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At least 811 records · Page 45Linked to original sources
PubMed1999

Distribution and developmental changes in metabotropic glutamate receptor messenger RNA expression in the rat lumbar spinal cord.

Using in situ hybridisation, the regional distribution of primary transcripts and splice variants of all metabotropic glutamate receptor subtypes (mGluR) currently known to be expressed in the spinal cord have been studied in the lumbar enlargement of the rat spinal cord. In adult animals, the messenger RNA of the mGluR subtypes 1, 5, 3, 4 and 7 were differentially expressed. The transcripts of mGluR1 and 5 were most abundant with mGluR5 messenger RNA being concentrated in the superficial dorsal horn. In contrast, the mGluR2 transcript was not detectable with the sensitivity of the method. Secondly, age related changes (postnatal days 1, 7, 12, 21) in the postnatal expression of mGluR1-5 and 7 transcripts have been investigated. mGluR1 and 7 messenger RNA showed a general decrease in spinal expression from postnatal day 1 to day 21. Quantitative densitometry showed high mGluR3 and 5 messenger RNA levels especially in the superficial dorsal horn at birth, however these levels decreased with age. In addition to changes in density, the regional distribution of mGluR3 messenger RNA was altered with postnatal development. Up to postnatal day 12, mGluR3 messenger RNA expression was almost exclusively restricted to the spinal grey matter, but with postnatal day 21 a strong additional expression in the white matter occurred. Distribution of mGluR4 messenger RNA showed little change in the dorsal horn, however motoneuronal expression emerged during development. These changes may suggest different roles for mGluRs in the maturation of spinal transmission of the rat nervous system.

Aging↗
PubMed1998

Coupling termination of transcription to messenger RNA maturation in yeast.

The direct association between messenger RNA (mRNA) 3'-end processing and the termination of transcription was established for the CYC1 gene of Saccharomyces cerevisiae. The mutation of factors involved in the initial cleavage of the primary transcript at the poly(A) site (RNA14, RNA15, and PCF11) disrupted transcription termination at the 3' end of the CYC1 gene. In contrast, the mutation of factors involved in the subsequent polyadenylation step (PAP1, FIP1, and YTH1) had little effect. Thus, cleavage factors link transcription termination of RNA polymerase II with pre-mRNA 3'-end processing.

Cytochrome c Group↗
PubMed1996

Comparative study of brain-derived neurotrophic factor messenger RNA and protein at the cellular level suggests multiple roles in hippocampus, striatum and cortex.

Brain-derived neurotrophic factor (BDNF) is important for the development and trophic support of several neuronal groups in the rat. In the present study, the distribution of BDNF messenger RNA was studied by in situ hybridization, and the cellular localization of BDNF protein was investigated with anti-peptide antibodies. Anatomical investigations were also made in animals with prolonged epileptic seizures which show an enhanced expression of BDNF messenger RNA. Major forebrain areas studied were the hippocampus, striatum and cortex. The messenger RNA coding for the putative high-affinity receptor, tyrosine kinase B, was also visualized using in situ hybridization with a probe specific for the full-length form. In the hippocampus, granule cells and pyramidal neurons expressed BDNF messenger RNA and BDNF-like immunoreactivity. Interneurons in dendritic layers did not show labelling with either method. Tyrosine kinase B messenger RNA was found within neurons in all these regions. In the medial septum-diagonal band, nucleus basalis and lateral hypothalamus, neurons with punctate cytoplasmic immunofluorescence were found, and neurons in the lateral septum were diffusely positive for BDNF. In striatum, positive labelling of medium-sized neurons was found with the antibody, whereas BDNF messenger RNA was only detectable during seizures. A laminar pattern of neuronal labelling for BDNF messenger RNA and protein was found in the neocortex. The analysis of the anatomical distribution of BDNF-producing cells suggests a number of possible cellular interactions. In the hippocampus, BDNF might act in an autocrine or paracrine manner for granule cells and pyramidal neurons, and, in addition, may serve as a signal from these principal cells to interneurons. BDNF could be a target-derived and a locally produced trophic factor for cholinergic neurons in the medial septum. The expression of BDNF in the striatum suggests that this factor could be a target-derived factor for dopaminergic neurons of substantia nigra and/or work as an autocrine/ paracrine factor within the striatum itself.

Animals↗
PubMed1986

Expression of albumin messenger RNA detected by in situ hybridization in preneoplastic and neoplastic lesions in rat liver.

The process of chemical hepatocarcinogenesis is characterized by the appearance of preneoplastic lesions showing changes in the expression of various marker enzymes. We have analyzed the phenotype of small preneoplastic foci and expansively growing nodules in liver sections obtained from rats treated with various carcinogens. Changes within the lesions in canalicular adenosine triphosphatase, gamma-glutamyl transpeptidase, NADPH-(cytochrome P-450) reductase, cytochrome P-450 PB2, epoxide hydrolase, and glycogen content were detected by means of enzyme histochemical and immunohistochemical staining procedures. In parallel sections the expression of albumin messenger RNA was investigated by in situ hybridization using a 35S-labeled albumin specific complementary DNA probe. In general, small preneoplastic lesions showed unchanged levels of albumin messenger RNA. In contrast, the expression of albumin messenger RNA was found to be reduced to varying degrees in large hepatic nodules. An expression of alpha-fetoprotein messenger RNA could not be detected in any of the nodules. No direct correlation between the enzyme phenotype of the lesions and the degree in reduction of albumin messenger RNA could be established except that the reduction was most pronounced in nodules which had lost their ability to store glycogen. Since the synthesis and excretion of albumin is a typical function of the differentiated hepatocyte in the adult animal, the observed decrease in albumin messenger RNA expression in large hepatic nodules is in accordance with the hypothesis of a gradual dedifferentiation or retrodifferentiation of the cell population during carcinogenesis. Hyperplastic nodules produced by continuous treatment of rats with 4-dimethylaminoazobenzene showed increased rather than decreased albumin levels. The analysis of albumin messenger RNA expression might therefore be used as a tool to discriminate between nodules of differing biological nature and fate.

Adenosine Triphosphatases↗
PubMed1977

Human globin messenger RNA: importance of cloning for structural analysis.

The sequence of most of the human beta globin messenger RNA and large sections of the alpha globin messenger RNA has been determined. Partly because of genetic polymorphism, it was necessary to clone globin complementary DNA in order to extend the analysis. Purified human fetal globin messenger RNA was isolated and used as a template by reverse transcriptase to produce duplex complementary DNA molecules. These molecules were linked in vitro to plasmid DNA by use of T4 ligase in the presence of Escherichia coli Pol 1. Several colonies transformed by these molecules have been shown to hybridize with labeled human globin complementary RNA.

Base Sequence↗
PubMed1983

Messenger RNA half-life in Dictyostelium discoideum.

Messenger RNA half-life in vegetatively growing cells of Dictyostelium discoideum was determined using a uridine pulse-chase procedure. In these experiments, mRNA decayed in a complex fashion, and consisted of at least two major components, one with a half-life of about 50 min and a second with a half-life of about 10 hr. These results independently confirm our previous studies on the decay of mRNA extracted from actinomycin D-treated cells. Since these results were in apparent conflict with half-life determinations obtained with a combination of actinomycin D and daunomycin (J. P. Margolskee and H. F. Lodish, 1980a, Dev. Biol. 74, 37-49), we have also studied mRNA half-life in cells treated with a combination of both drugs and found that simultaneous use of both drugs leads to accelerated mRNA decay and other noticeable side effects. In light of our observations, we have suggested an alternative to conclusions drawn by others with respect to mRNA synthesis and stability in Dictyostelium development.

Dactinomycin↗
PubMed1979

Defects in DNA and globin messenger RNA in homozygotes for hemoglobin Lepore.

Globin messenger RNA (mRNA) isolated from three patients homozygous for hemoglobin Lepore is shown to have a marked reduction of the amount of beta-like globin mRNA (Lepore-globin mRNA sequences) compared with alpha-globin mRNA by molecular hybridization. The relative amounts of alpha- and Lepore mRNA are similar to the amounts of alpha- and Lepore globin synthesized in intact cells and by isolated mRNA in a cell-free system. It is also demonstrated that Lepore-globin mRNA can completely hybridize to full-length or nearly full-length beta-globin specific complementary DNA and protect it from nuclease digestion, indicating close homology between the delta-mRNA sequences present in Lepore mRNA and the beta-complementary-DNA probe. We have also quantitated the numbers of beta-like globin gene sequences in genomic Lepore DNA by molecular hybridization and demonstrated a reduction in their number consistent with the Lepore gene being a delta beta-gene fusion product.

Adolescent↗
PubMed2000

Misdirected vimentin messenger RNA alters cell morphology and motility.

Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the (beta)-actin 3' untranslated region (3' UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or (beta)-galactosidase that localized through the (beta)-actin 3' UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.

Animals↗
PubMed2004

Application of ligation-mediated reverse transcription polymerase chain reaction to the identification of in vivo endonuclease-generated messenger RNA decay intermediates.

Most approaches to studying messenger RNA (mRNA) decay in vivo lack sufficient sensitivity to identify decay intermediates. The identification of such intermediates using in vitro decay systems can provide suggestive evidence for endonuclease-mediated degradation in vivo; to validate conclusions drawn from in vitro experiments one must demonstrate cleavage of the mRNA in vivo. Primer extension or S1 nuclease protection assays work best on relatively abundant mRNAs and even then require long exposure times. We describe a facile approach using ligation-mediated polymerase chain reaction to identify in vivo mRNA decay intermediates. In this procedure, total cellular RNA is ligated to a primer bearing a 5' phosphate and 3' amino group. Reverse transcription is primed using a complementary primer, and mRNA-specific decay intermediates are identified by polymerase chain reaction amplification using a 5'- [32P]-labeled gene-specific primer. Products generated in this manner are gel purified, reamplified, and the 3' end of each decay intermediate is identified by the sequence junction of the specific mRNA and the initial ligation primer. We show an example of the time-course of appearance of several specific decay intermediates of c-myc mRNA in differentiating murine erythroleukemia cells.

Animals↗
PubMed2003

Approaches to messenger RNA detection - comparison of methods.

Detection of messenger RNA is an important part of current biomedical research, although utilized for decades. This communication endeavors to compare three most commonly used techniques of mRNA detection, i.e. Northern blot, ribonuclease protection assay (RPA), and real-time polymerase chain reaction (RT-PCR). Principles and general procedures of these methods are described, and advantages and weaknesses of each are discussed in terms of their specificity, sensitivity, difficulty, time and material demands as well as health and environmental risks. We conclude that choice of any method discussed depends on particular purpose, experience of the researcher, and on laboratory equipment and organization.

Blotting, Northern↗
PubMed1992

[Isolation and characterization of prolactin messenger RNA from bovine anterior pituitary glands].

The messenger RNA was extracted from bovine anterior pituitary glands and was purified by oligo(dT)-cellulose chromatography. The length of the mRNA was 1,200 nucleotides measured by agarose gel electrophoresis containing methylmercury hydroxide. The bovine prolactin (PRL) mRNA was confirmed by Northern blot analysis of the mRNA with gamma-32P labelled synthetic oligonucleotide probes based on the partial amino acid sequences of bovine PRL and could stimulate the incorporation of 35S-methionine into protein in the rabbit reticulocyte cell-free system. The translation product of bovine PRL mRNA was immunoprecipitable by rabbit anti-ovine PRL anti-antibodies. The molecular weight of the translation product corresponding to the bovine PRL precursor was estimated to be approximately 25,000 by SDS polyacrylamide gel electrophoresis and autoradiograph.

Amino Acid Sequence↗
PubMed1989

Localization of VIP and PHI-27 messenger RNA in rat thalamic and cortical neurons.

Messenger RNA (mRNA) coding for vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI-27) were localized in cortical and thalamic neurons with synthetic DNA probes complementary to the PHI-27 and VIP exon coding sequence of the rat VIP precursor gene. Hybridization signal with these probes was found widely distributed in the thalamus, neocortex, and pyriform cortex, and the distribution of hybridization signal for each probe was identical. Furthermore, the distribution of each message was correlated closely with peptide distribution demonstrated immunohistochemically. Labeling of individual neurons with in situ hybridization histochemistry was characterized by a dense accumulation of silver grains in the cytoplasm of these cells with little or no label in the nucleus. Labeled neurons in the thalamus were observed in the ventrolateral, ventromedial, ventrobasal, and lateral reticular nuclei. In the neocortex, the distribution of labeled neurons was concentrated in layers II and III with scattered cells also apparent in deeper cortical layers. Hybridization signal was limited to nonpyramidal neurons in both the neo- and pyriform cortex. The coextensive distribution of immunoreactivity and mRNA coding regions for VIP and PHI-27 establishes that these peptides are synthesized from the same precursor mRNA in the same thalamic and cortical cell groups. Although the physiological role of these peptides in thalamocortical function remains unknown, these data provide an anatomical substrate which suggests that VIP and PHI-27 may be cotransmitters in thalamic and cortical neurons.

Animals↗
PubMed1986

Messenger RNA coding for argininosuccinate synthetase in citrullinemia.

Messenger RNA coding for argininosuccinate synthetase (ASS), extracted from the livers of some patients with citrullinemia, was analyzed using a cell-free translation system and dot and Northern blot hybridization with cDNA probe for ASS. In patients with quantitative-type citrullinemia, called type II here, previous studies have demonstrated that the hepatic content of the enzyme was about 10% of the control value, whereas the translatable mRNA level for the enzyme was similar to that of control livers. Here, we confirmed that the type II liver contained an almost normal amount of mRNA coding for ASS, judged by the dot-blot hybridization technique with cDNA. Northern blot hybridization of RNA indicated that there was hybridizable mRNA of approximately normal size (about 1.7 kilobase [kb]) in each, suggesting that large structural gene deletions had not occurred. These results indicate that in type II citrullinemia, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased or inhibited translation in the liver. Another type of citrullinemia was found and classified as type III. It is characterized by no detectable enzyme activity for ASS or translation activity for ASS mRNA. However, a smaller amount of RNA molecule hybridized for ASS cDNA was detected.

Adult↗
PubMed1999

A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export.

In order to identify additional factors required for nuclear export of messenger RNA, a genetic screen was conducted with a yeast mutant deficient in a factor Gle1p, which associates with the nuclear pore complex (NPC). The three genes identified encode phospholipase C and two potential inositol polyphosphate kinases. Together, these constitute a signaling pathway from phosphatidylinositol 4, 5-bisphosphate to inositol hexakisphosphate (IP6). The common downstream effects of mutations in each component were deficiencies in IP6 synthesis and messenger RNA export, indicating a role for IP6 in GLE1 function and messenger RNA export.

Biological Transport↗
PubMed1998

Regulation of nitric oxide synthase messenger RNA expression in the rat hippocampus by glucocorticoids.

Nitric oxide and glucocorticoids have been implicated in learning and memory, as well as in regulation of the stress response. By use of the in situ hybridization technique, we examined the role of glucocorticoids in the regulation of nitric oxide synthase messenger RNA in the hippocampus. In control animals, nitric oxide synthase subtype I (neuronal) messenger RNA was expressed in the CA1, CA3 and dentate gyrus of the hippocampus. Nitric oxide synthase subtype I expression was almost absent in CA2 pyramidal neurons. Neither subtype II (immunological) nor subtype III (endothelial) nitric oxide synthase messenger RNAs were observed in neurons of the hippocampal subfields. Bilateral removal of the adrenal glands resulted in a significant increase in nitric oxide synthase subtype I messenger RNA expression in the CA1 and CA3 pyramidal neurons and in granular cells of the dentate gyrus. To a lesser degree, the nitric oxide synthase subtype I messenger RNA signal was increased in CA2 pyramidal neurons. Daily administration of glucocorticoids for one week attenuated the adrenalectomy-induced increased level of expression of the messenger RNA encoding nitric oxide synthase subtype I in all areas studied. Because adrenalectomy, which suppresses the production of glucocorticoids, increases nitric oxide synthase expression, and replacement of adrenalectomized animals with glucocorticoids restores the basal levels of nitric oxide synthase subtype I expression, our results demonstrate an up-regulation of nitric oxide synthase subtype I messenger RNA in the absence of glucocorticoids in the hippocampus. The present findings suggest an involvement of the stress axis in the regulation of the synaptic plasticity process mediated by nitric oxide in the hippocampus.

Adrenalectomy↗
PubMed1996

Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells.

Glucocorticoid hormones increase voltage-gated Ca(2)+ current density in clonal pituitary cells. To test whether these steroids might stimulate expression of Ca(2)+ channel genes, messenger RNase protection assays were used to measure alpha IC and alpha ID RNAs that encode pore-forming subunits of L-type Ca2+ channels. We show here that dexamethasone rapidly increases alpha IC messenger RNA expression without affecting alpha ID messenger RNA level. This up-regulation of channel messenger RNA is also produced by natural glucocorticoids and is blocked by the glucocorticoid antagonist Ru48386. The up-regulation of the channel subunit messenger RNA expression is associated with an increase in high voltage-activated Ca(2)+ current density. Thus, glucocorticoids may produce a long-term effect on Ca(2)+ homeostasis in clonal pituitary cells by differentially regulating expression of Ca(2)+ channel subunit genes.

Animals↗
PubMed1995

Hemodynamic regulation of tyrosine hydroxylase messenger RNA in medullary catecholamine neurons: a c-fos-guided hybridization histochemical study.

Medullary catecholamine cell groups are involved in multiple modes of cardiovascular regulation and display indices of functional activation, including widespread c-fos expression, in response to hypotensive hemorrhage. Assessments of the impact of such challenges on transmitter-related gene expression are complicated by the biochemical and connectional heterogeneity that characterize these cell groups. Quantitative hybridization histochemical methods were used to follow the effects of 15% hemorrhage on levels of messenger RNA encoding tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in medullary aminergic neurons; concurrent staining for nuclear Fos-immunoreactivity permitted comparisons between cells that ostensibly were and were not targeted by the challenge. Increased levels of tyrosine hydroxylase messenger RNA were detected in Fos-immunoreactive neurons in all cell groups examined. Mean maximal increases ranged between 133 and 192% of control values, and were attained within 0.5-1 h post-hemorrhage in noradrenergic (A1 and A2) cell groups, and at 2 h in adrenergic ones (C1, C2, and C2d or dorsal strip). By 4 h after the challenge, tyrosine hydroxylase messenger RNA levels in Fos-immunoreactive neurons in all cell groups had returned to control values. By contrast, tyrosine hydroxylase messenger RNA in non-Fos-immunoreactive cells either did not change significantly over the course of the experiment (C2 and C2d), or showed a rapid and transient increase, whose magnitude tended to be less than that seen in Fos-immunoreactive cells. c-fos messenger RNA was prominently induced in catecholaminergic neurons in each of the medullary cell groups examined at 0.5 h after hemorrhage, suggesting that the early tyrosine hydroxylase messenger RNA response to hemorrhage in non-Fos-immunoreactive cells preceded the capacity of responsive neurons to manifest detectable Fos protein expression. These findings indicate that hemorrhage up-regulates tyrosine hydroxylase messenger RNA levels in medullary catecholaminergic cell groups which have access to adaptive neuroendocrine and/or autonomic control systems. The approach employed here should prove of general utility in assessing the impact of environmental events on messenger RNA expression in connectionally heterogeneous cell groups that share a common biochemical phenotype.

Adrenergic Fibers↗
PubMed1993

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat.

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, which is coupled to the inhibitory cyclic AMP cascade, was investigated in the central nervous system of the adult rat by in situ hybridization. Transcripts of mGluR2 were specifically localized to neuronal cells of the brain. Although the hybridization signals were widely distributed in the brain, the most prominent expression of mGluR2 messenger RNA was seen in Golgi cells of the cerebellum. Marked expression of mGluR2 messenger RNA was further observed in the mitral cells of the accessory olfactory bulb, neurons in the external part of the anterior olfactory nucleus, and pyramidal neurons in the entorhinal and parasubicular cortical regions. The granule cells of the accessory olfactory bulb, and many pyramidal and non-pyramidal neurons in the neocortical, cingulate, retrosplenial and subicular cortices, were moderately labeled. All of the granule cells in the dentate gyrus were also labeled moderately, whereas no significant hybridization signals were detected in Ammon's horn. In the basal forebrain regions, moderately labeled neurons were distributed in the triangular septal nucleus, in the lateral, basolateral and basomedial amygdaloid nuclei, and in the medial mammillary nucleus. Weakly labeled neurons were sparsely scattered in the striatum, globus pallidus, ventral pallidum and claustrum. The subthalamic nucleus was also labeled weakly. No significant labeling was found in the entopeduncular nucleus and substantia nigra. In the thalamus, moderately labeled neurons were distributed in the anterodorsal, anteromedial, ventromedial, intralaminar and midline nuclei; the ventrolateral part of the anteroventral nucleus and the rostral pole of the ventrolateral nucleus also contained moderately labeled neurons. No significant labeling was found in the thalamic reticular, submedius, ventroposterior, lateral geniculate and medial geniculate nuclei. In the lower brainstem, labeling was generally weak. No significant hybridization signals were found in the spinal cord. Some neurons in the inner part of the inner nuclear layer of the retina and some retinal ganglion cells were labeled moderately. The pattern of distribution of mGluR2 messenger RNA revealed in the present study indicates specific roles of mGluR2 in the glutamatergic system in the brain.

Animals↗