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PubMed1999

Differential expression of bcl-w and bcl-x messenger RNA in the developing and adult rat nervous system.

The bcl-2 family of proteins comprises both anti-apoptotic and pro-apoptotic members, which play a pivotal role in regulating cell death. Bcl-w is a recently identified member of this family, which was shown to inhibit apoptosis in haemopoietic cell lines. However, the function and expression patterns of bcl-w in the nervous system have so far not been described. We have cloned complementary DNA corresponding to rat bcl-w and analysed the expression of bcl-w messenger RNA during rat brain development, using RNA blotting and in situ hybridization techniques. We also compared the expression patterns of bcl-w with those of bcl-xL. During brain development, the levels of bcl-w messenger RNA were found to increase, with highest expression located to specific regions of the mature brain, such as hippocampus, cerebellum, piriform cortex and locus coeruleus. Bcl-w messenger RNA was expressed by neurons, as shown with double labeling with neuronal markers. In contrast to bcl-w, bcl-xL messenger RNA expression levels were highest during early development, particularly in cortex, hippocampus, thalamus, spinal cord and dorsal root ganglia. During postnatal development the expression of bcl-xL messenger RNA decreased and were only detected at low levels in the adult nervous system. As shown earlier for bcl-2, the expression of bcl-w and bcl-x messenger RNA in cultured cerebellar granule cells was not altered by the deprivation of neurotrophic factors. The present results suggest that bcl-w may play an important role in the mature nervous system.

Aging↗

PubMed1989

Ultrastructural detection of the vasopressin messenger RNA in the normal and Brattleboro rat.

The messenger RNA coding for vasopressin has been detected at the ultrastructural level in normal and Brattleboro rat neurons, by using an oligonucleotide rat AVP prove labelled with 35SdATP. Vibratome sections of rat hypothalamus fixed with a mixture of 4% formaldehyde and 0.1% glutaraldehyde were hybridized with the probe, osmicated, included in Araldite and cut in semi-thin and thin sections that were coated with emulsion. The results demonstrate that vasopressin mRNA can be visualized in the cytoplasm of normal and Brattleboro rat neurons with an acceptable preservation of the ultrastructural aspect of the tissue. In Brattleboro rat neurons, the vasopressin mRNA is preferentially located at the periphery of the cytoplasm and is less abundant than in normal rat, this suggesting that the single base gene deletion observed in the Brattleboro rat provokes an altered transcription and compartmentation of the corresponding mRNA.

Animals↗

PubMed1981

Regulation of nitrogenase messenger RNA synthesis and stability in Klebsiella pneumoniae.

Nitrogenase messenger RNA synthesis in Klebsiella pneumoniae was determined by labelling cells with (3H)uracil and isolating total RNA, which was then hybridized to filter-bound recombinant plasmid pSA30 DNA carrying the nitrogenase structural genes nifH, D, and K, Derepression of nitrogenase mRNA starts 1.5 h before the onset of nitrogenase activity (as measured by acetylene reduction). Exposure of nif-derepressed cultures to either NH4+, air, or high temperatures (39 degree C) results in a rapid decrease of the synthesis rates both of nitrogenase mRNA and nitrogenase polypeptides. Nitrogenase mRNA is remarkably stable. After blocking transcription with rifampicin, hybridizable and actively translatable nitrogenase mRNA survives with an average half-life of 18 min. Half-lives are considerably shorter when rifampicin-inhibited cultures are simultaneously shifted to conditions which are non-permissive for nitrogenase synthesis, pointing to some posttranscriptional influence on nitrogenase mRNA stability. In all experiments performed there was no evidence for uncoupling of nitrogenase mRNA synthesis from nitrogenase mRNA translation, indicating that nitrogenase synthesis is regulated solely by transcriptional control.

Genes, Bacterial↗

PubMed2006

Adenylation and exosome-mediated degradation of cotranscriptionally cleaved pre-messenger RNA in human cells.

In humans, polyadenylation of messenger RNA (mRNA) protects transcripts from degradation and enhances translation efficiency. Conversely, in bacteria, polyadenylation destabilizes mRNA. RNA adenylation was recently implicated in promoting degradation of some yeast RNAs by the exosome. The exosome complex of exoribonucleases is a major degradation machine in eukaryotes, and many of its components share significant homology with bacterial exonucleases. The human beta-globin pre-mRNA is cotranscriptionally cleaved within its 3' flank. Here, we show that some RNA ends, coinciding with these cotranscriptionally cleaved regions, contain short A tails on their 3' ends. Moreover, all of the pre-mRNA species detected accumulate in the absence of the exosome. We have also detected adenylation on RNA 3' ends originating within the mouse serum albumin (MSA) 3' flanking region RNA. This step in pre-mRNA degradation may represent an additional role for adenylation in mammals.

Animals↗

PubMed1997

Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling.

Intraventricular 192 IgG-saporin was used to induce a selective lesion of basal forebrain cholinergic neurons in rats. When subjected to 40 rapid hippocampal kindling stimulations with 5-min intervals, these animals exhibited increased number of generalized seizures and a higher mean seizure grade in response to the first five stimulations, and required fewer stimuli to develop focal behavioural seizures, as compared to non-lesioned rats. In contrast, both groups showed similarly enhanced responsiveness when test stimulated four weeks later. Using in situ hybridization, cholinergic denervation was found to cause a significant decrease of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex, whereas gene expression for nerve growth factor, neurotrophin-3, and TrkB and TrkC was unchanged. Four weeks after rapid kindling stimulations, basal levels of brain-derived neurotrophic factor messenger RNA in the dentate granule cells were restored to normal in the lesioned rats, whereas neurotrophin-3 messenger RNA levels were decreased. No differences in the seizure-evoked levels of neurotrophin and Trk messenger RNAs were detected, except in the dentate granule cell layer, which had significantly higher brain-derived neurotrophic factor messenger RNA expression in the lesioned animals at 2 h. In conclusion, the basal forebrain cholinergic system (i) dampens the severity of recurring seizures induced by rapid hippocampal kindling stimulations, but has no effect on the subsequent delayed phase of epileptogenesis; and (ii) exerts a tonic stimulation of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex. The findings also indicate that the cholinergic lesion does not affect neurotrophin and Trk gene expression after recurring seizures, and that the kindling process leads to long-term changes in basal brain-derived neurotrophic factor and neurotrophin-3 messenger RNA levels in the denervated animals.

Acetylcholinesterase↗

PubMed1980

Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.

Animals↗

PubMed2000

Transterm: a database of messenger RNA components and signals.

Transterm facilitates studies of messenger RNAs and translational control signals. Each messenger RNA (mRNA) from GenBank is extracted and broken into its functional components, its coding sequence, initiation context, termination context, flanking sequence representing its 5' UTR (untranslated region), 3' UTR and translational signals. In addition, numerical parameters characterising each coding region in Transterm, including codon and GC bias, are available. For each species in Transterm, the initiation and termination regions are aligned by their start or stop codons and presented as base frequency matrices and tables of the information content of the bases in the alignments. Users can obtain summaries of characteristics of the mRNAs for species of their choice and search for translational signals both in the Transterm database and in their own sequence. The current release contains data from over 10 000 species, including the complete genomes of 20 prokaryotes and three eukaryotes. Both flat-file and relational database forms of Transterm are accessible via the WWW at http://biochem.otago.ac.nz/Transterm/

Codon, Initiator↗

PubMed1988

Responses to GABA, glycine and beta-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain.

Poly (A)+ messenger RNA (mRNA) was extracted from rat and chick brains, and injected into oocytes of Xenopus laevis. This led to the expression of receptors that evoked membrane currents in response to gamma-aminobutyric acid (GABA), glycine and beta-alanine. These currents all inverted at about the chloride equilibrium potential in the oocyte, and showed a marked rectification at negative potentials. Oocytes injected with mRNA from chick optic lobe gave large responses to GABA and beta-alanine, but small responses to glycine. In contrast, one fraction of mRNA from rat cerebral cortex (obtained by sucrose density gradient centrifugation) caused oocytes to develop sensitivity to GABA, glycine and beta-alanine, but very little to GABA. The pharmacological properties of the three amino acid responses also differed. Barbiturate and benzodiazepines potentiated the responses to GABA and beta-alanine, but not to glycine. Strychnine reduced the responses to glycine and beta-alanine, but not to GABA, whereas bicuculline reduced the responses to GABA and beta-alanine, but not to glycine. We conclude that different species of mRNA code for receptors to GABA and glycine, and possibly also for separate beta-alanine receptors.

Amino Acids↗

PubMed1975

Translation of polysomal messenger RNA during epidermal differentiation.

Translation in vitro of messenger RNA obtained from preparations of active polysomes isolated from the epidermal basal, spinous, and granular cells of the newborn rat has provided evidence that in the context of differentiation in this tissue, control of protein synthesis is exerted at the level of transcription. The data supporting this hypothesis are reviewed.

Amino Acids↗

PubMed1980

Determination of alpha thalassaemia phenotypes by messenger RNA analysis.

The possibility of using alpha/beta globin messenger RNA (mRNA) ratios to distinguish between the carrier states for different forms of alpha thalassaemia has been explored. Alpha/beta globin mRNA ratios were determined in the red cells of a series of normal individuals and in members of four Cypriot families, one Thai and one Chinese family in which at least one person has haemoglobin H disease. It was found that there was a clear distinction in the ratios between normals, alpha thalassaemia 1 carriers, alpha thalassaemia 2 carriers, and those with haemoglobin H disease. This method should be a valuable addition to haematological analysis, haemoglobin synthesis and restriction mapping of DNA for the further elucidation of the genetics of alpha thalassaemia.

DNA↗

PubMed1990

Changes in angiotensinogen messenger RNA in differentiating 3T3-F442A adipocytes.

Angiotensinogen messenger RNA (mRNA) has been identified in both brown and white adipose tissue. Recently we have shown that when 3T3-L1 cells were treated with isobutylmethylxanthine (IBMX) to accelerate differentiation, angiotensinogen mRNA increased markedly in adipocytes as compared with preadipocytes. To determine if a correlation existed between the regulatory events associated with the differentiation process, we compared the change in angiotensinogen mRNA in spontaneously differentiating 3T3-F442A cells with two established parameters of differentiation in adipocyte cell lines. Differentiation was assessed by visual examination of cells for lipid droplets, fluorescent staining of the F-actin fibers, and increases in glycerol phosphate dehydrogenase mRNA. F-actin fibers were highly structured in preadipocytes, becoming disassembled and very disorganized as cells differentiated into adipocytes. The quantity of angiotensinogen mRNA increased as the number of lipid-containing cells increased within a culture. Glycerol phosphate dehydrogenase mRNA accumulated in differentiated adipocytes to about the same extent as angiotensinogen mRNA. Thus, increases in angiotensinogen mRNA were associated with the morphological and biochemical changes that occur during the phenotypic modulation of 3T3-F442A cells.

Actins↗

PubMed1985

Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.

Amino Acid Sequence↗

PubMed1997

Increases in thyrotropin-releasing hormone messenger RNA expression induced by a model of human temporal lobe epilepsy: effect of partial and complete kindling.

Thyrotropin-releasing hormone and its receptor are differentially distributed throughout the limbic forebrain. In addition to its neuroendocrine function, several non-endocrine central nervous system effects of thyrotropin-releasing hormone and its analogs have been reported, including anticonvulsant effects in animals and humans. Kindling, as a model of temporal lobe epilepsy, produces elevations of endogenous thyrotropin-releasing hormone specifically in seizure-prone limbic regions. The present study used semi-quantitative in situ hybridization to characterize changes in thyrotropin-releasing hormone messenger RNA that occurred during the kindling process (partial kindling), as well as after fully kindled seizures. No significant change in thyrotropin-releasing hormone messenger RNA was detected 1 h postictally, whereas significant elevations were detected in the granule cell layer of the hippocampal dentate gyrus, diffuse nuclei of the amygdala and in layers II and III of piriform and entorhinal cortices from 3 to 48 h after a single generalized seizure in fully kindled rats. Peak messenger RNA expression occurred from 6 to 12 h postictally, with a decline at 24 h, followed by a precipitous return to undetectable levels by 48 h, except in the dentate gyrus. In marked contrast, partial kindling produced no detectable change in thyrotropin-releasing hormone messenger RNA by 6 h after the first occurrence of stage 1-5 seizures. Electrode placement, a single afterdischarge, or a 20-microA stimulation of the amygdala was not associated with accumulation of thyrotropin-releasing hormone messenger RNA. Thus, only full kindled generalized seizures increased thyrotropin-releasing hormone messenger RNA expression in identical limbic regions which also showed postictal elevations in thyrotropin-releasing hormone. However, this enhancement followed a more immediate and shorter lasting time-course than previously demonstrated increases in the tripeptide. These results support the hypothesis that thyrotropin-releasing hormone is an important neuromodulator in epileptic foci.

Animals↗

PubMed1992

Cortical transynaptic activation of tyrosine kinase receptor trkB messenger RNA expression in rat hippocampus.

The trkB gene encodes a tyrosine kinase receptor which is an essential component of the high-affinity cell surface receptor for the neurotrophin brain-derived neurotrophic factor. In this report we have used quantitative in situ hybridization to study the expression of trkB messenger RNA in the rat hippocampus following stimulation of afferents in the entorhinal cortex. A bilateral three-fold increase of trkB messenger RNA levels in the hippocampus was seen 4 h after quisqualate injection into the left entorhinal cortex. The increase was confined to the granule layer of the dentate gyrus. A small increase, however, was also seen bilaterally in the pyramidal cell layer. The increases in all hippocampal areas were completely prevented by pretreatment of the animals with systemic injection of diazepam but not with scopolamine. We suggest that glutamate release from cortical afferents to the hippocampus has the capacity to increase neuronal expression of trkB messenger RNA within the hippocampus. The results from the present study extend the interpretation of our previous evidence of cortical transynaptic activation of brain-derived neurotrophic factor messenger RNA and indicate the presence of a concomitant activation of trkB messenger RNA expression in the hippocampus.

Animals↗

PubMed1997

Cholinergic sprouting in the rat fascia dentata after entorhinal lesion is not linked to early changes in neurotrophin messenger RNA expression.

After unilateral entorhinal cortex lesion cholinergic septohippocampal fibres sprout in the denervated fascia dentata. This process is dependent on neurotrophin changes following the lesion. Thus, there is an up-regulation of nerve growth factor and brain-derived neurotrophic factor messenger RNA expression in the denervated granule cells which is detectable 4 h postlesion and returns to control levels by 24 h. Here, using a competitive polymerase chain reaction and in situ hybridization, a transient neurotropin messenger RNA increase could be demonstrated bilaterally following unilateral electrolytic entorhinal cortex lesion. Treatment of the animals with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate blocked this messenger RNA increase, suggesting an involvement of this receptor type in the neurotrophin changes. However, in spite of this blockade, the typical cholinergic sprouting response as visualized with acetylcholinesterase histochemistry was present in animals four weeks after entorhinal cortex lesion. These data suggest that brief initial changes in neurotrophin messenger RNA expression in dentate granule cells are not responsible for the induction of the cholinergic sprouting. Changes in neurotrophin messenger RNA expression occurring immediately postlesion may be linked to glutamate release from entorhinal terminals resulting from the electrolytic lesion of the projection cells in the entorhinal cortex. We hypothesize that later changes in neurotrophin expression, for example in glial cells, are more likely to be related to the cholinergic sprouting process.

Acetylcholinesterase↗

PubMed1964

BETA-GALACTOSIDASE: INACTIVATION OF ITS MESSENGER RNA BY ULTRAVIOLET IRRADIATION.

A brief exposure of Escherichia coli cells to an indutcer for beta- galactosidase results in the production of a messenger RNA which subsequently expresses itself as beta-galactosidase. Ultraviolet irradiation of cells after exposure to the inducer results in a decrease in the amount of beta-galactosidase formed. The messenger RNA formed during the brief exposure to inducer is evidently inactivated by the ultraviolet radiation. The decay of messenger RNA activity in irradiated cells has the same kinetics as that observed in unirradiated cells.

Chloramphenicol↗

PubMed1993

Food stimulation of histidine decarboxylase messenger RNA abundance in rat gastric fundus.

1. Histidine decarboxylase in the enterochromaffin-like cells of the gastric corpus mucosa converts histidine to histamine which in turn stimulates gastric acid secretion. The control of histidine decarboxylase activity is poorly understood. We have examined how fasting and refeeding influence the abundance of the messenger RNA encoding histidine decarboxylase in the gastric corpus of the rat. 2. The polymerase chain reaction was used to generate a probe for detection of histidine decarboxylase messenger RNA in Northern and slot blots of total RNA from the gastric corpus of rats fasted for up to 48 h, or fasted and then refed. A gastrin monoclonal antibody was used to neutralize the action of endogenous gastrin. 3. Fasting progressively reduced histidine decarboxylase messenger RNA abundance by 3- to 4-fold after 48 h. Refeeding induced a rapid increase in histidine decarboxylase messenger RNA abundance which was detectable after 30 min. 4. There was a significant correlation between histidine decarboxylase messenger RNA abundance and plasma gastrin. Administration of gastrin antibody inhibited the increase in histidine decarboxylase activity after 6 h refeeding, but not after refeeding for 30 min. 5. The results suggest that histamine-mediated changes in postprandial acid secretion depend on control of histidine decarboxylase mRNA levels, and that gastrin regulates production of this enzyme in the rat over periods of a few hours.

Animals↗

PubMed1975

Use of a specific probe for ovalbumin messenger RNA to quantitate estrogen-induced gene transcripts.

DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

Animals↗