Nuclear Run-off Transcription Assays Research Articles

Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells.

In vivo, endothelial cells (EC) are subjected to hemodynamic forces which may influence the production of nitric oxide. This study was designed to examine the effect of cyclic strain on the expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic EC. EC were grown on flexible membranes which were subjected to deformation at 60 cycles/min with -5 or -20 kPa of vacuum. This results in an average strain of 6 and 10%, respectively, which is transmitted to the attached cells. Northern blot analysis of total cytosolic RNA demonstrated an increase in eNOS gene expression with both strain regimens but the increase with 10% average strain was greater than that at 6%. Nuclear runoff transcription assays confirmed the induction of eNOS transcripts. Western blot analysis showed an increase in eNOS level after 24 h of cyclic 10% average strain compared with controls or 6% average strain. Immunohistochemical staining of EC for eNOS was increased in the high strain periphery (7-24% strain) of membranes deformed with -20 kPa vacuum. These results demonstrate that cyclic strain upregulates the expression of eNOS transcripts and protein levels in bovine aortic EC thus emphasizing the importance of hemodynamic forces in the regulation of eNOS in vivo.

Increased Expression of Acidic Ribosomal Protein (P0) mRNA After Phorbol Ester Treatment of Cultured Rat Thyroid (FRTL-5) Cells

P0, an acidic protein component of the ribosomal protein in eukaryotic 60 S ribosomal subunit, plays an important role in polypeptide chain elongation during translation. To investigate the role of protein kinase C in thyroid cell protein synthesis, we examined the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of P0 mRNA and protein. RNA slot blot hybridization revealed that TPA induced the accumulation of P0 mRNA in FRTL-5 cells in a time- and dose-dependent manner. A maximal increase of 2-fold was observed 18 h after addition of TPA. Cycloheximide markedly inhibited the TPA-induced accumulation of P0 mRNA. Nuclear runoff transcription assays using nuclei prepared from TPA-treated FRTL-5 cells revealed that TPA increased the transcription of P0 mRNA but not of β-actin. Immunoblotting experiments using anti-P protein antibody showed that TPA also increased the protein amount of P0. These results suggest that TPA activates protein synthesis in thyroid cells by inducing the expression of ribosomal proteins.

The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection.

We have previously shown that the herpes simplex virus immediate-early regulatory protein ICP27 acts posttranscriptionally to affect mRNA processing (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). Specifically, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. Here, we have investigated the effect of ICP27 during herpes simplex virus type 1 (HSV-1) infection on accumulation of spliced cellular mRNAs. ICP27 viral mutants have been shown to be defective in host shutoff (W. R. Sacks, C. C. Greene, D. P. Aschman, and P. A. Schaffer, J. Virol. 55:796-805, 1985). Therefore, we examined whether ICP27 could contribute to this complex process by decreasing cellular mRNA levels through its effects on host cell splicing. It was found that in infections with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs were higher than those seen with wild-type HSV-1. The differences occurred posttranscriptionally as shown by nuclear runoff transcription assays. The stabilities of the spliced products during infection with wild-type or ICP27 mutant viruses were similar, and unspliced precursor mRNA for a viral spliced gene was detected in infections with wild-type HSV-1 but not in infections in which ICP27 was not expressed. These results suggest that the reduction in cellular mRNA levels and the accumulation of pre-mRNA are related and may be caused by an impairment in host cell splicing. These data further show that ICP27 is required for these effects to occur.

Induction of pulmonary Mn superoxide dismutase mRNA by interleukin-1

We have previously demonstrated that intratracheal (IT) but not intraperitoneal (IP) administration of 5 micrograms tumor necrosis factor (TNF) or interleukin-1 (IL-1) selectively enhances pulmonary Mn superoxide dismutase (Mn SOD) mRNA, leading to increased Mn SOD protein and enzyme activity, and protects rats against O2 toxicity. In this study, we demonstrated that enhancement of pulmonary Mn SOD mRNA by TNF or IL-1 was highly dependent on the route of administration. IT insufflation of 5 micrograms TNF or IL-1 selectively enhanced levels of pulmonary but not splenic or renal Mn SOD mRNA. In contrast, IP or intravenous (i.v.) administration of TNF (5 micrograms) or IL-1 (5, 20, or 50 micrograms) had little or no effect on levels of Mn SOD mRNA in the lung, spleen, or kidney. Both TNF and IL-1, whether given by IT, IP, or i.v. administration, had no effect on levels of Cu, Zn SOD mRNA. IP administration of 2 mg/kg actinomycin D 2 h before IT insufflation of IL-1 paradoxically increased the level of pulmonary Mn SOD mRNA without affecting the level of Cu,Zn SOD or beta-actin mRNA in IL-1-treated rats. Nuclear runoff transcription assay revealed that IT insufflation of IL-1 enhanced the rate on MN SOD but not Cu,Zn SOD mRNA synthesis. We conclude that IL-1-induced increase in pulmonary Mn SOD mRNA is at least in part regulated at the transcriptional level.

Identification of Newly Transcribed RNA

AbstractNewly transcribed RNA can be identified using the nuclear runoff transcription assay. Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Cell lysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmic material often results in poor incorporation of 32P‐labeled UTP into nascent transcripts. Although there is no way to predict what cell types present this problem, many adherent cell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, very few nonadherent cell lines have posed this problem. As described in this unit, isolating nuclei by detergent lysis of cells works well for many tissue culture cell lines but may not be appropriate for all cell lines and many tissues. Protocols for detergent lysis and Dounce homogenization or cell lysis in an isoosmotic solution and centrifugation through a sucrose cushion are described along with a for the preparation of the cDNA nitrocellulose filter strips that are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.

Modulation of surfactant protein D expression by glucocorticoids in fetal rat lung.

The production of pulmonary surfactant protein D (SP-D) increases abruptly during late gestation, and the accumulation of this protein in lung tissue is increased following the exposure of fetal rats to glucocorticoids in utero. To examine the regulation of these events, we administered dexamethasone (Dex; 1 mg/kg/day intramuscularly for 3 days), or saline, to timed-pregnant rats and harvested the lungs on days 19 to 21 of gestation. Samples of pooled fetal lungs were analyzed for SP-D protein, mRNA, and gene transcription by immunoblot, Northern hybridization, and nuclear run-off transcription assays. Lungs from 19 day controls showed barely detectable levels of SP-D gene transcription and negligible accumulation of SP-D message. However, SP-D transcription and the accumulation of SP-D mRNA and protein were readily detected in lungs from 19 day Dex-treated rats. Dexamethasone also caused dose- and time-dependent increases in SP-D protein and mRNA accumulation in 19 day fetal lung explants. Immunohistochemistry of control 19 day lung using antibodies to rat SP-D showed only weak labeling of a small number of airway epithelial cells. By contrast, Dex-exposed rats showed strong staining of columnar and cuboidal epithelial cells lining airways and epithelial tubules and cuboidal cells lining primitive air sacs. In situ hybridization assays showed similar alterations in the number, intensity, and distribution of labeled epithelial cells in 19 day Dex-exposed lungs and demonstrated labeling of alveolar type II and nonciliated columnar cells in adult lung. These data indicate that the accelerated lung maturation accompanying glucocorticoid exposure in utero is associated with a precocious increase in SP-D gene transcription and protein production by pulmonary epithelial cells.

Regulation of apolipoprotein A-I gene expression by phenobarbital in the human hepatocarcinoma cell line, Hep3B

Apolipoprotein (apo) A-1 is the major protein constituent of plasma high density lipoprotein (HDL), which has been suggested to play a protective role against the development of atherosclerosis. The effect of phenobarbital on apo A-1 mRNA and protein levels was studied in the human hepatoma cell line, Hep3B. Exposure of Hep3B cells to the drug (200 μg/ml) for 16 h resulted in a 4-fold and 8-fold increase in apo A-I mRNA and secreted protein levels, respectively. The induction of apo A-I mRNA level caused by phenobarbital could be due to increased rates of transcription and/or alteration in mRNA stability. To test these possibilities, nuclear run-off transcription assays and pulse-chase deinduction experiments were performed. We have demonstrated that phenobarbital treatment is associated with a 2-fold induction in apo A-1 transcriptional activity. The estimated half-lives for apo A-I mRNA are 2 h and 3,6 h in the absence or presence of phenobarbital, respectively. The combination of increase in apo A-1 transcription rate and mRNA stabilization could explain the 4-fold induction in apo A-1 mRNA levels caused by phenobarbital treatment. However, these events could not be solely responsible for the 8-fold increase in secreted apo A-I protein level observed. The results suggest that the mechanism(s) by which phenobarbital induces apo A-I production operate at both pre- and either co- or post-translational mechanisms. The induction of apo A-1 is specific since no significant alteration in apo E mRNA and proteins was observed in drug-treated cells.

Diurnal Variation of the Serine Dehydratase mRNA Level in Rat Liver

Serine dehydratase mRNA in the livers of rats maintained on laboratory chow (containing 22% protein) under a 12-h light (7:00-19:00)/12-h dark (19:00-7:00) cycle showed a daily oscillation that was maximal at 19:00 and minimal at 7:00 with an amplitude of more than 20-fold. The feeding of high-protein diets (60 and 91% casein) did not affect the oscillation phase. Virtually identical oscillatory phases for the mature serine dehydratase mRNA and its precursor RNAs, and the results of nuclear run-off transcription assays indicated that the oscillation was generated at the level of transcription. Similar oscillation patters were seen in adrenalectomized, starved, or diabetic rats. However, the mRNA oscillation was not precisely reflected in the oscillation of enzyme activity or enzyme protein in both the phase and the amplitude. Rats maintained on laboratory chow under constant light for 2 weeks still showed mRNA oscillation with a peak at 3:00, whereas rats exposed to constant light and fed between 7:00 and 9:00 exhibited an oscillation peak at 3:00-7:00. Under the conditions of a 12-h light/dark cycle and a 2-h restricted feeding between 7:00 and 9:00, a broad peak was seen at 23:00-3:00. These results indicate that the rhythmic expression of serine dehydratase gene is controlled by both meal-responsive and photoresponsive regulators.

Impaired gene transcription and nuclear protein kinase C activation in the brain and liver of aged rats

The expression of the hsp70 and c- fos genes and the activation of nuclear protein kinase C (PKC) were studied in young and aged whole rats under heat-shock conditions. The induction of hsp70 and c- fos genes by heat shock were decreased several fold in the brain as well as in the liver of senescent animals. Nuclear run-off transcription assay indicated that this age-related impairment could be attributed to a block at the level of transcription. Nuclear PKC activation by heat shock was not apparent in old animals. Nuclear PKC involvement in the repression of transcription during senescence is postulated.

Regulation of endothelial cell tissue factor expression by minimally oxidized LDL and lipopolysaccharide.

Tissue factor (TF) is the predominant physiological initiator of coagulation, and its regulation is a critical aspect of endothelial cell hemostatic function. This report describes the regulation of TF mRNA expression by two physiological agonists: minimally oxidized low-density lipoprotein (MM-LDL), which may modulate endothelial hemostatic function in atherosclerosis, and lipopolysaccharide (LPS), which is a mediator of septic shock. Northern blot analysis of total RNA from human endothelial cells exposed to either MM-LDL or LPS for varying times showed that TF mRNA increased sharply at 1 hour, peaked at 2 to 3 hours, and declined to basal levels by 6 to 8 hours after treatment. The half-life of TF mRNA in MM-LDL- and LPS-exposed endothelial cells was approximately 45 minutes and 40 minutes, respectively. The rate of TF mRNA degradation was similar at 1 and 4 hours after exposure in either MM-LDL- or LPS-stimulated endothelial cells. Nuclear runoff transcription assays showed a significantly increased rate of TF gene transcription in both MM-LDL- and LPS-exposed endothelial cells. Cycloheximide inhibited the induction of TF protein activity, but it enhanced the accumulation of TF mRNA in MM-LDL- and LPS-induced endothelial cells. These results indicated that regulation of TF expression by MM-LDL and LPS in human endothelial cells occurs principally at the level of gene transcription.

Interleukin-2 increases production and secretion of parathyroid hormone-related peptide by human T cell leukemia virus type I-infected T cells: possible role in hypercalcemia associated with adult T cell leukemia.

Although parathyroid hormone-related peptide (PTHRP) is produced by adult T cell leukemia (ATL) cells and causes hypercalcemia in ATL patients, very little is known about the regulation of PTHRP gene expression in the leukemic cells. The present study was undertaken to clarify the role of T cell growth factor, interleukin-2 (IL-2), in the expression of PTHRP gene, using a human T cell leukemia virus type I (HTLV-I)-infected T cell line, MT-2. Recombinant human IL-2 caused a transient increase in the steady state level of PTHRP messenger RNA (mRNA) in MT-2 cells, and a maximal effect was observed at 3-6 h. The effect of IL-2 was dose dependent, with a maximal response being observed at 10(-10) M. A monoclonal antibody against IL-2 receptor (anti-Tac antibody) inhibited the IL-2-induced increase in PTHRP mRNA level. Recombinant human IL-1, IL-3, IL-4, and IL-6 failed to increase PTHRP mRNA level. Nuclear run-off transcription assay showed that the transcription rate of the PTHRP gene was modestly increased by IL-2. In addition, IL-2 caused a substantial increase in the stability of PTHRP mRNA, compared with control cells in which the apparent half-life of PTHRP mRNA was less than 30 min after RNA synthesis was inhibited by the RNA polymerase II inhibitor, dichlorobenzimidazole riboside. The secretion of PTHRP, as determined by both a newly established immunoradiometric assay using recombinant human PTHRP(1-87) as the standard and an RIA using an antibody against PTHRP(109-141), was increased by IL-2 but not by IL-1, IL-3, IL-4, or IL-6. The IL-2-induced increase in PTHRP secretion was completely inhibited by the addition of anti-Tac antibody. These results demonstrate that IL-2 stimulates the production and secretion of PTHRP by HTLV-I-infected T cells through specific binding to IL-2 receptor and that the effect of IL-2 is mediated by a posttranscriptional as well as a transcriptional mechanism. It is suggested that IL-2 may be involved in an auctocrine/paracrine fashion not only in the proliferation of HTLV-I-infected T cells but also in the enhanced production and secretion of PTHRP and thus the development of hypercalcemia in ATL patients.

Role of histocompatibility antigen gene and protooncogene expressions in intracerebral tumorigenicity of mouse neuroblastoma.

The role of N-myc, c-src, and major histocompatibility complex (MHC, H-2 in the mouse) class I antigen gene expressions in dimethyl sulfoxide (DMSO)-induced differentiation and intracerebral tumorigenicity was examined using a mouse MNB85 neuroblastoma cell line. A fluorescence-activated cell sorter disclosed cell-surface MHC enhancement by DMSO, causing an increase in cytotoxic T-lymphocyte sensitivity. Southern blot analysis verified a single copy of the proto-oncogenes and MHC deoxyribonucleic acids in both untreated and DMSO-treated MNB85 cells. Northern blot analysis indicated that DMSO treatment induced a decrease in N-myc and an increase in c-src and MHC messenger ribonucleic acids. Nuclear run-off transcription assay revealed down-regulation of N-myc at a posttranscriptional level, contrasted with primary up-regulation of c-src at a transcriptional level. Immunoprecipitation after treatment with enzyme endo-beta-N-acetyl-glycoseamidase H proved that the terminal glycosylation of MHC heavy-chain gene products normally occurs in the Golgi apparatus of MNB85 cells. Intracerebral tumorigenicity assay showed that cells highly MHC-expressed by DMSO were less tumorigenic than untreated cells in association with DMSO-augmented cytotoxic T-lymphocyte susceptibility. These results suggest that proto-oncogenes may be linked to cellular differentiation, while cell-surface MHC gene expression influences intracerebral immunosurveillance.

Transcription of the gene for parathyroid hormone-related peptide from the human is activated through a cAMP-dependent pathway by prostaglandin E1 in HTLV-I-infected T cells.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL), and hypercalcemia frequently associated with ATL is mediated by parathyroid hormone-related peptide (PTHRP). The present study was undertaken to clarify the role of cAMP second messenger system in the regulation of human PTHRP gene expression in ATL cells, using an HTLV-I-infected T-cell line, MT-2. Forskolin and dibutyryl cAMP (Bt2cAMP) caused a marked and transient increase in the steady-state level of PTHRP mRNA. The effects of these agents were dose-dependent, and the maximal effects were observed at 3 h. Nuclear runoff transcription assay showed that forskolin and Bt2cAMP caused an increase in the transcription rate of the human PTHRP gene. In contrast, the stability of PTHRP mRNA was only modestly increased by these agents. Forskolin and Bt2cAMP also increased the secretion of PTHRP by MT-2 cells, as determined by both a newly established immunoradiometric assay using two antibodies against human PTHRP-(1-34) and PTHRP-(50-83) and a radioimmunoassay using an antibody against human PTHRP-(109-141). Prostaglandin E1 (PGE1) caused a marked stimulation of intracellular cAMP production in MT-2 cells, whereas PGE2 and PGF2 alpha had only modest effects. The ability of these PGs to stimulate cAMP production correlated well with their ability to increase PTHRP mRNA level and the secretion of PTHRP. Indomethacin did not affect the basal level of cAMP production or PTHRP mRNA, suggesting that endogenous PG was not involved in the basal production of cAMP or PTHRP. When PGE1 was given to MT-2 cells together with interleukin 2, which is another stimulator of PTHRP gene expression, PTHRP secretion was synergistically stimulated. These results suggest that the transcription of the human PTHRP gene is enhanced through a cAMP-dependent pathway by PGE1 and that PGE1, as well as interleukin 2, plays an important role in the overexpression of the human PTHRP gene in HTLV-I-infected T cells leading to the development of hypercalcemia in ATL patients.

Developmental regulation of arylphorin gene activity in fat body cells and gonadal sheath cells of Galleria mellonella

The arylphorin gene of Galleria mellonella is permanently shut off in fat body cells within 48 h following the larval-pupal molt. Experiments were performed to determine the signal for this irreversible inactivation. Interruption of the normal pupal hormonal program, by debraining at day 0 or by head-ligation of pharate pupae, resulted in the presence of arylphorin transcripts for at least 10 days. Nuclear run-off transcription assays showed that persistence of these transcripts in debrained pupae was due to continued transcription rather than increased mRNA stability. Treating debrained pupae with 20-hydroxyecdysone (20-HE) resulted in a rapid decline (within 15 h) of arylphorin transcript levels. Taken together, the available data indicate than normal arylphorin gene inactivation is programmed by the release of 20-HE in the absence of juvenile hormone, which occurs beginning 24 h post-pupation and which initiates adult development. We also revisited the question of the tissue-specificity of Galleria arylphorin gene expression: arylphorin transcripts were detected by northern blot analysis in larval gonadal tissue, albeit at lower levels than in the fat body. Sensitive in situ hybridizations demonstrated that arylphorin transcripts were present in peripheral somatic sheath cells, but not in the enclosed germ line cells. Furthermore, about half of the gonads in batch preparations failed to stain at all, suggesting that unlike fat body cell expression, the gonadal expression of the arylphorin gene may be sex-limited.

The use of a wild-type dihydrofolate reductase-encoding cDNA as a dominant selectable marker and induction of expression by methotrexate

The cDNA ( DHFR) encoding the wild-type (wt) dihydrofolate reductase (DHFR) was used as a dominant selectable marker in the transfection of murine hybridoma Sp2/0-Ag14 cells by protoplast fusion. The initial clones contained 100–400 copies of integrated plasmid DNA, and the high level of wt DHFR protein produced enabled the cells to survive the drug selection at 100 nM methotrexate (MTX). The expression of the gene of interest was several fold higher than when the mutant DHFR with decreased MTX binding was used as the selection marker, presumably because the clones were more sensitive to the stress induced by MTX. When the clones were propagated at higher concentrations of MTX, expression of both DHFR and the gene of interest increased. This induction is freely reversible, and we have shown that it is controlled at the transcriptional level, by nuclear run-off transcription assays.

Transcriptional regulation of calcium-activated neutral protease in cardiomyocytes of hypertensive rats

Increased enzymatic activity of calcium-activated neutral protease II (CANP II) has been reported previously in cardiac tissues of rats with 2 kidney, 1 clip Goldblatt hypertension (2K, 1C-HT); this was associated with elevated intracellular free Ca ++. Because it was suggested that increased levels of the enzyme were responsible for the enhanced activity, CANP II mRNA expression was assessed in cardiomyocytes isolated from 2K, 1C-HT rats and from a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Utilizing a rabbit probe for the large subunit of CANP II (pLM 1006 cDNA), a 3.7 kilobase mRNA band was visualized in Northern blots of poly A+ RNA. Densitometric analysis of the blots revealed that there was a significant ( p < 0.005) increase in the levels of CANP II large subunit mRNA in cardiomyocytes of 2K, 1C-HT rats when compared with controls. Interestingly, CANP II mRNA levels were comparable in cardiomyocytes of SHR and Wistar Kyoto (WKY) rats. Results of nuclear runoff transcription assays indicated that enhanced expression of CANP II mRNA in 2K, 1C-HT rat hearts was regulated at the transcriptional level. The data support specific CANP II gene activation in the hearts of renal hypertensive rats.

Nerve growth factor employs multiple pathways to induce primary response genes in PC12 cells.

Nerve growth factor (NGF) leads to neuronal differentiation of PC12 cells and promotes their survival in serum-free medium. Past studies have shown that purine analogues block some of the effects of NGF but not others and thus that they can be used to dissect the mechanistic pathways of its action. In the present work we used 2-aminopurine (2-AP) and 6-thioguanine (6-TG) to examine whether NGF causes activation of primary response genes through a single signaling pathway or via multiple pathways. Northern blot analysis and nuclear run-off transcription assays were used to assess the activation of c-fos, c-jun, TIS1, TIS8, and TIS11 after exposure of PC12 cells to NGF in the presence or absence of 2-AP and 6-TG. Our findings indicate that NGF appears to employ at least three distinct pathways to induce early genes in PC12 cells. This suggests that the NGF signaling mechanism diverges at an early point after interaction of NGF with its receptor.

Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response.

Eucaryotic organisms possess natural defense processes triggered by stress factors such as injury, infection, and inflammation. The acute phase response is an early defense mechanism during which striking changes in protein synthesis occur in the liver and other tissues. The altered expression of many acute phase protein genes is at the transcriptional level. Some of these genes have DNA binding sites for the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. We report here that in vivo expression of three isoforms of C/EBP is dramatically changed during the acute phase response. The steady-state mRNA levels of C/EBP alpha decreased significantly in the liver, lung, and fat tissues of lipopolysaccharide (LPS)-treated mice; moreover, nuclear run-off transcription assays indicated a decrease in the rate of C/EBP alpha gene transcription in isolated liver nuclei. The steady-state levels of C/EBP beta and a new isoform, C/EBP delta, were dramatically increased in many tissues within 4 h following LPS treatment. The rates of transcription of these two genes were only minimally altered in liver but significantly increased in kidney nuclei isolated from stimulated animals. Thus, the C/EBP isoforms exhibit differential mechanisms in their responses to LPS in various tissues and are likely to play an important role in mediating the transcriptional activation of genes involved in the acute phase response.

Regulation of globin gene expression in human K562 cells by recombinant activin A

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

Absence of c- fos induction in neonatal rat brain after seizures

Induction of the proto-oncogene c- fos is often considered to be a marker of increased neuronal activity. We have used in situ hybridization to study the pattern of c- fos expression in limbic structures following kainic acid-induced seizures during the postnatal period in the rat. Prior to postnatal day 13 (P13), seizure activity did not result in c- fos induction in any limbic structure. Between P13 and P25, a gradual increase in c- fos expression was observed in hippocampus and cortical structures. These results were corroborated by nuclear run-off transcription assay. Thus, alterations in c- fos transcription that may facilitate stimulus-transcription coupling occur during postnatal development. The possible relationship between the postnatal maturation of c- fos expression and the increase in susceptibility of specific neuronal populations to seizure-induced cell damage is discussed.