Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA, Complementary”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources
PubMed2006

Additive regulation of GluR1 by stargazin and serum- and glucocorticoid-inducible kinase isoform SGK3.

The serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) and stargazin have both been shown to enhance the synaptic expression level of GluR1. The present study was performed to elucidate whether SGK3 and stargazin interact or are effective through different pathways in the regulation of GluR1. Proteins were expressed in Xenopus oocytes by injection of complementary RNA (cRNA) encoding GluR1, SGK isoforms, and/or stargazin. In oocytes expressing GluR1 6 days after cRNA injection, glutamate induced an inward current (IGlu), which was increased approximately fourfold following coexpression of SGK3. Coexpression of stargazin similarly enhanced IGlu. Coexpression of both SGK3 and stargazin stimulated the current by a factor of 15.5. Replacement of the serine by alanine at the only SGK consensus sequence (RXRXXS/T) in stargazin enhanced the efficacy of stargazin but did not prevent further stimulation of IGlu by additional coexpression of SGK3. Western blotting showed that stargazin accelerated membrane insertion of GluR1 protein leading to enhanced GluR1 plasma membrane protein abundance 2 days, but not 6 days, after cRNA injection, while SGK3 increased plasma membrane protein abundance 6 days after cRNA injection. In conclusion, SGK3 and stargazin regulate GluR1 independently, and thus, their effects on glutamate-induced currents are additive.

Animals↗
PubMed1996

Glass-funnel technique for the recording of membrane currents and intracellular perfusion of Xenopus oocytes.

In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e., extra- and intracellular, compartments. The technique is fairly simple to use, provides a much higher clamp speed compared to the double-microelectrode voltage-clamp technique, and allows effective control of the composition of the intracellular milieu. To elucidate the performance of the technique with respect to various membrane currents we present data relating to the recording of Ca-channel currents expressed in X. laevis oocytes by means of mRNA extracted from the rat cerebellum and heart, as well as currents induced by cRNA for the skeletal muscle micro1 Na+ channel and the dog heart NCX1 Na+-Ca2+ exchanger. Due to effective elimination of intra- and extracellular Cl- it became possible to measure not only Ba2+ but also Ca2+ current through the expressed Ca channels, and to record the activity of the Na+-Ca2+ exchanger following dialysis of the oocyte with high-Ca2+ intracellular solutions. Corresponding currents showed properties identical to those obtained with other techniques, suggesting the adequacy of the glass-funnel technique for critical analysis of membrane ionic currents in Xenopus oocytes.

Animals↗
PubMed2003

Classification of Dukes' B and C colorectal cancers using expression arrays.

PURPOSE: Colorectal cancer is one of the most common malignancies. Substaging of the cancer is of importance not only to prognosis but also to treatment. Classification of substages based on DNA microarray technology is currently the most promising approach. We therefore investigated if gene expression microarrays could be used to classify colorectal tumors. METHODS: We used the Affymetrix oligonucleotide arrays to analyze the expression of more than 5,000 genes in samples from the sigmoid and upper rectum of the left colon. Five samples were from normal mucosa and five samples from each of the Dukes' stages A, B, C, and D. Expression data were filtered based on either covariance or a selection of the most significantly varying genes between tumor stages. RESULTS: A nearest neighbor classifier was used to classify normal, and Dukes' B and C samples with less than 20% error, whereas Dukes' A and D could not be classified correctly. A number of interesting gene clusters showed a discriminating difference between Dukes' B and C samples. These included mitochondrial genes, stromal remodeling genes, and genes related to cell adhesion. CONCLUSION: Molecular classification based on gene expression of one of the most common malignancies, colorectal cancer, now seems to be within reach. The data indicates that it is possible at least to classify Dukes' B and C colorectal tumors with microarrays.

Adenocarcinoma↗
PubMed2000

Coordinate expression of NGF and alpha-smooth muscle actin mRNA and protein in cutaneous wound tissue of developing and adult rats.

Nerve growth factor (NGF) is synthesized in cutaneous wound tissue, and its higher levels in the neonate may contribute to more efficient wound healing. We used in situ hybridization and immunohistochemistry to define NGF mRNA and protein expression in intact skin and following excision wounding in neonatal and adult rats. To determine whether NGF is associated with wound contractile fibroblasts (myofibroblasts), we also examined expression of alpha-smooth muscle actin (alpha-SMA) mRNA and protein, established markers for these cells. In intact skin, NGF mRNA and protein were present in vascular and arrector pili smooth muscle, hair follicle sheath cells, keratinocytes, and hypodermal fibroblasts. Neonatal adipocytes and Schwann cells also expressed NGF mRNA and protein, while adult adipocytes and Schwann cells displayed only NGF-ir. Following wounding, NGF mRNA expression was exuberant in these cell types, and increased similarly at both ages and appeared de novo in skeletal muscle cells. Additionally, both NGF mRNA and protein were present in macrophages and myofibroblasts, and expression in myofibroblasts was significantly greater in neonates. Wound myofibroblasts also expressed alpha-SMA. Surprisingly, after wounding alpha-SMA mRNA and protein were present in essentially all cells in which NGF mRNA was detected. We conclude that NGF expression is enhanced in many cell types after wounding, but greater NGF synthesis in neonates appears to be due to a more robust myofibroblast response. In addition, cell types which demonstrated NGF mRNA also expressed alpha-SMA, and staining for both markers increased following wounding, suggesting synthesis of both proteins is regulated in a coordinated fashion.

Actins↗
PubMed2006

SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses.

Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.

Animals↗
PubMed2005

Dynamic histone acetylation of late embryonic genes during seed germination.

Histone acetylation is involved in the regulation of gene expression in plants and eukaryotes. Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from histones, which is associated with the repression of gene expression. To study the role of histone acetylation in the regulation of gene expression during seed germination, trichostatin A (TSA), a specific inhibitor of histone deacetylase, was used to treat imbibing Arabidopsis thaliana seeds. GeneChip arrays were used to show that TSA induces up-regulation of 45 genes and down-regulation of 27 genes during seed germination. Eight TSA-up-regulated genes were selected for further analysis - RAB18, RD29B, ATEM1, HSP70 and four late embryogenesis abundant protein genes (LEA). A gene expression time course shows that these eight genes are expressed at high levels in the dry seed and repressed upon seed imbibition at an exponential rate. In the presence of TSA, the onset of repression of the eight genes is not affected but the final level of repressed expression is elevated. Chromatin immunoprecipitation and HDAC assays show that there is a transient histone deacetylation event during seed germination at 1 day after imbibition, which serves as a key developmental signal that affects the repression of the eight genes.

Acetylation↗
PubMed2005

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.

Gene Expression Regulation, Plant↗
PubMed1987

Normalization of multiple RNA samples using an in vitro-synthesized external standard cRNA.

A method for the normalization of multiple RNA samples is described. This method exploits the recently developed technology which allows the synthesis of single-stranded, specific RNA molecules in vitro using either SP6 or T7 RNA polymerase to prepare an external standard cRNA. When this external standard cRNA is added to cell samples at the time of lysis, it becomes a stable, integral part of the RNA content of each sample, which can easily and reproducibly be detected and quantitated by either Northern blot or RNase protection. The feasibility of this approach to normalization has been tested in a mouse 3T3 cell model system. In multiple samples, the relative levels of this externally added standard transcript are shown to closely parallel the relative levels of an internal standard control transcript as well as the amount of RNA determined by spectrophotometric analysis. The data obtained demonstrate that an externally added, in vitro-synthesized transcript can serve as an accurate, universal means of normalizing multiple RNA samples, since it is not dependent on sample RNA concentration, species, cell or tissue type, or experimental manipulation.

Animals↗
PubMed1986

The 3'-noncoding region of the chick myosin light-chain gene hybridizes to a family of repetitive sequences in the slime mold Dictyostelium discoideum.

During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light-chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium.

Animals↗
PubMed1995

Gene expression of epimorphin in rat incisor ameloblasts.

Epimorphin has been recently identified as an important factor in the morphogenesis of epithelial cells. A cDNA encoding epimorphin from skin of newborn mice was cloned by the polymerase chain-reaction technique before the preparation of digoxigenin-labelled cRNA probes. In situ hybridization of longitudinal sections of rat incisors revealed a distinct pattern of expression of epimorphin mRNA in the ameloblast layer. Epimorphin mRNA was detected from the presecretory stage up to the beginning of the maturation stage of amelogenesis. With the identification of this expression by epithelial-derived cells, i.e. ameloblasts, it is thought likely that epimorphin is one of the factors that modulate the differentiation cascade of ameloblasts in the course of amelogenesis.

Ameloblasts↗
PubMed1976

Analysis of the human genome by complementary RNA/DNA gradient hybridization and relaxes cesium sulfate-silver ion density centrifugation.

3H-Labeled complementary RNA (cRNA) transcribed from total nuclear human DNA was hybridized to homologous DNA and the hybrids formed localized in CsC1 density gradients. Radioactive peaks indicative of cRNA/DNA hybrids identified ten density components. The experimental conditions used indicated that these components correspond to DNA rich in repeated sequences. In addition, cRNAs transcribed from five Cot fractions (31% of total DNA; Cot less than 100) were hybridized to total DNA in CsC1 gradients. The hybrids sedimented at similar densities to those seen in total cRNA but showed a differential distribution along the five fractions. To isolate native double-stranded DNA components containing the various families of repeated sequences observed, nuclear DNA was fractionated using relaxes Cs2SO4/Ag+ density gradient centrifugation. These fractions revealed the presence of seven major components a densities of 1.715, 1.711, 1.708, 1.705, 1.702, 1.700 and 1.698 g/cm3 and three minor components at densities of 1.696, 1.693 and 1.687 g/cm3. The density components corresponded to those observed by cRNA/DNA hybridization, thus suggesting that the bulk of the human genome is made up of ten density-biased components containing sequences of varying degrees of repetitiveness.

Base Sequence↗
PubMed1978

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.

Antigens, Viral↗
PubMed1978

An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels.

A new procedure is developed to isolate DNA from agarose gels. Using a kind of blotting technique, DNA is isolated from the gel. It is shown that the isolated DNA can be used for fragmentation by restriction endonucleases, synthesis of complementary RNA by DNA-dependent RNA polymerase from Escherichia coli and nick translation. The procedure gives a high recovery and is easy to perform.

Buffers↗
PubMed1981

Buoyant density and hybridization analysis of human DNA sequences, including three satellite DNAs.

Total human DNA was fractionated from the three types of Cs2SO4 gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.6888 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml, comprising about 1% of the total an satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 m/gl, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellite I an II.

Animals↗
PubMed1987

On antisense peptides: the parathyroid hormone as an experimental example and a critical theoretical view.

We followed an approach which predicts that translation of two complementary RNA strands into protein generates pairs of "antisense" peptides which bind each other with specific and high affinity (Bost et al. Proc. Natl. Acad. Sci. (1985) 82, 1372). We used human parathormone as an experimental example, and we analysed by computer homologies between antisense peptide sequences and their published receptor sequences. We conclude that there is no experimental indication that parathormone binds to a synthetic peptide, the sequence of which was derived from the antisense RNA sequence. Based on homology scores and antigenicity indexes (Hopp) the analysis shows that the peptide ligand itself, or a random artificial peptide, are as good candidates as the antisense peptide in producing antibodies, presumably recognizing the receptor. We therefore question the general applicability of this approach.

Amino Acid Sequence↗
PubMed1987

Developmental expression and regional distribution of the scrapie-associated protein mRNA in the rat central nervous system.

The scrapie associated protein (SAP) has been shown to be a normal brain protein of yet undefined function. This study demonstrates that rat brain SAP mRNA levels undergo a transcriptionally dependent increase during normal development. The interregional variation in the adult rat central nervous system (CNS) is roughly 10 fold, with highest levels in the basal ganglion/thalamus and lowest levels in the spinal cord.

Animals↗
PubMed1987

Expression of the murine homeo box gene Hox 1.5 during embryogenesis.

The spatial pattern of expression of the murine homeo box-containing gene Hox 1.5 was studied during embryogenesis. In situ hybridization of single-stranded RNA probes to mouse embryo sections revealed a specific spatial distribution of the Hox 1.5 transcripts in mouse embryos 8.5 to 12.5 days postcoitum (p.c.). Analysis of mouse embryos 8.5 days p.c. showed that the gene is expressed in a spatially restricted manner. Expression appears to be limited to the central nervous system with an anterior boundary in the hindbrain and extending posteriorly through caudal regions of the spinal cord. The same spatial pattern of expression was observed in embryos 9.5 to 12.5 days p.c. These results show that the murine Hox 1.5 gene is expressed in a spatially restricted manner during embryonic development similar to the patterns observed in Drosophila homeotic genes.

Animals↗
PubMed1988

Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells.

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.

Animals↗