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Recent studies have established that glial cells are important targets of the neurotransmitter dopamine (DA), but the regulatory effects of DA on glial cells have not been extensively studied. In the present study, we have investigated the influence of DA on gene transcription in glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes cultured from rat cerebral tissues in standard media or in culture media treated with DA. PCR-select differential screening was used to further verify the differentially expressed cDNA clones, positive clones were sequenced, and the mRNAs were re-examined on Northern blots. Fourteen sequences were identified of which eleven are homologous to known genes, three are homologous to expressed sequence tags (ESTs). Three novel full-length cDNAs were isolated using the EST fragments as probes to screen a cDNA library constructed from human brain. Analysis of these sequences suggested that complex intracellular signaling pathways, involving crosstalk with growth factor pathways, steroid hormone pathways, and an interferon-regulated 2-5 A pathway, are responsive to DA in astrocytes. The responsive proteins downstream from the signaling pathways were found to fall into at least three groups, including a series of metabolic enzymes, stress proteins, transfer proteins, etc. In addition, several of them have established their relationships with specific neurodegenerative diseases, showing that there is overlap in the pathogenic mechanisms of different diseases. Our results have provided a foundation for better understanding of the molecular basis of glial cell functions in dopaminergic transmission and an approach to find possible medication for the related disorders.

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978+/-0.0035 from 0.941+/-0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10(-14)) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K(+) channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration.

Chronic ethanol exposure produces changes in behavior that may result from effects of ethanol on gene expression. To identify potentially ethanol-regulated genes, mRNA differential display was used to screen the expressed genes in whole brain of mice chronically exposed to ethanol vapors. Mice genetically selected for susceptibility (Withdrawal Seizure-Prone; WSP) or resistance (Withdrawal Seizure-Resistant; WSR) to ethanol withdrawal convulsions were exposed to either ethanol vapor (ETOH group) or air (CTL group) for 72 h. A putative ethanol-regulated product was isolated; nucleotide sequence analysis of this product revealed >85% nucleotide identity to human neuroendocrine-specific protein (NSP) gene. Northern analysis of the expression of this product revealed hybridization to two transcripts ( approximately 3.0 kb and 1.4 kb) on blots containing whole brain RNA, consistent with the transcript sizes of hNSP. Ethanol-induced regulation of mNSP was suggested in whole brain of WSP mice, but not in WSR mice, by Northern blot analysis. One transcript (3.0 kb) suggests a 26% increase in relative abundance in whole brain of ethanol-exposed WSP mice, while there was no effect of ethanol on abundance of the 1.4-kb transcript in WSP mice. No effects of ethanol were observed for WSR mice. These preliminary findings suggest that mNSP represents a novel ethanol-induced gene in mice selected for genetic susceptibility to severe ethanol withdrawal.

Diabetic nephropathy is characterised by an accumulation of extracellular matrix proteins in the glomerular mesangium. Hyperglycaemia is a major factor promoting this progressive expansion of the mesangial matrix. We have used the technique of mRNA differential display to investigate changes in gene expression in cultured human mesangial cells following long-term (21 days) exposure to either physiologic (4 mM) or pathologic (30 mM) D-glucose concentrations. Approximately 12,000 mRNA species were screened for evidence of altered expression and several hundred candidate cDNA fragments were obtained. Northern blot and RT-PCR analysis of ten randomly chosen candidate cDNA fragments revealed three exhibiting increased mRNA expression under elevated D-glucose levels. Nucleotide sequence analysis identified two of the cDNA fragments as encoding prolyl 4-hydroxylase alpha-subunit and thrombospondin-1. The third cDNA fragment represents a novel glucose-regulated gene, encoding a putative zinc finger protein. Upregulated expression of these genes in response to high levels of D-glucose may contribute significantly to the disease process. mRNA differential display is a useful tool to investigate the mechanism of diabetic nephropathy.

The calbindin D9k (CaBP9k) gene is under strict estrogen control in the rat uterus. This tissue contains two CaBP9k messenger RNA (mRNA) species. We have used primer extension analysis, reverse transcriptase associated with polymerase chain reaction, and RNase H digestion to show that these two mRNA species have the same structural features, including 5'- and 3'-ends, and poly(A) tail length. Our results suggest that the difference in electrophoretic mobilities of the two mRNA species might be due to interaction with another factor. We also analyzed the imperfect estrogen-responsive element (ERE) present on the first 5'-splice site of the rat CaBP9k gene. The oligonucleotide corresponding to the CaBP9k ERE was cloned in the plasmid pBLCAT2 (where the thymidine kinase promoter governs the expression of the chloramphenicol acetyl transferase gene) and transfected into MCF7 cells. This CaBP9k ERE was found to be a hormone-inducible enhancer that worked in an orientation-independent manner on a heterologous promoter and was functional at physiological hormone concentrations. One CaBP9k ERE conferred only weak (about 2-fold) estrogen induction, but two EREs cloned in tandem were strongly synergistic (14- to 16-fold). The CaBP9k ERE also bound to the partially purified estrogen receptor (ER) and to ER expressed in COS cells by gel shift assay. Methylation interference showed that all the guanine residues in both half-sites of the CaBP9k ERE were protected by ER binding. Thus, ER binds to the CaBP9k ERE in a way similar to other EREs. The gel shift assay results indicate that the strong synergistic effect of two EREs cloned in tandem is not due to cooperative binding between the two elements. As the CaBP9k gene is under strong estrogenic control in the uterus in vivo, the imperfect CaBP9k ERE may cooperate with another trans-acting factor to become fully efficient.

Precise neural mechanisms underlying the pathophysiology and pharmacotherapy of psychotic disorders remain largely unknown. Present studies investigated the effects of various antipsychotic drugs on expression of the gene encoding the purported endogenous antipsychotic-like peptide neurotensin (NT) in striatal regions of the rat brain. The results demonstrate that several clinically efficacious antipsychotic drugs selectively and specifically increase expression of NT/neuromedin N (NT/N) mRNA in the shell of the nucleus accumbens, a region of the forebrain associated with limbic systems. On the other hand, only typical antipsychotics that cause a high incidence of acute motor side effects increased the expression of NT/N mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. In addition, it appears that distinct mechanisms may be involved in the effects of antipsychotics on NT/N gene expression in the dorsolateral striatum versus the accumbal shell. Thus neuroleptic-induced increases in NT/N mRNA expression in the dorsolateral striatum were preceded by a rapid and transient activation of c-fos mRNA, whereas none of the antipsychotics affected c-fos mRNA expression in the accumbal shell. The anatomical characteristics of NT/N gene expression induced by typical versus atypical antipsychotics raise the possibility that increased activity of specific NT neurons may contribute to the therapeutic effects (NT neurons in the accumbal shell) or motor side effects (NT neurons in the dorsolateral striatum) of these drugs.

Since cadmium exposure results in neuropathological alterations in central nervous system, we investigated the effects of cadmium on the gene expression of neuroblastoma (GOTO) cells. We observed an increase in mRNA levels of heat-shock protein (hsp) 70, hsp 90, hsp 32 and metallothionein after treatment of GOTO cells with cadmium, although the time courses of the changes of individual mRNA of the heat-shock proteins and metallothionein were somewhat different from each other. An accumulation of N-myc and multidrug-resistance gene (MDR1) mRNA was detected in the presence of cadmium. This is contrary to the previous report, in which an inverse correlation between the expression of MDR1 gene and N-myc oncogene in human neuroblastoma had been described. However, the increase of N-myc and MDR1 mRNA in the present study is not likely due to the loss of regulatory mechanism of these genes by cytotoxic effects of cadmium, because active protective mechanisms such as heat-shock proteins and metallothionein could be induced under these conditions.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Tissue factor (TF) is the membrane-bound glycoprotein whose cofactor activity with factor VIIa causes activation of the extrinsic pathway of coagulation. The transition of endothelium to a procoagulant state by agents such as bacterial lipopolysaccharide (LPS) is the result of TF expression by these cells. The mechanism of TF induction in human umbilical vein endothelial cells (HUVEC) was investigated in response to LPS and phorbol 12-myristate 13-O-acetate (PMA). Northern blot analysis of total RNA from HUVEC showed a rapid rise in TF mRNA levels which was maximal at 2 h and had fallen to low levels by 6 h following both LPS (10 micrograms/ml) and PMA (10 ng/ml) stimulation. Nuclear-run on experiments showed at most a 2-fold increase in transcription of the TF gene following LPS stimulation but a 10-fold increase following PMA stimulation. In addition 24-h pre-incubation with PMA desensitized HUVEC to further PMA exposure, but caused no alteration in the response to LPS. Cycloheximide (10 micrograms/ml) alone caused induction of TF mRNA. Treatment of cells previously exposed to LPS for 1 or 4 h with actinomycin D indicated a 12-fold difference in the TF mRNA half-life. Therefore the rapid accumulation of TF mRNA in HUVEC stimulated by LPS is largely a result of an increase in mRNA stability rather than an increased rate of transcription of the gene.

The Na,K-ATPase alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes exhibit a complex pattern of expression during heart development. To identify possible molecular signals that regulate the differential expression of these genes, isolated neonatal rat myocardial and non-myocardial cells were cultured in chemically defined medium and the responses of the multiple Na,K-ATPase subunit mRNAs to various hormones were tested. Myocardiocytes in control cultures express primarily alpha 1 and beta mRNAs. Triiodothyronine (T3) induced the expression of alpha 2, alpha 3, and beta mRNAs without influencing alpha 1 mRNA levels. Dexamethasone (DEX) treatment similarly induced alpha 2 mRNA levels, but the abundance of the other subunit transcripts remained unaltered. T3 and DEX together caused increases in alpha 2 and beta mRNA, increments similar to that observed with T3 alone. However, DEX specifically repressed the induction of alpha 3 mRNA by T3. Both hormones stimulated corresponding changes in the sarcolemma concentration of these Na,K-ATPase isozymes. Addition of norepinephrine to the cultures had little appreciable effect on expression of the alpha isoform and beta mRNAs. Although characterized less extensively, control cultures of non-myocardiocytes expressed alpha 1, alpha 3, and beta mRNAs, of which only the beta mRNA was stimulated by T3. These data indicate that thyroid and glucocorticoid hormones differentially regulate the expression of multiple alpha isoform and beta subunit mRNAs of Na,K-ATPase in cardiocytes in vitro and, therefore, may also be important physiological modulators in vivo.

A combined approach of cDNA cloning and direct oligonucleotide mapping of TGF-beta 1 mRNA from several human cell lines has revealed that the major human TGF-beta 1 transcript is 381 bases shorter than originally reported, and that the reduced mRNA size is due to polyadenylation from an ATTAAA signal at position 2136 rather than use of the expected AATAAA signal at position 2517. Moreover, there is no evidence for a significant amount of structural heterogeneity, as a result of alternative polyadenylation, in the human TGF-beta 1 transcripts. Considering that the 381-base domain is not part of the major human TGF-beta 1 mRNA, we analyzed this sequence for potential transcriptional regulatory elements. We have identified a 16-base pair domain which contains three putative phorbol ester responsive elements (TREs) based on homology to the TRE consensus sequence. We also show that this 16-base pair fragment confers phorbol ester responsiveness to the chloramphenicol acetyltransferase gene after transient transfection of the heterologous construct in NIH-3T3 cells. The identification of a TRE immediately downstream of the last TGF-beta 1 exon suggests that a 3' enhancer may play an important role in human TGF-beta 1 gene transcription, and suggests a basis for growth factor-mediated regulation of TGF-beta 1 expression by activation of protein kinase C.

Dexamethasone rapidly stimulated transcription of the tyrosine aminotransferase and metallothionein-I genes--but not of the phosphoenolpyruvate carboxykinase gene--in rat hepatocytes cultured in serum-free medium. This differential response was not observed for cyclic AMP. The results suggest that the phosphoenolpyruvate carboxykinase gene--but not the tyrosine aminotransferase and metallothionein-I genes--requires a factor which is permissive for stimulation of transcription by the glucocorticoid receptor.

Previous studies of sucrase-isomaltase (SI) activities have shown this complex to be absent in the suckling rat and to appear during the weaning period. We describe here the cloning of a heterologous SI cDNA and its use for the quantitation of SI mRNA as a first step toward understanding the molecular basis of SI development. A survey of RNA from 12 tissues of mature rats by Northern blot analysis showed a 6-kb band of SI mRNA only in the small intestine. Within the latter, both sucrase activity and SI mRNA peaked in the jejunum. Assay of jejunal tissue from developing rats showed sucrase activity and SI mRNA to be first detectable at 18 days, to rise in parallel through 24 days, and then to diverge a little (enzyme activity being lower) by 36 days. When glucocorticoid was administered to 10-day-old rats, neither sucrase activity nor SI mRNA was detectable 12 h later. Both parameters were readily detected 24 h postinjection, although the mRNA had risen relatively more than the enzyme activity. The two parameters increased in concert through 5 days postinjection and then plateaued. We conclude that, with respect to distribution along the intestine and to normal and precocious development, activities of SI in the rat are determined primarily by the abundance of its mRNA.

Interleukin 3 (IL-3) is transiently produced by murine bone marrow-derived mast cells in response to antigen stimulation of the high-affinity immunoglobulin E receptors. We have studied the postreceptor signaling pathways involved in regulating expression of the IL-3 gene in the murine mast cell line PB-3c. Large amounts of IL-3 mRNA accumulated after exposure of cells to calcium ionophore A23187, a reagent that increases intracellular Ca2+. Phorbol 12-myristate 13-acetate, which stimulates protein kinase C, did not induce IL-3 mRNA accumulation, although it did potentiate the effect of A23187. Nuclear run-on analysis showed that the IL-3 gene is constitutively transcribed in unstimulated cells and that treatment with A23187 and/or phorbol ester has no influence on its transcription rate. The effect of A23187 was found to be due to stabilization of the IL-3 mRNA. In cells maintained in the presence of A23187 the IL-3 mRNA was stable during 3 hr of incubation with actinomycin D, whereas removal of A23187 under the same conditions resulted in rapid degradation of the mRNA. These results indicate that control of expression of the IL-3 gene in mast cells is primarily at the posttranscriptional level and that the Ca2(+)-dependent signal-transduction pathway plays an important role in this process. Synthesis of granulocyte/macrophage colony-stimulating factor mRNA in response to A23187 and phorbol ester was found to be subject to both transcriptional and posttranscriptional regulation.

Control of ferritin synthesis by iron at the level of transcription is potentially hazardous to DNA because of the iron-catalyzed degradation of DNA. The induction of ferritin synthesis in reticulocytes of embryos (bullfrog tadpoles) occurs by two types of translational control i.e. increased availability of stored ferritin mRNA, in response to iron, coupled with a high translational efficiency. Since erythroid cell nuclei have large amounts of heterochromatin and may be relatively inactive genetically, the translational control of ferritin by iron observed in red cells was studied in other tissue by isolating poly (A+) RNA from tadpole liver and analyzing protein synthesis in vitro. Liver ferritin mRNA directed the synthesis of 7.0% of the protein in a wheat germ system, compared to 1.2% in vivo, suggesting that tadpole liver contained a large amount of stored ferritin mRNA. At levels of poly (A+) RNA which were saturating for total protein synthesis, ferritin synthesis was still linearly dependent upon RNA concentration, indicating a high efficiency of translation of ferritin mRNA. The results are analogous to those previously observed in red cells and confirm the storage of ferritin mRNA deduced from studies of the polysomal and nonpolysomal distribution of the mRNA in rat liver. The results indicate that the increased availability for translation of stored ferritin mRNA, in response to iron, and the high translational efficiency of ferritin mRNA are a general characteristic of ferritin synthesis rather than a specific feature of red cell maturation. This novel form of regulation of ferritin gene expression can be attributed to a need to protect DNA from degradation by iron and oxygen. The normal barrier between DNA and iron is apparently breached by the iron-oxygen complex of the drug bleomycin, an antitumor agent thought to act in vivo by iron-catalyzed cleavage of DNA.

One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3'-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.

Eighteen cDNAs, cloned from interferon-treated T98G neuroblastoma cells, correspond to seven different mRNAs induced up to 40-fold by interferon. One codes for metallothionein II and another for a class I HLA. The others do not code for proteins of known sequence. In the continued presence of interferon, accumulation of the mRNAs continues for about 1 day but ceases whenever interferon is removed. Once induced, the mRNAs are stable. Synthesis of new proteins is not required for induction. The rate of transcription of one of the genes doubles 5 min after treatment with interferon and reaches a maximum by 60 min. This rate begins to fall after 4-6 hr, reaching the uninduced level by 8-12 hr. Since the mRNA continues to accumulate after 8-12 hr, posttranscriptional events must also play a role in increasing its level.

beta-Adrenergic receptor expression and receptor mRNA levels are down-regulated by beta-adrenergic agonists and up-regulated by glucocorticoids. The interaction between these two opposing regulatory pathways was investigated at the levels of receptor and receptor mRNA in DDT1 MF-2 hamster vas deferens cells. Dexamethasone blunted a marked decrease in receptor expression induced by isoproterenol alone, as made visible by indirect immunofluorescence using antireceptor antibodies. Receptor mRNA levels were quantified by DNA-excess solution hybridization. Dexamethasone stimulated a sharp increase in receptor mRNA at 4 h following the addition of steroid in either the absence or the presence of isoproterenol. By 12 h, dexamethasone treatment resulted in a new steady-state level of receptor mRNA double that observed in untreated cells. Isoproterenol blunted the dexamethasone effect observed at 12 h. Cells treated with isoproterenol and dexamethasone in combination displayed a new steady-state level only 30% greater than untreated cells. Measured by nuclear run-on assays, transcription rates of the receptor gene were unaffected in cells challenged with isoproterenol alone. Dexamethasone, in contrast, stimulated a 4-fold increase in beta 2-adrenergic receptor gene transcription. Isoproterenol and dexamethasone in combination promoted a transcription rate comparable to dexamethasone alone. The half-life of receptor mRNA in untreated and dexamethasone-treated cells was 12 h. In contrast, beta-adrenergic receptor mRNA half-life declined to 5 h in cells that were treated with isoproterenol in the presence or absence of dexamethasone. Agonist-promoted destabilization and steroid-induced transcription provide mechanisms for the interplay of two opposing pathways controlling receptor mRNA levels.

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function. To define further both the specificity of CsA and the level at which it interferes with lymphokine gene expression, we have studied its effects on TCGF mRNA accumulation as well as TCGF gene transcription. These studies were performed with a cloned human leukemic T-cell line (Jurkat, subclone 32), which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of TCGF. In these cells, high levels of TCGF mRNA were present in induced but not in uninduced Jurkat cells as judged by hybridization to a cloned human TCGF cDNA probe. CsA completely inhibited induced TCGF mRNA accumulation at concentrations of 0.3-1.0 microgram/ml, whereas low levels of appropriately sized TCGF mRNA were present at 0.01 microgram/ml. In nuclear transcription experiments, CsA inhibited the synthesis of TCGF transcripts in a dose-dependent manner with complete inhibition at a concentration of 1 microgram/ml. In contrast, CsA did not inhibit the expression of two other inducible genes, TCGF receptor and HT-3. Further, HLA gene expression was also less affected than TCGF in CsA-treated cells. These data suggest a relatively selective action of CsA on TCGF gene transcription.

The single-copy RP2 gene in mice produces three major mRNAs, the abundances of which are significantly increased in the kidneys by the administration of testosterone. S1 nuclease analysis of the kidney mRNAs indicated that they differ in the lengths of their 3' untranslated regions as a result of the use of different polyadenylation sites. When the mRNAs from different inbred mouse strains were examined by Northern blot analysis, it was observed that the largest mRNA varies in size, whereas the sizes of the other mRNAs remain the same. In DBA/LiHa and DBA/2J mice, the largest mRNA is approximately 2,150 nucleotides long, whereas the corresponding mRNA in C57BL/6J and BALB/cJ mice is only 1,950 nucleotides in length. All of these strains also have RP2 mRNAs that are 1,450 and 1,350 nucleotides long. By S1 nuclease mapping and comparison of the sequence of cDNA clones representing these mRNAs in DBA/LiHa and C57BL/6J mice, we determined that this size difference or polymorphism observed in the largest mRNA is the result of the insertion of a member of the B1 family of repeats into the 3' untranslated region of the RP2 gene in DBA mice. This particular B1 repeat is transcribed by RNA polymerase III in vitro, and its transcriptional orientation is opposite to that of the RP2 transcript. The polymorphism described here is evidence for the mobility of B1 repetitive elements within the genome.

In rat liver, triiodothyronine (T3) and dietary carbohydrate induce the expression of the genes coding for malic enzyme (ME) (EC 1.1.1.40) and S14 protein. The mRNAs for both ME and S14 are elevated under circumstances associated with augmented lipogenesis. Since the lag time in the induction of mRNA coding for S14 is short (20 min) and the lag time in the induction of the mRNA for ME is relatively long (2-6 h), the possibility arose that the induction of the ME gene by T3 was mediated by S14 protein. To test this hypothesis we examined the temporal relationship between the accumulation of the hepatic S14 protein and the mRNAs coding for ME. In confirmation of previous reports, we found that two mRNAs coded for ME, one 27 S and the other 21 S in size. The level of enzyme activity generated appeared to be determined by both mRNA species. Sequencing of the 27 S fragment established that this mRNA is generated as a consequence of the use of an alternate polyadenylation site downstream to that used in the 21 S mRNA. Unanticipated from the earlier descriptions was the finding of a markedly asynchronous response of these mRNAs to T3 in hypothyroid animals. The lag time following T3 administration was 90 min for the 27 S and fully 8-12 h for the smaller 21 S sequence. Despite the rapid rise of mRNA S14, the S14 protein could not be detected for approximately 12 h after T3 administration. This ruled out the possibility that S14 is an obligate mediator in the induction of the ME gene. A contrasting pattern was observed in the euthyroid state where both ME mRNAs had indistinguishable lag times of 2-3 h, and the S14 protein rose within the same time frame. The delayed response of the 21 S mRNA for malic enzyme in hypothyroid animals thus appears to be due to a reversible defect in the transcription of the ME gene.

The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in alpha 1-acid glycoprotein (alpha AGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron alpha AGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, alpha AGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A)+ RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some alpha AGP sequences in smaller, more heterogeneous poly(A)- RNA, and leakage of some alpha AGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature alpha AGP mRNA to the nucleus. In contrast to alpha AGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest alpha AGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A)+ RNA preparations which may represent 3'-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of alpha AGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of alpha AGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of alpha AGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of alpha AGP in the acute phase response.

The enzymes of the DNA synthesizing machinery constitute a group of gene products that are generally expressed co-ordinately at the G1/S boundary of the cell cycle. We have investigated how growth factors regulate the steady-state mRNA levels of two of these genes, the PCNA (proliferating cell nuclear antigen)/cyclin and the thymidine kinase genes. To detect the PCNA/cyclin mRNA, we isolated a cDNA clone from a human library. Two different cell lines were used for these studies: BALB/c3T3 cells, which are exquisitely sensitive to growth factors, and ts13 cells, a temperature-sensitive (ts) mutant of the cell cycle, which arrests in G1 at the restrictive temperature. The steady-state levels of the RNAs for these two genes under different growth conditions were also compared with the levels of histone H3 RNA which are good indicators of the fraction of cells in S phase. Both PCNA/cyclin and thymidine kinase genes share two fundamental characteristics, i.e. they are not inducible in a G1-specific ts mutant of the cell cycle at the restrictive temperature and their expression is inhibited by cycloheximide, indicating that unlike early growth-regulated genes, they require the previous expression of other growth-regulated genes. However, the two genes also show differences, the most notable being that PCNA/cyclin is inducible by epidermal growth factor alone, while thymidine kinase is not.

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

Levels of mRNA for lipoprotein lipase (LPL) in guinea pig epididymal adipose tissue, heart and liver were determined by dot blot analysis of total RNA using a cDNA probe complementary to the coding region, and compared to the LPL activity. For adipose tissue we also measured the incorporation of radioactivity into immunoprecipitable LPL after pulse-labeling with [35S]methionine. LPL activity was 93%, LPL mRNA 82% and LPL synthesis 85% lower in epididymal fat pads from animals fasted for 48 h compared to rigorously fed animals. In contrast, neither LPL activity nor LPL mRNA levels differed in heart. A single dose of tumor necrosis factor (TNF) decreased LPL activity and LPL mRNA in fat pads with no effects in heart. In the liver, TNF caused a marked increase in LPL mRNA levels, which are normally very low. Northern-blot analysis confirmed a previous observation that the patterns of mRNA species differ between heart, in which a 3.8-kb mRNA dominates, and adipose tissue, in which the LPL mRNAs of 3.3 and 2.1 kb occur in similar abundance as the 3.8-kb species.

This report directly documents that a polypeptide hormone can regulate specific gene expression as a consequence of increasing the levels of a primary genomic transcript. The regulation and expression of the prolactin gene was studied in a cell line (GH4) derived from a rat pituitary tumor. These cells respond to addition of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH; thyroliberin), by elevation of the levels of mature prolactin mRNA and increase in prolactin biosynthesis. The message induction is preceded by, and apparently consequential to, a comparable rapid increase in the nuclear prolactin RNA precursors, including the putative primary transcript.

It is demonstrated that the accumulation of 42 K casein mRNA in mammary tissue from adrenalectomized, virgin rats is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25 K casein mRNA in this tissue is totally dependent on the steroid. The results indicate a much greater dependency on hydrocortisone than was appreciated previously, and also show that this dependency does not reflect a loss of cell viability in the absence of the steroid.

We have applied techniques of multivariate analysis to the characterization and comparison of the effects of various pathophysiological and hormonal stimuli on the expression of the rat hepatic genome at a pretranslational level. In vitro translated products were resolved by two-dimensional gel electrophoresis. We analyzed 10 pathophysiological states brought about by variation in thyroidal status, starvation, administration of high carbohydrate diet, and the production of experimentally induced diabetes mellitus. Each state differed significantly from every other state in the two-dimensional electrophoretic profiles. The set consisting of the minimal number of products necessary for maintaining the distinctive patterns was identified. The analysis also defined those clusters of products that behaved in a coordinate fashion in response to the various stimuli. Lastly, the similarity and dissimilarity of hepatic mRNA activity profiles to each other could be geometrically represented in three-dimensional space. Our finding that the hepatic mRNA activity profile could distinguish reliably between closely related hormonal and pathophysiological stimuli indicates the specificity of hepatic genomic expression. A systematic analysis of such profiles may be useful as an overall index of the biologic response at the hepatocellular level.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.

Hepatic microsomal cytochrome P-450 was elevated in 17-day chick embryos by combined administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Increased apoprotein levels were the result of de novo protein synthesis; in vitro obtained translation data suggested that cytochrome P-450 mRNA levels were elevated. A 1000-base cDNA sequence for the drug-induced cytochrome P-450 mRNA was isolated from a chick embryo cDNA 'library' and this was used as a specific probe to investigate drug-mediated induction of cytochrome P-450 mRNA. RNA-DNA 'dot' hybridisation studies demonstrated that drug treatment led to a 3-5-fold increase in the level of this mRNA and that the mRNA was predominantly associated with membrane-bound polyribosomes. Treatment of embryos with the drugs individually demonstrated that both of them induced synthesis of the same mRNA. These studies show directly that treatment of chick embryos with 2-allyl-2-isopropylacetamide or 3,5-diethoxycarbonyl-1,4-dihydrocollidine caused increased levels of cytochrome P-450 mRNA and suggest that this involved increased transcription of the gene.

Calcium's role in a variety of cellular processes has been well documented. The storage, distribution, and delivery of calcium are regulated by a family of binding proteins including troponin C, calmodulin, parvalbumin, and vitamin D dependent calcium binding protein (CaBP-28), all of which have evolved from a common ancestral gene. To evaluate vitamin D regulation of gene transcription, a CaBP-28 cDNA (767 base pairs) was isolated from a chicken intestine lambda gt11 library utilizing a polyvalent CaBP-28 antibody as a probe. Coincident with the identification of the CaBP-28 cDNA, a group of cDNAs also was isolated (with the anti-CaBP-28 antibody) that demonstrated 84% nucleotide homology and 99% deduced amino acid homology with chicken brain calmodulin (CaM). This new CaM-like cDNA was named neoCaM. There is little nucleotide homology between the CaBP-28 cDNA and neoCaM. The CaBP-28 cDNA hybridizes with three transcripts of 2000, 2900, and 3300 bases which are dramatically induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], while the neoCaM cDNA recognizes three distinct (from CaBP-28) transcripts. Two of these mRNAs are 1400 and 1800 bases as described for brain CaM, but another larger 4000-base transcript is detected with neoCaM. Neither the CaM nor the neoCaM transcript reveals any modulation by 1,25(OH)2D3. Herein, we discuss the possible significance of not only the isolation of both cDNAs with a single antibody but also the relation of neoCaM to other well-characterized CaM cDNAs.

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.

Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.

Medium of untreated human FS-4 foreskin fibroblasts contained a factor which, upon the addition of exogenous tumor necrosis factor (TNF), inhibited encephalomyocarditis virus replication when neither medium alone nor TNF alone were effective. This antiviral activity was abolished by a monoclonal antibody to human interferon (IFN)-beta, suggesting that the active component in the medium from untreated FS-4 cells was IFN-beta, present at subeffective concentrations. In addition, we show that untreated FS-4 cells contain IFN-beta mRNA, demonstrable by the highly sensitive polymerase chain reaction after reverse transcription. Treatment of FS-4 cells with TNF produced an approximately 16-fold increase in the steady-state level of IFN-beta mRNA. Our results support the conclusion that autocrine IFN-beta is secreted by untreated normal fibroblasts and that TNF can enhance the production of autocrine IFN-beta by increasing the level of IFN-beta mRNA. Our study also demonstrates that subeffective concentrations of autocrine IFN-beta, which escape detection in conventional assays, are sufficient to produce a strong synergistic action with TNF.

We have previously shown that insulinlike growth factor I (IGF-I) inhibits growth hormone (GH) secretion and messenger RNA (mRNA) levels in pituitary cells. The effects of IGF-I on new GH mRNA synthesis rates in primary monolayer rat pituitary cells were therefore examined by nuclear runoff transcription assays. IGF-I (1.3 nM) treatment for 1 h inhibited GH gene transcription to 60% of controls. IGF-I (3.25 nM) maximally suppressed GH gene transcription to 30% of control values after 4 h. After 24 h treatment, GH transcription was suppressed to 48% of controls by 3.25 nM IGF-I. IGF-I (3.25 nM) also inhibited the twofold growth hormone-releasing hormone (GHRH) (10 nM)-stimulated GH gene transcription by 30% after 4 h. Transcription of the prolactin (PRL) gene was not suppressed in these cells by IGF-I. Relatively high doses of insulin (200 nM) also suppressed GH gene transcription, but epidermal growth factor and fibroblast growth factor did not change GH mRNA synthesis. The results show that IGF-I exerts a rapid and selective suppression of basal and GHRH-stimulated GH gene transcription. These data indicate a role for IGF-I in negative feedback of GH gene expression and provide evidence for the direct transcriptional regulation of the GH gene by IGF-I in primary rat anterior pituitary cells.

We have investigated the linkage between CD4/CD8 phenotype and programming for specific responses in primary T-cell populations. In situ hybridization has been used to determine the frequency of cells competent to express the interleukin 2 (IL-2) gene after short-term stimulation with various polyclonal activators. The effects of the T-cell receptor ligands Con A and anti-CD3 monoclonal antibody were compared with those of a calcium ionophore that bypasses membrane receptors altogether. Induction with a calcium ionophore and phorbol ester revealed that potential IL-2 producers not only constitute greater than 85% of the cells with a CD4+ "helper/inducer" phenotype but also constitute over half of the cells with a CD8+ "killer/suppressor" phenotype. There is no defect in the ability of these CD8+ cells to accumulate IL-2 transcripts under these conditions. By contrast, in response to phorbol ester and either Con A or anti-CD3, the CD8+ cells show an abortive IL-2 production response with rapid disappearance of IL-2 mRNA. This results in substantially lower yields of IL-2 per cell than is made by CD4+ cells in response to the same stimuli. The extent to which these populations appear to have diverged in function thus depends on the stimulus used to trigger the response. The results suggest that differences in signal transduction or posttranscriptional regulatory mechanisms, rather than effector gene inducibility per se, may initially underlie the commitment of CD4+ and CD8+ cells to distinct functional roles.

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.

Thyroid hormones exert "permissive effects" on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling.

The studies described in this report were carried out as a first step towards the elucidation of mechanisms underlying the tissue specificity of regulation of gene expression by estrogen. Using a procedure established earlier in our laboratory for primary culture of Xenopus hepatocytes, we have characterized how estradiol-17 beta and progesterone affect the rate of synthesis of total RNA and that of accumulation of two oviduct-specific mRNAs in Xenopus oviduct cells in primary culture. In cells that had recovered from 'culture shock' 3 days after they were plated out, both hormones had only a slight or no effect on the overall rate of labelling of newly synthesized RNA over 24 h. Cloned cDNA probes for two mRNAs, termed 7F and 6G and specifying as yet unknown proteins expressed in the oviduct and not in the liver, were used to quantify the two mRNAs. The levels of both mRNAs declined for the first 2 days in culture after which they were stabilized. When added to the oviduct cell cultures 3 days after they were plated out, estradiol increased the steady-state concentration (relative to total RNA) of 7F and 6G mRNAs by 3- to 7-fold after 60-80 h, but with different time-course and dose-response kinetics for the two messages. The antiestrogen tamoxifen also exerted different degrees of antagonist effect on the estrogen-induced accumulation of 7F and 6G mRNAs. Although the protein products of these two oviduct-specific mRNAs have not yet been characterized, these studies set the stage for comparing the regulation by estrogen of their genes with that of vitellogenin genes in primary cultures of Xenopus oviduct cells and hepatocytes.

BSF-2/IL-6, GM-CSF and IL-1 beta mRNAs were induced by recombinant IL-1 in human astrocytoma cell line U373MG. The induction of BSF-2/IL-6 and IL-1 beta mRNAs did not require de novo protein synthesis while that of GM-CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL-1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL-1 and glucocorticoids, respectively, in astrocytes.