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PubMed1975

The separation of 9 S RNA from avian immature red blood cells into f2c-histone mRNA and globin mRNA.

9 S RNA from avian immature red blood cells was isolated from polysome-released ribonucleoprotein particles by sucrose-gradient techniques. Translation of this RNA in an Ehrlich ascites cell-free system and product analysis revealed that globin mRNA was contaminated by f2c-histone mRNA. When 9 S RNA was applied to oligo(dT)-cellulose columns a partial separation could be achieved. Poly (A)-containing globin mRNA did not contain f2c-histon mRNA, whereas the RNA which was not absorbed to oligo(dT)-cellulose contained all the f2c-histone mRNA besides substantial amounts of globin mRNA.

Animals↗

PubMed1976

Modified nucleosides and 5'-end groups in purified mouse immunoglobulin light chain mRNA and rabbit globin mRNA detected by borohydride labelling.

The borohydride reaction has been used to investigate modified nucleosides and end groups in purified immunoglobulin light chain mRNA and rabbit globin mRNA. 1. The light chain mRNA was isolated from the microsomal fraction of MOPC 41A mouse myelomas, which secrete kappa chains, by two cycles of oligo(dT) cellulose chromatography and glycerol gradient centrifugation. The 12 S mRNA was active in a Krebs II ascites cell-free system and appeared to be homogenous as judged by gradient centrifugation, polyacrylamide gel electrophoresis in 98% formamide and fingerprint analysis of 125I-labelled mRNA. 2. End group labelling of the light chain and globin mRNAs by oxidation with periodate and reduction with boro[3H]hydride showed that the RNAs have a 5'-terminal 7-methyl guanosine in 5'-pyrophosphate linkage with the next nucleoside. 3. To detect any modified residues in the interior of chains, nucleosides in complete digests of the mRNAs were converted by the borohydride reaction to 3H-labelled nucleoside trialcohols, which were fractionated by two dimensional chromatography (the Randerath technique). The light chain mRNA was found to contain N6-methyl adenosine (1 mole) but the rabbit globin mRNAs lacked this nucleoside. Deficiencies in this technique for analysis of minor constituents in large RNAs were noted.

Animals↗

PubMed1989

Phorbol ester regulates the abundance of enkephalin precursor mRNA but not of amyloid beta-protein precursor mRNA in rat testicular peritubular cells.

Cultured peritubular cells prepared from the testes of 20-day-old rats contained both preproenkephalin (A) mRNA (1.5 kb) and amyloid beta-protein precursor mRNA (3.6 and 2.8 kb). The phorbol ester TPA and forskolin (an adenylate cyclase activator) increased the preproenkephalin mRNA abundance to 9.0 and 5.8 times the control, respectively. TPA alone had no effect on the intracellular cAMP level. A combination of TPA and forskolin elicited a synergistic increase in the ppEnk mRNA abundance over 30-fold. Dexamethasone potentiated the effect of forskolin but not of TPA. These results suggest that TPA regulates the preproenkephalin mRNA abundance through a cAMP-independent pathway. In contrast, TPA, forskolin, and dexamethasone showed little or no effect on the abundance of amyloid beta-protein precursor mRNA.

Amyloid↗

PubMed1991

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA.

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

Animals↗

PubMed1993

Retinoic acid-induced decrease of DNA synthesis and peroxidase mRNA levels in human thyroid cells expressing retinoic acid receptor alpha mRNA.

In order to clarify the effect of retinoids on thyroid cell growth and function, the presence of retinoic acid receptors (RARs) and the action of retinoic acid (RA) on DNA synthesis and on thyroid peroxidase (TPO) and thyroglobulin (TGB) mRNA expression were investigated in primary cultures of human thyroid follicular cells. A time and dose-dependent reduction in 3H-thymidine (3H-thy) incorporation was found in cells exposed for 48 h to all-trans-RA up to 1 microM. A cytotoxic effect was found only with the higher dose of 50 microM. The RA-induced decrease of 3H-thy incorporation was reflected by parallel change in DNA content of cell monolayers. The inhibitory effect of 1 microM RA on 3H-thy incorporation ranged from 28.5 +/- 4.6% in normal cells to 42 +/- 3.2% in adenomatous cells. In addition, 1 microM RA significantly reduced basal and TSH-induced TPO mRNA levels in normal, goitrous and adenomatous cells, but did not alter TGB mRNA levels. Furthermore, in these cells the study of RAR alpha and beta mRNA showed the presence of two major RAR alpha mRNA transcripts of approximately 3.5 and 2.8 Kb in size, whereas RAR beta mRNA was undetectable. Overall, our data indicate that RAR alpha gene is expressed in human thyrocytes and that RA may be involved in the regulation of the human thyroid by reducing proliferation and function of follicular cells.

Adenoma↗

PubMed1983

mRNA species and proteins of adenovirus type 12 transforming regions: identification of proteins translated from multiple coding stretches in 2.2 kb region 1B mRNA in vitro and in vivo.

Analysis of cytoplasmic RNA in Ad12-infected and -transformed cells showed that more than 20 mRNA species are transcribed from regions 1A and 1B. These mRNA species could be classified into four classes according to the extent to which they were expressed in infected and transformed cells under various conditions. The proteins synthesized from the major species of each of these mRNAs were identified by in vitro translation. In region 1A, the 40K (and 38K) proteins were synthesized from three mRNA species that differed from one another in their 5' ends, and in region 1B three proteins with molecular weights of 19,000 (19K), 50K, and 17K were synthesized from a single species of 2.2 kb mRNA. Only the 19K protein was immunoprecipitated by anti-T serum (G serum) from rats bearing GY1 cell tumors, [GY1 cells are rat cells transformed by the Ad12 HindIII-G fragment (0-6.8 map units).] The 19K protein was also detected by immunoprecipitation in extracts from both transformed and infected cells. The 17K protein was immunoprecipitated by anti-T serum (C serum) derived from rats bearing CY1 cell tumors, but was not by G serum. [CY1 cells are rat cells transformed by the Ad12 EcoRI-C fragment (0-16.5 map units).] The 17K protein was also translated in vitro from the 0.5 kb region 1B mRNA. These results suggest that the 19K and 17K proteins correspond to Ad12 T antigen f and polypeptide IX, respectively, and that these two proteins are translated in vivo from different coding regions in 2.2 kb mRNA in CY1 cells.

Adenoviruses, Human↗

PubMed1990

Biosynthesis of reovirus-specified polypeptides. 2-aminopurine increases the efficiency of translation of reovirus s1 mRNA but not s4 mRNA in transfected cells.

The effect of 2-aminopurine (2AP), an inhibitor of the RNA-dependent P1/eIF-2 protein kinase, on the expression of the reovirus serotype 1 Lang strain S1 and S4 genes in transfected simian COS cells was examined. In the absence of 2AP, the s4-encoded sigma 3 gene product was expressed about five times more efficiently than the s1-encoded sigma 1 gene product. When COS cells were treated with 2AP, the synthesis of the sigma 1 polypeptide was increased about fivefold compared to that in untreated cells even though s1 mRNA levels were not detectably altered. In contrast to the increased translational efficiency of the s1 mRNA observed in 2AP-treated cells, the translational efficiency of the s4 mRNA was not affected by 2AP treatment. However, the cytoplasmic accumulation of s4 mRNA was transiently decreased by 2AP treatment. These results demonstrate that the expression of the reovirus S1 and S4 genes in transient transfection assays is differentially affected by 2AP. Furthermore, when considered together with the prior observation that the reovirus s1 mRNA is a potent activator of the RNA-dependent protein kinase relative to the s4 mRNA which is a very poor activator, the results are consistent with the suggestion that the differential translational efficiency of the reovirus s1 and s4 mRNAs in vivo may be attributed in part to their differential ability to activate the P1/eIF-2 protein kinase.

2-Aminopurine↗

PubMed1982

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA.

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.

Actins↗

PubMed1989

A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA.

Iron regulates human transferrin receptor (hTR) expression by modulating the stability of cytoplasmic hTR mRNA. This regulation requires a distinct secondary structure in the mRNA 3' untranslated region. We identified a specific cytoplasmic factor that binds simultaneously to four homologous palindromes within the regulatory domain. Iron chelator induced the RNA binding activity 25-fold in parallel with mRNA. Upon the addition of iron salts, a rapid decay of factor activity closely preceded hTR mRNA degradation, indicating a causal relation. Induction and decay occurred posttranscriptionally. Binding of the factor to hTR mRNA palindromes was competed by 5' regulatory sequences of ferritin mRNA, which are responsible for iron-dependent translational control. These results suggest that cellular iron maintains its homeostasis by coordinate regulation of hTR and ferritin expression via a common factor.

Animals↗

PubMedDate not supplied

Target cell-induced perforin mRNA turnover in NK3.3 cells is mediated by multiple elements within the mRNA coding region.

We have previously shown that in cytolytic cells exposed to sensitive targets the mRNA of the cytolytic protein perforin undergoes rapid downregulation. We now demonstrate that perforin message undergoes accelerated turnover in NK3.3 cells exposed to sensitive TC. This inducible mRNA decay phenomenon is specific for cytolytic protein messages, as levels of the constitutive message beta-actin are unchanged. This TC-induced perforin mRNA turnover cannot be attributed to a blockage of RNA synthesis, or to a rapid half-life (t 1/2). To determine the region(s) within the perforin transcript responsible for governing this TC-mediated turnover event, various segments of the perforin cDNA were cloned and inserted into the 3' UTR of rabbit beta globin (RG). Constructs containing perforin coding region cDNA, but not 3' UTR cDNA, mediated TC-induced mRNA turnover. These data indicate that multiple elements governing perforin mRNA stability reside within the coding region, a novel type of mRNA regulation not previously described.

Animals↗

PubMed1990

Perforant path stimulation differentially alters prodynorphin mRNA and proenkephalin mRNA levels in the entorhinal cortex-hippocampal region.

The regulatory effect of the perforant path on opioid gene expression in the entorhinal cortex-hippocampal region was investigated. The left perforant path was electrically stimulated at the angular bundle under conditions which elicit wet dog shakes but no motor seizures in rats. Animals were given either an acute stimulation composed of several consecutive stimulation trials, or daily stimulations with a single trial every day for 6 days. Rats were then sacrificed at 24 h or 6 days after the last trial. The amounts of prodynorphin mRNA (DYN mRNA) and proenkephalin A mRNA (EK mRNA) in the hippocampus and entorhinal cortex were measured by RNA blot analysis. Dynorphin A(1-8) and [Met5]enkephalin immunoreactivities were determined by radioimmunoassay. A decrease in DYN mRNA level of approximately 50-80% was found on both sides of the hippocampus 24 h after both acute and daily stimulation. Hippocampal dynorphin A(1-8) immunoreactivity was also reduced at 24 h, and persisted for at least 6 days. In contrast, bilateral increases in EK mRNA level were observed in the hippocampus (54-101%) and entorhinal cortex (97-165%) 24 h after the acute stimulation. Also, [Met5]enkephalin immunoreactivity in the hippocampus tended to be increased at this time. These results indicate that activation of the perforant path inhibits the gene expression of prodynorphin, but enhances that of proenkephalin in the entorhinal cortex-hippocampal region.

Animals↗

PubMed1992

Glucocorticoid-mediated down regulation of c-fos mRNA in C6 glioma cells: lack of correlation with proenkephalin mRNA.

We investigate the linkage between the transcriptional factor, c-fos, and expression of proenkephalin in rat C6 glioma cells. C6 cells contained abundant levels of c-fos mRNA. Treatment of cells with dexamethasone resulted in a 10-fold decline in c-fos transcripts and a small increase in proenkephalin mRNA. Combined exposure to dexamethasone and isoproterenol also induced a decrease in c-fos mRNA while proenkephalin mRNA increased 8-fold. Treatment of the C6 cells with phorbol 12-myristate 13-acetate caused a 13-fold increase in c-fos expression 0.5 h after administration and a decrease in proenkephalin mRNA. These data indicate that c-fos and proenkephalin mRNA are not regulated in a sequential, parallel manner, that newly synthesized c-fos is not the determining factor controlling proenkephalin gene regulation, and that c-fos expression is under negative control by glucocorticoids.

Animals↗

PubMed1993

Co-expression of dopamine transporter mRNA and tyrosine hydroxylase mRNA in ventral mesencephalic neurones.

Radioactive in situ hybridization was used to map the cellular localization of dopamine (DA) transporter mRNA-containing cells in the adult rat central nervous system. The distribution of DA transporter mRNA-containing cells was compared to adjacent sections processed to visualize tyrosine hydroxylase (TH) mRNA, a marker of catecholamine containing neurones. TH mRNA-containing cells, visualized using an alkaline phosphatase labelled probe, were detected in the hypothalamus, midbrain and pons; the strongest hybridization signals being detected in the substantia nigra, ventral tegmental area and locus coeruleus. The distribution of DA transporter mRNA-containing cells was more restricted; a strong signal being detected in the substantia nigra pars compacta and ventral tegmental area only. No hybridization signal was detected in the locus coeruleus. By simultaneously hybridizing mesencephalic tissue with both the alkaline phosphatase-labelled TH probe and the 35S-labelled DA transporter probe we were able to demonstrate that both DA transporter and TH mRNAs are expressed by the same cells in the substantia nigra and ventral tegmental area. The restricted anatomical localization of DA transporter mRNA-containing cells and the lack of expression in the locus coeruleus and other adrenergic and noradrenergic cell groups confirms the DA transporter as a presynaptic marker of DA containing nerve cells in the rat brain.

Animals↗

PubMed1996

Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

Animals↗

PubMed1991

In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout.

We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.

Animals↗

PubMed1991

Glycerol 3-phosphate dehydrogenase gene expression in cultured 3T3-L1 adipocytes: regulation by insulin, dexamethasone and dibutyryl cAMP at the level of mRNA abundance, transcription and mRNA stability.

In fully differentiated 3T3-L1 adipocytes, glycerol 3-phosphate dehydrogenase (G3PDH:Sn-glycerol 3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) is subject to regulation by hormones and dibutyryl cAMP. An increase by insulin (4-fold) and decrease by dexamethasone (by 50%) and dibutyryl cAMP (by 70%) was observed for G3PDH mRNA abundance as analyzed by Northern blot hybridization. In addition, incubation of adipocytes with dibutyryl cAMP resulted in 3-fold increase in G3PDH gene transcription as measured by nuclear transcript elongation assay. The effects of these modulators on the control of G3PDH mRNA stability were also investigated. The G3PDH mRNA has a half-life of about 125 min. Dibutyryl cAMP caused an increase in G3PDH mRNA degradation by greater than 2-fold (t1/2 = 55 min) whereas insulin had an opposite effect (t1/2 = 240 min) and dexamethasone was without any effect on G3PDH mRNA stability. Taken together, our results directly demonstrate that in fully differentiated 3T3-L1 adipocytes the regulation of G3PDH gene expression by dibutyryl cAMP and insulin is exerted by alterations in transcription as well as mRNA stability.

3T3 Cells↗

PubMed1993

Platelet derived growth factor-A chain gene expression in cultured mesangial cells: regulation by phorbol ester at the level of mRNA abundance, transcription and mRNA stability.

In human renal mesangial cells, platelet derived growth factor (PDGF)-A chain is subject to regulation by protein kinase C (PKC) activator, phorbol ester (phorbol 12-myristate 13-acetate, PMA). Treatment of mesangial cells with PMA increases PDGF-A chain mRNA abundance as analyzed by Northern blot hybridization. In contrast to the effect of PMA, the inactive analog phorbol had no effect on PDGF-A chain mRNA levels, while the PKC inhibitor H7 markedly reduced the PMA-induced increment in PDGF-A chain mRNA. To determine the mechanism by which PMA increases the abundance of this gene, transcription rate was measured by nuclear transcript elongation assay. Treatment of mesangial cells with PMA resulted in a 2-fold increase in PDGF-A chain gene transcription. In addition, we analyzed the effects of PMA on PDGF-A chain mRNA half-life as measured directly by pulse-chase method. PDGF-A chain mRNA has a half-life of about 106 min. The PDGF-A chain mRNA half-life was reduced by 30% (t1/2 = 74 min) when mesangial cells were incubated with PMA. Our results demonstrate that in human renal mesangial cells, the regulation of PDGF-A chain gene expression by PMA is primarily at the level of transcription.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Dopaminergic regulation of striatal proenkephalin mRNA and prodynorphin mRNA: contrasting effects of D1 and D2 antagonists.

In situ hybridization was used to measure the levels of proenkephalin mRNA and prodynorphin mRNA in regions of rat striatum and nucleus accumbens. Chronic administration of haloperidol (2.4 mg/kg/day for 7 days) increased the levels of proenkephalin mRNA in both striatum and nucleus accumbens. In contrast, the levels of prodynorphin mRNA were not significantly affected in any region. Chronic administration of the D1 antagonist SCH 23390 (2.4 mg/kg/day for 7 days) decreased the striatal content of proenkephalin mRNA. A similar effect was seen in the lateral nucleus accumbens. The levels of prodynorphin mRNA were unaffected by SCH 23390 treatment in all regions examined. These results suggest that there is no major tonic dopaminergic regulation of prodynorphin synthesis in the basal ganglia. However, it appears that there is a tonic suppression, via D2 receptors, and a tonic enhancement, via D1 receptors, of proenkephalin synthesis, in the striatum and nucleus accumbens.

Animals↗