The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

Glutamine synthetase expression in perinatal spiny mouse liver.

The pronounced increase in the protein/mRNA ratio of ammonia-metabolising enzymes in rat liver in the last prenatal week represents a clear example of a post-transcriptional level of control of gene expression. Both the underlying mechanism, namely an increase in translational efficiency of the mRNA and/or enhanced stability of the protein, and its importance for perinatal adaptation are unknown. We investigated this process in spiny mouse liver, because the comparison of rat and spiny mouse can discriminate adaptively from developmentally regulated processes in the perinatal period. We focused on glutamine synthetase (GS) because of the conveniently small size of its mRNA. Prenatally, GS enzyme activity slowly accumulated to approximately 1.3 U x g-1 liver at birth and postnatally more rapidly to 5.5 U x g-1 at 2 weeks. Both phases of enzyme accumulation obeyed exponential functions. Western-blot analysis showed that changes in GS activity reflected changes in GS protein content. GS mRNA content of the liver was 45 fmol x g-1 at 2 weeks before birth and slowly declined to approximately 25 fmol x g-1 at 2 weeks after birth. The GS protein/mRNA ratio increased 2.5-fold prenatally and sixfold postnatally. Analysis of prenatal and postnatal polysome profiles revealed no evidence of GS mRNA-containing ribonucleoprotein particles. Instead, GS mRNAs were (fully) occupied by 12 ribosomes, indicating regulation at the level of elongation. The kinetics of GS protein accumulation, in conjunction with GS mRNA content, are consistent with an approximately sixfold increase in its rate of synthesis at birth as the result of a corresponding stimulation of the rate of elongation.

The spatio-temporal control of the expression of glutamine synthetase in the liver is mediated by its 5'-enhancer.

In previous studies of the glutamine synthetase gene, the promoter and two enhancer elements, one in the upstream region and one within the first intron, were identified. To analyze the role of the far-upstream enhancer element in the regulation of the expression of the glutamine synthetase gene, two classes of transgenic mice were generated. In GSK mice, the basal promoter directs the expression of the chloramphenicol acetyltransferase reporter gene. In GSL mice reporter gene expression is driven, in addition, by the upstream regulatory region, including the far-upstream enhancer. Whereas chloramphenicol acetyltransferase expression was barely detectable in GSK mice, high levels were detected in GSL mice. By comparing chloramphenicol acetyltransferase expression with that of endogenous glutamine synthetase in GSL mice, three groups of organs were distinguished in which the effects of the upstream regulatory region on the expression of glutamine synthetase were quantitatively different. The chloramphenicol acetyltransferase mRNA in the GSL mice was shown to be localized in the pericentral hepatocytes of the liver. The developmental changes in chloramphenicol acetyltransferase enzyme activity in the liver were similar to those in endogenous glutamine synthetase. These results show that the upstream region is a major determinant for three characteristics of glutamine synthetase expression: its organ specificity, its pericentral expression pattern in the liver, and its developmental appearance in the liver.

Identification and functional characterization of regulatory elements of the glutamine synthetase gene from rat liver.

Hepatic glutamine synthetase (GS) shows a unique expression pattern limited to a few hepatocytes surrounding the terminal hepatic veins. Starting from the genomic clone of the rat GS gene, lambda GS1 [Van de Zande, L. P. G. W., Labruyère, W. T., Arnberg, A. C., Wilson, R. H., Van den Bogaert, A. J. W., Das, A. T., Frijters, C., Charles, R., Moorman, A. F. M. & Lamers, W. H. (1990) Gene (Amst.) 87, 225-232] additional genomic clones containing up to 9 kb of 5'flanking region were isolated in order to characterize cis-acting elements involved in the regulation of GS expression. Sequence analysis of the 5'flanking region up to -2520 bp revealed a putative AP2-binding site at -223 bp and a second GC box at -2343 bp in addition to the canonical TATA, CCAAT and GC boxes found proximal to the transcription-start site. A possible negative glucocorticoid-responsive element (GRE) and regions with very weak similarity to a GRE and to a known silencer element were noted at -506 bp, -406 bp and at -798 bp, respectively. Within the sequenced part of the 5'flanking region no known regulatory elements associated with liver-specific gene expression were found except for a putative HNF3-binding site at -896 bp. Functional analysis by transient transfection assays using constructs with the pSSCAT or the pXP1 vector revealed that the elements present within the first 153 bp and particularly the first 368 bp of upstream sequence constitute an active promoter the activity of which is decreased by additional sequences up to -2148 bp. The presence of dexamethasone led to a 2-4-fold increase in the promoter activity of all these constructs. Using the heterologous truncated thymidine-kinase-gene promoter of the plasmid pT81-luc a strong enhancer element was located between -2520 bp and -2148 bp. Its activity was not affected by dexamethasone but was negatively influenced by flanking sequences in both directions. This enhancer was also effective with the homologous GS promoter (-153 to +59 bp) and the heterologous full thymidine-kinase-gene promoter (pT109luc). No further enhancers were found up to -6200 bp. Using the same approach, a second enhancer was found between +259 bp and +950 bp within the first intron. Deoxyribonuclease-I hypersensitivity studies confirmed the presence of a hypersensitive site between +350 bp and +550 bp and suggested a second site between +850 bp and +1200 bp.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression patterns of mRNAs for ammonia-metabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity.

The expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver by in situ hybridization. In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity. Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.

Isolation and characterization of the rat glutamine synthetase-encoding gene.

From a rat genomic library in phage lambda Charon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5-10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-length cDNAs containing a short poly(A) tract were identified. The sequences of the entire mRNA and of the exon-intron transitions were determined. The smaller mRNA is identical to the 5' 1375 nt of the long mRNA and contains the entire protein-coding region. The position of the transcription start point was mapped. Within the first 118 bp of promoter sequence, a (T)ATAA-box, a CCAAT-box and an SP1-binding site were identified.

Creatine kinase isozyme expression in prenatal rat heart.

The distribution pattern of creatine kinase (E.C 2.7.3.2) isozymes in prenatal rat heart and skeletal muscle was studied by immunohistochemistry. Between embryonic day (ED) 12-18, creatine kinase M (CK-M) is heterogeneously expressed in the heart: a pronounced staining of CK-M is first observed in the outflow tract and the trabeculae of the right ventricle (ED12-14), and subsequently in the venous valves, the interatrial septum and the sinoatrial node. From ED18 onwards, a homogeneous expression of CK-M is observed due to an increase in isozyme concentration in the remaining part of the myocardium. By contrast, the developmental appearance of creatine kinase B (CK-B) occurs almost homogeneously throughout the heart between ED11-14. Thereafter, a decrease of the CK-B is first observed in the inflow tract (in particular in the sinoatrial node), in the inner part of those atrial walls that are adjacent to the atrioventricular junction, and temporarily in a band in the upper part of the interventricular septum. From ED18, a selective disappearance of CK-B is found in the papillary muscle of the left ventricle. At birth, a considerable amount of CK-B remains present in the ventricular walls. Although some of the stage-dependent regional differences in expression of the creatine kinase isozymes, in particular those of the M-subunit, are shared by other mammalian and avian species, their significance for the developmental changes in the physiology of the heart is speculative at present.

The effects of alpha M-foetoprotein, an acute phase protein, and BaSO4-induced injury on IgE-mediated, systemic anaphylaxis in the rat.

A recently developed method for inducing fatal, IgE-mediated, bronchial and cardiovascular anaphylaxis in the rat was used to compare the effects of exogenously administered, purified alpha M-foetoprotein (alpha M FP) and BaSO4 pretreatment (as mean to induce an acute phase reaction with increased alpha M FP serum levels) with regard to mortality, bronchoconstriction and cardiovascular events. The BaSO4 pretreatment protected the rats almost completely against mortality, whereas exogenously administered alpha M FP offered no protection at all. With respect to the antigen-induced bronchoconstriction alpha M FP greatly inhibited the increase of the pulmonary resistance (RI), whereas the BaSO4 pretreatment suppressed either the dynamic lung compliance (Cdyn) or RI considerably. The cardiovascular events were only influenced by the BaSO4 pretreatment demonstrating a small but highly significant reduction of the initial fall in blood pressure together with a remarkable recovery within almost I h in the majority (91%) of the animals. Both exogenously administered alpha M FP and BaSO4 pretreatment increased the alpha M FP serum levels from a normal value of 59 +/- 4 micrograms/ml (n = 22), to 2732 +/- 252 micrograms/ml (n = 9) and 855 +/- 200 micrograms/ml (n = 22), respectively. From these data we conclude that the antianaphylactic activity of alpha M FP is limited to bronchoprotection of the more central parts of the lungs, whereas BaSO4 pretreatment covers a much broader antianaphylactic profile. This implies that BaSO4 pretreatment does not only induce alpha M FP but also other endogenous antianaphylactic factors.

Depletion of total acetylcholine by hemicholinium-3 in isolated rat diaphragm is less in the presence of dexamethasone.

Low concentrations of dexamethasone (Dex) stimulate the initial rate of radioactive choline (Ch) accumulation in the endplate-rich area (EPA) of indirectly stimulated hemidiaphragms, while higher concentrations (greater than 0.6 microM) inhibit. This biphasic concentration-effect curve is found even in the presence of 26 microM hemicholinium-3 (HC-3), an inhibitor of Ch accumulation. In incubations (3 min) where the total hemidiaphragm acetylcholine (ACh) content is not altered by 26 microM HC-3, the inhibition by HC-3 of both the Ch accumulation rate and the incorporation of radioactive Ch into ACh in the EPA of stimulated tissues is less in the presence of 0.2 microM Dex. In 120 min incubations with 15 microM HC-3 and without added Ch, the tissue ACh content is depleted in both stimulated and unstimulated hemidiaphragms. In both cases the depletion of ACh is significantly less in the presence of 0.2 microM Dex. In stimulated tissues a comparable depletion of ACh due to 15 microM HC-3 is also found with 1 and 10 microM Ch added to the medium. It is significantly less when 0.2 microM Dex and 1 microM Ch are added to the medium. In 120 min incubations with stimulated tissue, the amount of "bound' ACh is increased by addition of 30 microM Ch to the medium, decreased in the presence of 0.2 microM Dex, and greatly decreased in the presence of 15 microM HC-3. In the presence of Dex plus HC-3, the decrease in the amount of "bound' ACh due to either Dex or HC-3 alone, is abolished provided that 30 microM Ch is also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic, facilitatory effects of the corticosteroid dexamethasone in rat diaphragm: modulation by beta-bungarotoxin.

Low concentrations of dexamethasone (up to 200 nM) increase the accumulation of choline (Ch) and its incorporation into acetylcholine (ACh) in the endplate rich area (EPA) of stimulated and unstimulated diaphragms in the presence of 10 microM Ch. Tissue ACh is not significantly altered, even after 140 min incubation. The specific radioactivity of the ACh in the EPA is thus increased by dexamethasone (Dex). The corticosteroid has no effects on acetylcholinesterase or choline acetyltransferase in diaphragm extracts. In the same medium, the amplitudes of the MEPPs, MEPCs and EPCs are also increased by Dex. Neither the quantal content of the EPCs nor the MEPP frequency, nor the half decay time of the MEPCs are altered. Therefore Dex (200 nM) increases both the resting and evoked output, and turnover of ACh in rat diaphragm. Beta-bungarotoxin (beta-BuTx) antagonizes the Dex-induced increase in Ch accumulation and its incorporation into ACh, and abolishes the increases in MEPC- and EPC-amplitudes, providing further argument for a presynaptic effect of Dex. In continuously-stimulated diaphragms, beta-BuTx causes an accumulation of ACh which is much greater than in unstimulated tissue. This accumulation of ACh is less in the presence of Dex, provided that Dex is added before beta-BuTx. The interaction of Dex and beta-BuTx is discussed in terms of their possible presynaptic sites of action.

Characterization of two paralysing protein toxins (A-MTX and B-MTX), isolated from a homogenate of the wasp Microbracon hebetor (Say).

Two paralysing toxins (A-MTX and B-MTX) from extracts of Microbracon hebetor (Say) wasps were isolated and purified by gel chromatography, ion exchange chromatography and gel electrophoresis. Both toxins are labile proteins with molecular weights of 43,700 and 56,700, respectively, as estimated by gel chromatography, and with isoelectric pH of 6.85 and 6.62, respectively. Both toxins are inactivated by proteolytic enzymes and by dithiothreitol, but A-MTX appears to be more resistant than B-MTX. The relative amino acid compositions of both toxins show great similarity. The biological effects of the two toxins were identical to those previously found for crude toxin preparations.