A simplified polymerase chain reaction (PCR) technique for the detection, semiquantitation and cloning of low-abundance RNAs is described. This assay involves first-strand cDNA synthesis by reverse transcription of mRNA with specific oligonucleotide primers, followed by second-strand synthesis and PCR amplification, in the same tube with only the addition of TaqI DNA polymerase. The assay is sufficiently sensitive to detect target RNA from as little as 1 ng of total RNA. The beta-actin transcripts may also be simultaneously reverse transcribed, amplified and used as an internal standard to determine relative expression of specific RNAs. Using this simple technique, expression of the multidrug resistance (mdr 1) gene can easily be detected in human breast tumors. The technique is also applicable for the cloning of rare transcripts.

Actins↗
PubMed1992

Sugarbeet minicircular mitochondrial DNAs: high-resolution transcript mapping, transcript abundance and copy number determination.

Three minicircular mitochondrial DNAs have been studied to address several aspects of transcription in sugarbeet mitochondria. High-resolution transcript mapping experiments have shown that sequences at the 5' termini of minicircle transcripts are highly homologous and resemble sequences at the 5' termini of sugarbeet mainband mitochondrial genes (atpA, atp6). In addition, they show homology to transcript termini of mitochondrial genes from other dicotyledonous plants, suggesting they may function as promoter sequences. Conserved sequences, which most probably act as RNA processing signals, were also identified at the 3' termini of minicircle transcripts. An oligonucleotide probe to a 14 base conserved sequence was used to determine the relative copy numbers of the three minicircle components in male-fertile mitochondria. Copy numbers were roughly equivalent, suggesting minicircles are replicated and/or transmitted with nearly equal efficiency, at least in sugarbeet taproots. Mc.a and Mc.c transcript levels are equivalent, consistent with their template copy number, however; Mc.d transcript levels were significantly lower than expected, implicating additional factors such as promoter strength and/or transcript stability in determining transcript levels in sugarbeet mitochondria, as recently demonstrated in maize.

Base Sequence↗
PubMed1988

A herpes simplex virus transcript abundant in latently infected neurons is dispensable for establishment of the latent state.

We have previously reported that a novel herpes simplex virus RNA transcript partially overlapping the gene encoding ICPO and expressed from the opposite DNA strand is abundant in sensory neurons of mice harboring a latent infection [J.G. Stevens, E.K. Wagner, G.B. Devi-Rao, M.L. Cook, and L.T. Feldman, Science 235, 1056-1059 (1987)]. This finding suggested that this transcript might be involved in establishment, maintenance, or reactivation of latent virus. To determine the function of this latency-associated transcript (LAT), we have examined the latency characteristics of a deletion mutant which is unable to express the LAT gene. Although no viral transcripts could be found in the lumbosacral ganglia of mice surviving rear footpad infection with this deletion virus, a latent infection had been established since infectious virus could be induced and detected after explanation and cocultivation of ganglia with permissive cells in culture. These results indicate that HSV-1 LAT expression is not an absolute requirement for establishment of the latent state.

Animals↗
PubMed2026

Abundance-transcription decoupling reveals functional partitioning in bioelectrochemical denitrification biofilms.

Bioelectrochemical denitrification (BED) is often attributed to electroactive microorganisms that access electrode-derived electrons, yet the relative functional contribution of electroactive taxa and denitrifying populations within complex BED biofilms remain unclear. Here, we integrated reactor measurements with genome-resolved metagenomics and metatranscriptomics to examine microbial community structure, functional potential, and gene transcription across contrasting BED operational regimes differing in dissolved oxygen (DO), hydraulic retention time (HRT)/loading, and poised potential. Nitrate removal exceeded 90% across all tested conditions, but nitrogen intermediate accumulation, current generation, and theoretical electron balance differed substantially. Electroactive taxa such as Geobacter dominated (>80% abundance) under longer HRT and stronger poised potential, but contributed minimally to the transcription of canonical denitrification genes. Weaker cathodic potential enriched transcriptionally active denitrifying taxa such as Stutzerimonas, Acidovorax, and MR-S7, while oxygen exposure induced redox-stress responses and reshaped nitrogen metabolism beyond being a competing electron acceptor. Together, these results reveal a decoupling between taxonomic abundance, genomic functional potential, and transcriptional contribution in BED biofilms, indicating that nitrate-removal performance cannot be inferred from current generation or electroactive-taxon abundance alone.

Bioelectrochemical system↗
PubMed1988

Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts.

Transfection of an activated rat oncogene into NIH3T3 fibroblasts leads to transformation and induction of a metastatic phenotype. To identify genes whose activation might mediate these processes, we used a differential screening strategy. A 1.5-kb transcript is induced fiftyfold, constitutes 1% of ras transformed cell messenger RNA (mRNA) and is the most abundantly induced message in these cells. Our sequence data shows that it encodes murine cathepsin L, a potent collagenolytic and elastinolytic lysosomal enzyme. The murine clone was used to isolate human cathepsin L complementary DNA (cDNA) clones. The complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L are presented and compared to other papain family cysteine proteinases. Northern analysis shows that both human and murine cathepsin L probes hybridize to a 1.5-kb transcript in several tissues, but also to a 4-kb transcript in human kidney. These clones will facilitate studies of the structure, expression, and function of cathepsin L, including its unexpected upregulation in transformation.

Amino Acid Sequence↗
PubMed1990

Measurement of histone mRNA transcript abundance in Xenopus oocytes by a quantitative primer extension assay.

A quantitative primer extension method was used to measure the mass of histone gene transcripts in mature oocytes of the amphibian Xenopus laevis. The procedure, using a large excess of gene-specific oligonucleotide primer and continuous incorporation of a radiolabeled deoxynucleoside triphosphate precursor, is more sensitive and quantitative than primer extension assays employing end-labeled primers. It was determined that there are stoichiometric amounts, approximately 2 X 10(8) copies, of mRNA for each of the five major histone gene classes in mature Xenopus oocytes. These observations are consistent with a model whereby transcription of these genes is coordinately regulated in a cell cycle-independent manner during amphibian oogenesis.

Animals↗
PubMed1991

Isolation and characterization of cDNA clones for NADP-malic enzyme from leaves of Flaveria: transcript abundance distinguishes C3, C3-C4 and C4 photosynthetic types.

To study the control of enhanced synthesis of enzymes associated with C4 photosynthesis relative to non-C4 plants, we investigated the expression of NADP-malic enzyme (NADP-ME) in different photosynthetic types of Flaveria. Complementary DNA clones encoding NADP-ME were constructed using poly(A)+ RNA from leaves of Flaveria trinervia (C4) and F. linearis (C3-C4) and identified by homology to a cDNA clone (500 bp) encoding NADP-ME from maize (Zea mays L. [39]). The sequence of one clone from each species was determined. The Flaveria clones were 90% homologous over a 564 nucleotide region encoding the carboxy terminal end of the derived polypeptide; sequence similarity to the maize transcript in this region was 71%. Both Flaveria clones detected a 2/3 kb transcript by hybridization to poly(A)+ RNA from expanding leaves of F. trinervia, F. linearis and F. pringlei (C3). The level of transcripts paralleled previously observed NADP-ME activity and abundance differences determined in these species, suggesting that control of the expression of NADP-ME in different photosynthetic types is predominantly at the transcriptional/post-transcriptional level. Southern analysis of genomic DNAs from F. trinervia, F. linearis and F. pringlei indicated a low copy number for this gene in all three species.

Amino Acid Sequence↗
PubMed1991

Glycerol 3-phosphate dehydrogenase gene expression in cultured 3T3-L1 adipocytes: regulation by insulin, dexamethasone and dibutyryl cAMP at the level of mRNA abundance, transcription and mRNA stability.

In fully differentiated 3T3-L1 adipocytes, glycerol 3-phosphate dehydrogenase (G3PDH:Sn-glycerol 3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) is subject to regulation by hormones and dibutyryl cAMP. An increase by insulin (4-fold) and decrease by dexamethasone (by 50%) and dibutyryl cAMP (by 70%) was observed for G3PDH mRNA abundance as analyzed by Northern blot hybridization. In addition, incubation of adipocytes with dibutyryl cAMP resulted in 3-fold increase in G3PDH gene transcription as measured by nuclear transcript elongation assay. The effects of these modulators on the control of G3PDH mRNA stability were also investigated. The G3PDH mRNA has a half-life of about 125 min. Dibutyryl cAMP caused an increase in G3PDH mRNA degradation by greater than 2-fold (t1/2 = 55 min) whereas insulin had an opposite effect (t1/2 = 240 min) and dexamethasone was without any effect on G3PDH mRNA stability. Taken together, our results directly demonstrate that in fully differentiated 3T3-L1 adipocytes the regulation of G3PDH gene expression by dibutyryl cAMP and insulin is exerted by alterations in transcription as well as mRNA stability.

3T3 Cells↗