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Results for “In-vitro transcription”

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At least 19 recordsLinked to original sources

PubMed2026

Molecular characterization of vitellogenin and its receptor with CRISPR-based sgRNA validation in the legume pod borer, Maruca vitrata (Geyer) (Lepidoptera: Crambidae).

Maruca vitrata, the legume pod borer, causes yield losses of up to 80% in grain legumes. Increasing insecticide resistance and environmental concerns necessitate sustainable pest management alternatives. In the present study, the complete vitellogenin (Vg) coding sequence (CDS), a key reproductive gene involved in oogenesis and embryonic development, was cloned and molecularly characterised from M. vitrata. The assembled Vg CDS (∼5.3 kb) shared 99.04% sequence identity with the reported M. vitrata Vg sequence (MG799570.1). Phylogenetic analysis demonstrated close evolutionary association with related Lepidopteran species, while protein domain analysis identified three conserved domains, namely LPD_N, DUF1943, and VWD. Among these, the single exon-encoded LPD_N domain was selected as the target region for CRISPR/Cas9-mediated editing. Homology models of Vg and vitellogenin receptor (VgR) (Global Model Quality Estimation (GMQE): 0.58 and 0.51) showed a favourable interaction by protein-protein docking (score: -295.66). Three single-guide RNAs (sgRNAs) were designed, synthesised through in-vitro transcription, and evaluated using in vitro cleavage assays. sgRNA1 targeting the LPD_N domain and sgRNA2 targeting the signal peptide region exhibited efficient site-specific cleavage activity, whereas sgRNA3 failed to induce cleavage because of an unfavourable secondary structure that likely impaired Cas9-sgRNA complex formation. Overall, this study provides the first CRISPR-oriented functional characterisation and sgRNA validation of the M. vitrata Vg gene, together with structural characterisation of VgR and Vg-VgR interaction analysis, providing preliminary molecular resources for future CRISPR/Cas9 studies and supporting future embryo microinjection and heritable genome editing for sustainable management of M. vitrata.

CRISPR/Cas9↗

PubMed1992

Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.

Base Sequence↗

PubMed1990

Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-beta subunit: influence of mutation on hCG production.

The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the alpha and beta subunits. The beta subunit is specific for each hormone and is responsible for the biological specificity, but the beta subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different beta subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-beta subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the beta subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.

Amino Acid Sequence↗

PubMed1992

RNA-protein interactions in the 5' untranslated region of preproinsulin mRNA.

A comparison between species of the 5' untranslated region of preproinsulin mRNA revealed conserved sequences associated with a potential stem-loop structure. The present study was undertaken to determine whether specific protein interactions exist with mRNA sequences involved in the formation or stabilization of this structure in the 5' untranslated region. 32P-Labelled RNA probes corresponding to sequences from this region were synthesized by an in-vitro transcription reaction and used in electrophoretic mobility shift and u.v.-crosslinking studies with cytoplasmic protein extracts from a number of cell lines. Specific protein-RNA interactions were mapped to a sequence located between nucleotides -21 and -50 upstream of the AUG start codon. A number of proteins of molecular mass 25 kDa, 40kDa, 46kDa, 58kDa, 69kDa, 97kDa, 110kDa and 160kDa were specifically crosslinked to this sequence. The observed specific protein-RNA interactions in the 5' untranslated region may affect the activity of preproinsulin mRNA.

Animals↗

PubMed1990

In-vitro synthesis of functional varicella zoster and herpes simplex viral thymidine kinase.

The varicella zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) genes were cloned into the transcription vector pGEM4. In-vitro translation (ivt) of RNA transcribed from these genes showed prominent expression of functional TK proteins with the expected molecular weights of 36 kD for VZV and 43, 39, and 38 kD for HSV-1. The TK proteins were recognized by rabbit anti-VZV and anti-HSV-1 antibodies, respectively. Analysis of the ivt products by thin-layer chromatography revealed the conversion of thymidine to its phosphorylated forms (TMP, TDP, and TTP) by both the VZV and HSV-1 TK genes. The estimated specific activities of the in-vitro translated VZV and HSV-1 TKs were comparable. VZV TK templates were linearized at internal restriction sites and RNAs transcribed from these templates directed the synthesis of polypeptides with sizes consistent with the colinearity of the VZV TK gene. Deletion of 107 amino acids at the carboxy terminus of the VZV TK gene abolished the in-vitro TK activity. In addition, immunoprecipitation of truncated proteins synthesized in vitro suggested the possible involvement of the region between amino acid residues 101 and 168 from the amino terminus of the VZV TK molecule in the formation of structures necessary for antigenicity.

Amino Acid Sequence↗

PubMed2027

Production of Viral Particles from a Chikungunya Virus Infectious Clone.

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.

Chikungunya virus↗

PubMed2025

Intrathecally expanded GZMK+/GZMH+ CD8 T cells targeting EBV antigens may reduce severity of Multiple Sclerosis.

Combining cerebrospinal fluid B cell receptor and T cell receptor repertoire analysis with transcriptional/ flow cytometry cellular profiles in hundreds of deeply-phenotyped people with Multiple Sclerosis (pwMS) and controls, we identified intrathecal expansion of anti-viral, cytotoxic, granzymes H/K (GZMH+/GZMK+) double positive (DP) CD8+ T cells that recognize EBV epitopes in pwMS. DP CD8+ T cells are activated and expanded by, and kill autologous, EBV-infected CSF B cell lines in-vitro. Correlations of surrogate transcriptional profiles with clinical and imaging outcomes infer a beneficial role for EBV-targeting DP CD8+ T cells, as untreated pwMS with proportionally higher DP CD8+ T cells to intrathecal B cells accumulate neurological disability slower. MS therapies also increase ratios of beneficial CD8+ T cell responses to intrathecal B cells, consistent with their ability to inhibit disability progression. This study provides indirect evidence that intrathecal EBV infection participates in disability accumulation in pwMS.

Journal Article↗

PubMed1990

In-vitro translation of cucumoviral satellites. III. Translational efficiencies of cucumber mosaic virus-associated RNA 5 sequence variants can be related to the predicted secondary structures of their first 55 nucleotides.

The cucumber mosaic virus (CMV) satellites D- and S-CARNA 5 (CARNA 5 = Cucumber mosaic virus-Associated RNA 5), their full-length cDNA clone transcripts, and DNA clone transcripts of their open reading frames (ORFs) were used as mRNAs in the wheat-germ in-vitro translation system. Natural D-CARNA 5 yielded an anomalously large polypeptide, while transcripts made from cDNA clones of D-CARNA 5 or its first ORF had no mRNA activity. Transcripts made from the second major ORF in D-CARNA 5 yielded a smaller product, consistent with its size. Natural S-CARNA 5 and its cDNA clone transcripts both yielded the two polypeptides previously reported, while transcripts of its only major ORF yielded exclusively the smaller of the two products. The potential for an alternate initiation codon, 36 nucleotides upstream, being the source of the larger of the two polypeptides was tested. The differences in the translational properties of D- and S-CARNA 5 were related to the predicted secondary structures of the first 55 nucleotides in these CARNA 5 sequence variants. The calculated free energies of the predicted hairpins correlated inversely with their in-vitro translational activities.

Base Sequence↗

PubMed1992

A reverse polymerase chain reaction method for detection of human cytomegalovirus late transcripts in cells infected in vitro.

A rapid and simple application of the polymerase chain reaction is described for the detection of human cytomegalovirus (HCMV) mRNAs in cells infected in-vitro. The method was first used to study the transcription of two HCMV genes, and confirm the link between the transcription of one, encoding for the major capsid protein, and viral replication. The oligonucleotides chosen in this region were specific for HCMV genome and sensitivity experiments showed that a single infected cell in 5 x 10(5) can be detected. Detection of this transcript should be suitable for diagnostic purposes, permitting the distinction between latency and active infection.

Base Sequence↗

PubMed1992

Increased steady-state levels of several mitochondrial and nuclear gene transcripts in rat hepatoma with a low content of mitochondria.

Cells from a rapidly growing rat Zajdela hepatoma were shown to contain (on a protein basis) five-times less mitochondria than hepatocytes from resting or regenerating rat liver. Transcripts of four nuclear genes for representative mitochondrial membrane proteins (beta-F1 subunit and N,N'-dicyclohexyl-carbodiimide-binding protein of ATP synthase, subunit IV of cytochrome oxidase and ADP/ATP translocase) were present in 2-4 times higher amounts in the poly(A)-rich RNA of the hepatoma than in the corresponding RNA fraction from resting or regenerating rat liver. The liver and hepatoma transcripts for the beta-F1 subunit were translated in an in-vitro system with equal efficiency. Pulse-chase labeling of isolated Zajdela hepatoma cells and hepatocytes from resting and regenerating liver revealed a relative excess of the newly synthesized beta-F1 subunit in the tumor cells. The half-life of the beta-F1 subunit was significantly shorter in the hepatoma cells than in hepatocytes from resting and regenerating liver. The contents of transcripts of three mitochondrial genes examined (cytochrome oxidase subunits I and II and NADH-ubiquinone reductase subunit 2) in Zajdela hepatoma mitochondria were about five-times higher than in the mitochondria of the resting cells and 3-4 times higher than in the organelles of the regenerating organ. The results indicate that events other than transcription (most likely post-translational) may be responsible for the reduced content of mitochondria in tumor cells.

Animals↗

PubMed1992

A novel calcium-binding protein from Euglena gracilis. Characterisation of a cDNA encoding a 74-kDa acidic-repeat protein targeted across the endoplasmic reticulum.

We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73,542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23-amino-acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF-hand type calcium-binding motif. Evidence is presented that a fusion protein of this novel protein with beta-galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In-vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120-125 kDa. The protein is processed into a smaller, protease-protected form (110-120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium-binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic-repeat protein.

Amino Acid Sequence↗

PubMed1992

Expression of functional growth hormone receptors in human granulosa cells.

Both clinical and experimental evidence suggest that growth hormone may be of importance for ovarian function. The present study investigated whether growth hormone receptors are expressed in human granulosa cells. Granulosa cells were isolated either from natural cycles or from stimulated cycles in the course of in-vitro fertilization. Total RNA hybridized with a 32P-labelled rat growth hormone receptor cRNA probe revealed one major transcript with an estimated size of 4.5 kb and one minor transcript with an estimated size of 1.3 kb. Biotinylated growth hormone was used to analyse growth hormone binding. Competitive growth hormone binding was detected in freshly isolated granulosa cells, as well as in cultured cells. Growth hormone augmented basal and/or follicle stimulating hormone-stimulated steroidogenesis in granulosa cells obtained from patients with natural cycles, but the response to growth hormone stimulation showed considerable variation. We conclude that functional growth hormone receptors are present in human granulosa cells and that growth hormone, therefore, may have an important role in ovarian function.

Analysis of Variance↗

PubMed1992

Alpha 5 integrin subunit expression changes during myogenesis.

Fibronectin and its cellular receptor, the alpha 5 beta 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the alpha 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the alpha 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the alpha 5 integrin subunit in muscle development.

Alternative Splicing↗

PubMed1990

A cis-acting sequence, located at -450 in the promoter of the human interferon-inducible gene 6-16, binds constitutively to a nuclear protein and decreases the expression of a reporter interferon-inducible promoter.

An interferon-inducible synthetic promoter was constructed with oligonucleotides which correspond to two regions of the promoter of the human interferon-inducible gene 6-16 that interact in-vitro with nuclear proteins. Firstly, we cloned a direct repeat sequence that was necessary for interferon regulation and that interacted with a 55 kilodalton nuclear protein. 5' to the direct repeat we introduced a 45 bp long oligonucleotide located at -450 in the native 6-16 promoter that interacted in-vitro with an 80 kilodalton nuclear protein. Expression after transfection of these plasmids showed that the direct repeat is necessary for interferon-inducible control and the -450 oligonucleotide by itself has no effect on transcriptional activity while in conjunction with the direct repeat it decreased the basal and interferon-inducible transcriptional activity of the reporter promoter in human cells.

Animals↗

PubMed2026

Integrated metabolomics, transcriptional, and physicochemical analysis reveals key metabolites and genes associated with somatic embryogenesis in Phyllostachys pubescens.

Phyllostachys pubescens (Moso bamboo) is a significant perennial crop species that provides valuable nutritional and industrial uses, as well as carbon sequestration. Due to its remarkable growth rate, bamboo offers an ideal system for studying organogenesis, particularly in monocots. Somatic embryogenesis (SE) serves as a useful technique for crop breeding and improvement. SE in moso bamboo (Phyllostachys pubescens) remains challenging due to limited knowledge of its transcriptional and metabolomic reprogramming. To address this, we optimized callus initiation (MS + 18.1 µM 2,4-D + 8.5 µM picloram), callus proliferation (MS + 12.5 µM 2,4-D + 8.5 µM picloram), and somatic embryogenesis (MS + 1.1 µM 2,4-D + 3.3 µM metatopolin), using nodal segments as explants. UHPLC-Q-TOF-MS-based metabolite profiling revealed distinct biochemical trajectories across developmental stages of P. pubescens. NEC (non-embryogenic callus) was enriched in flavonoids, alkaloids, and saponins, while in-vitro shoots showed flavonoids and glycosides enrichment, and ex-vitro shoots showed high accumulation of glycosides and terpenoids. In contrast, EC (embryogenic callus) showed elevated levels of fatty acid derivatives (α-ESA, 26-Methyl Nigranoate), phytoalexins (Wyerone acid), sesquiterpene (Alpha-santalal, Beta-guaiene), flavonoid glycosides, and plant hormones (Cis-Zeatin, Gibberellin A45), indicating a metabolically active state supporting somatic embryogenesis. Similarly, genes and transcription factors controlling cell differentiation and embryogenesis were upregulated during SE. This study provides a comprehensive resource to facilitate future genomic and genetic investigations aimed at deciphering the molecular basis of organogenesis and advancing research on somatic embryogenesis in bamboo.

Plant Somatic Embryogenesis Techniques↗

PubMed1992

Intraventricular administration of thyrotrophin-releasing hormone (TRH) suppresses prolactin secretion and synthesis: a possible involvement of dopamine release by TRH from rat hypothalamus.

The administration of thyrotrophin-releasing hormone (TRH) causes a variety of dopamine-related biological events. To understand the specific role of TRH on rat hypothalamic dopamine neurones, we examined the in-vivo effects of intraventricular (i.c.v.) infusion of TRH on the release and synthesis of prolactin in the rat pituitary gland and on the changes in binding of [3H]MeTRH and dopamine turnover rates in rat hypothalamus. We have also examined the in-vitro effects of TRH on the release of [3H]dopamine from dispersed tuberoinfundibular dopamine neurones. Female rats were treated with i.c.v. infusions of 1 mumol TRH/1 daily for 1, 3 and 7 days using Alzet osmotic pumps. Following 7 days of treatment the serum prolactin concentrations were significantly decreased. A reduction in hypothalamic TRH-binding sites (Bmax) was also apparent but the dissociation constant (Kd) was unaffected. Northern blot analysis of total RNA isolated from the pituitary glands of control animals using 32P-labelled prolactin cDNA as a probe indicated the presence of three species of prolactin gene transcripts of approximately 3.7, 2.0 and 1.0 kb in size, and these were decreased by TRH treatment. We examined the turnover rate of dopamine in the rat hypothalamus when TRH was administered i.c.v. for 7 days. There was a significant increase in 3,4-dihydroxyphenylacetic acid/dopamine ratio with TRH treatment. Moreover, exposure to TRH stimulated [3H]dopamine release from rat tuberoinfundibular neurones in a time- and dose-dependent manner. Dopamine receptor antagonists such as SCH23390 and (-)sulpiride, and other neuropeptides such as vasoactive intestinal peptide and oxytocin did not affect TRH-stimulated [3H]dopamine release.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

PubMed1975

[Properties of ribosomes of normal and tumor cells (author's transl)].

On the basis of results showing that the extent and direction of protein biosynthesis essential for cell growth and cell differentiation can be regulated, aside from the transcription level, also on the level of ribosomes (translation), the main results of various properties of ribosomes from normal and tumor tissues are described. The main established difference between the two tissues concerns the organization and localization of the ribosomes within the cell. In tumors, polysomes and inactive dimers and monomeric particles are encountered in considerable amounts freely in the cytosol, i.e. not attached to the membranes. Furthermore, the polysomal profile of tumor cells compared with the normal tissue is characterized by a shift towards fractions with lower sedimentation coefficients. Monomeric ribosomes were not found to differ in physical and chemical properties. In smaller oligonucleotide fragments of ribosomal RNA, differences in the frequency of certain dinucleotides appear likely. Hybridization-competition experiments with DNA do suggest, however, that the ribosomal RNA from tumors is being synthesized at the same cistrons as ribosomal RNA from normal tissues, thus proving its overall sequence homology. Electrophoretic and immunologic methods have not so far revealed any clear differences in the properties of ribosomal proteins from normal and tumor tissues. In in-vitro systems, ribosomes from various tumors show approximately the same incorporation rates for amino acids as ribosomes from normal tissues. The enhanced protein synthesis of the cells, on the basis of data obtained from in-vitro experiments with cell free systems, is to be attributed mainly to the increased content of translation factors in the cytosol.

Animals↗

PubMed1992

Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.

Binding Sites↗