BLOTTO-MF, an inexpensive and reliable hybridization solution in northern blot analysis using complementary RNA probes.
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Blotting, Northern↗
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Results for “RNA, Complementary”
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2'-5'-linked oligonucleotides form stable complexes with complementary RNA and DNA.
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Base Sequence↗
Modulation of eukaryotic gene expression by complementary RNA or DNA sequences.
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Animals↗
Interference by a non-defective variant of influenza A virus is due to enhanced RNA synthesis and assembly.
Mouse-adapted influenza A virus, FM-MA, interferes with the replication of wild-type strains on co-infection. The interference phenotype was previously mapped to FM-MA segment 2 encoding a mutant PB1 protein, the catalytic component of the RNA polymerase complex. To identify the point at which FM-MA interferes with wild-type A/HK/1/68 (HK), the relative levels of transcription and genome replication of the PB1, NP and M1 genes were determined for FM-MA and HK viruses in co-infected cells using RT-PCR. All stages of HK macromolecular synthesis (primary and secondary transcription, genomic RNA, complementary RNA and protein synthesis) were suppressed relative to FM-MA. Infection with HK virus alone resulted in the accumulation of similar or greater amounts of RNA at late times post-infection relative to FM-MA thus indicating that the presence of FM-MA specifically compromised HK transcription and replication in co-infected cells. However early in infection FM-MA was ten times more active in mRNA transcription than HK or its parental strain FM. FM-MA's ability to interfere was primarily due to an increased capacity for primary transcription. FM-MA genomes were also selectively assembled into progeny virus from cells co-infected with HK and FM-MA, a step which was distinct from the capacity for enhanced RNA synthesis. This suggests that interference of HK growth by FM-MA in mixed infections results from two distinct events: a preferential synthesis of FM-MA-specific macromolecules which is then augmented by a preferential assembly of FM-MA genomes.
Animals↗
Physical properties of the complementary T4 RNA.
The complementary transcribed T4 RNA after self-annealing and RNAase treatment was isolated by gel chromatography and then used for further studies. From salt-dependent RNAase resistance and melting studies it is evident that this RNA represents a genuine double-stranded structure. The base content of the isolated double-stranded RNA was found to be the same as total T4 mRNA. Sucrose gradient analysis and hydroxyapatite chromatography of T4 RNA, annealed early and late RNA, and of the isolated double-stranded RNA, gave results indicating that the complementary RNA is part of a RNA molecule and further that the size of the complementary regions are independent of the RNA molecules. Partial digestion of pulse-labelled late RNA with phosphodiesterase I prior to annealing with unlabelled early RNA, showed that the complementary regions on the mRNA are not located to the 5'- or 3'-end but randomly distributed along the T4 RNA molecules.
Escherichia coli↗
Similarity between the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA.
An interesting pattern in the genetic code was recently observed: Codons for hydrophilic and hydrophobic amino acids on one strand of nucleic acid are complemented by codons for hydrophobic and hydrophilic amino acids on the other strand, respectively. The average tendency of codons for "uncharged" (slightly hydrophilic) amino acids is to be complemented by codons for "uncharged" (slightly hydrophilic) amino acids. We have postulated that this pattern can result in the binding of peptides that are encoded by complementary RNA strands and we have presented supporting evidence. In this report we demonstrate the specific and high-affinity binding of naturally occurring peptides [corticotropin (ACTH) and gamma-endorphin] to synthetically derived counterparts that were specified by RNA sequences complementary to the mRNA for ACTH and gamma-endorphin, respectively. That this binding might result from one peptide being an "internal image" of the other was strongly suggested by the observation that antibody to the peptide that was encoded by the complementary RNA for ACTH recognized the adrenal cell ACTH receptor. Based on these findings, a theory on the evolution of peptides and their receptors is suggested.
Adrenocorticotropic Hormone↗
Effect of mic gene structure on repressor activity in the OmpA system.
It has been previously established that micRNA (mRNA-interfering complementary RNA) complementary to an individual mRNA specifically represses the expression of the target mRNA. We have constructed several plasmids which produce micRNAs which are complementary to different regions of the ompA mRNA. The repressor activity of these different micRNAs has been compared to determine the role of mic gene structure in effective micRNA function. The results indicate that micRNAs complementary to regions of the ompA mRNA likely to encounter ribosomes have the highest repressor activities. A clear effect of mic gene dosage was also observed. This was demonstrated using both identical and different mic(ompA) genes.
Bacterial Outer Membrane Proteins↗
Expression of the Drosophila 70,000 Dalton heat shock protein is translationally controlled in yeast.
Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.
Animals↗
Production of large amounts of 35S RNA and complementary DNA from avian RNA tumor viruses.
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Animals↗
Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.
UNLABELLED: Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 102 copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome. IMPORTANCE: The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for guiding strand-resolved diagnostics and antiviral evaluation.
CRISPR-Cas12a↗
Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b.
bI1 RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II-MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand-specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon-intron fragment and labeling it by the same procedure; mRNA and pre-mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm + 20 degrees C). Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self-complementarity of the RNA. A pre-mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe. The amount of the non-sense bI1 RNA strand is very low, in cells from two wild-type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression. bI1 RNA is resistant to RNase. Very little self-complementarity is seen by computer analysis of the sequence. Purified bI1 RNA is seen by electron microscopy under non-denaturing conditions as a mixture of double-stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single-stranded circular and linear molecules is seen as reported previously (Cell, 19, 321-329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules. The ratio of non-sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
Base Sequence↗
RNA-catalysed synthesis of complementary-strand RNA.
The Tetrahymena ribozyme can splice together multiple oligonucleotides aligned on a template strand to yield a fully complementary product strand. This reaction demonstrates the feasibility of RNA-catalysed RNA replications.
Animals↗
A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis.
Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.
Animals↗
Complementary nuclear RNA's of murine sarcoma-leukemia virus complex in transformed cells.
The high molecular weight RNA (S(20,w) = 69S) of murine sarcomaleukemia virus (MSV-MLV) complex dissociated into 37S subunits when heated to 95 degrees C for 2 minutes. Specific annealing tests revealed that the viral RNA had complementarity with two heterogeneous RNA species from the nuclei of cells (78 A1) transformed by and chronically infected with this virus complex. The two nuclear RNA species that hybridized with the viral RNA had sedimentation constants ranging from 31-36S and 18-22S, respectively. There was no specific annealing between viral RNA and the cytoplasmic RNA of the 78 A1 cells. The detection of the complementary RNA strands of MSV-MLV suggests that this oncogenic virus complex may replicate in a fashion similar to that of the non-oncogenic RNA viruses, but may differ in that the complementary strands are found in the nucleus rather than in the cytoplasm of the transformed cells.
Animals↗
Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads.
The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific sequence on the rRNA nascent transcript was labeled in a pattern consistent with its location; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was systematically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simultaneously maintaining sufficient levels of specific labeling.
Animals↗
Localization of urokinase-type plasminogen activator mRNA in the adult mouse brain.
Urokinase-type plasminogen activator (uPA) is an inducible serine protease, secreted by a variety of cell types, that functions in fibrinolysis and has been implicated also in events such as cell migration and tissue remodeling and repair. To explore the role of uPA in the adult brain we have now screened the whole mouse brain for cells expressing the uPA gene through in situ hybridization using 35S-complementary RNA. uPA mRNA was visualized predominantly in three regions: (1) the subicular complex, (2) the entorhinal cortex, (3) the parietal cortex, where the signal was somewhat lower and confined to layers IV and VI. Weaker signals were seen in the basolateral nucleus of the amygdala and in the anterodorsal thalamic nucleus, and also in the hilus of the dentate gyrus where labeling was slightly over background. Cells exhibiting uPA mRNA signaling were large neurons according to morphological criteria. These results support the view of uPA being involved in neuronal functions of the adult brain, specifically in the hippocampal formation and the parietal cortex.
Animals↗
A syntaxin-related protein controls acetylcholine release by different mechanisms in Aplysia.
Polyclonal antibodies raised against rat syntaxin-1B and an affinity-purified fraction have been used to study the functional role of this protein in transmitter release from Aplysia neurons. In a ganglionic protein extract, this fraction recognized a 37,000 molecular weight protein which therefore might be the Aplysia homologue of rat brain syntaxin-1B. Immunoglobulins were injected in the presynaptic cell of an identified cholinergic synapse of the buccal ganglion of Aplysia californica. This treatment decreased the postsynaptic response due to a reduction of the number of quanta released in relation to a decline of presynaptic Ca2+ current. When antibodies were applied extracellularly, transmitter release also decreased. In contrast to intracellular injection, this reduction was not accompanied by a decrease of the Ca2+ current but by an increase of presynaptic outward current. When injected in the presynaptic neuron, syntaxin complementary RNA also depressed Ca2+ current and transmission. This work provides evidence that Aplysia neurons express a syntaxin-like protein which is involved in the control of the presynaptic Ca2+ influx triggering acetylcholine release from terminals. This protein appears to have an extracellular segment which might interact with outward current.
Acetylcholine↗
32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid.
Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5-10 pg of viroid obtained with 32P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.
Biotin↗