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Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability.Fernandez J, Yaman I, Mishra R, Merrick WC, Snider MD, Lamers WH, Hatzoglou M.Departments of Nutrition and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, USA.The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (−12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from −161 to +138) and enhancer (469 bp around −6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5′-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein–protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.

Two methods are described for directing the expression of genes to the livers of animals using retroviral vectors containing the predominantly liver-specific promoter from the gene for phosphoenolpyruvate carboxykinase (PEPCK)-linked to the structural gene for either amino 3'-glycosyl phosphotransferase (neo) or bovine growth hormone (bGH). Replication-incompetent retrovirus was used to infect the livers of fetal rats by intraperitoneal injection of animals in utero or to infect adult rats by direct injection into the portal vein after partial hepatectomy. The proviruses were integrated into the hepatic DNA, and the chimeric genes were expressed from the PEPCK promoter for as long as 8 months after infection. The expression of the PEPCK-bGH gene was regulated by diet and hormones in a manner similar to the regulation of the endogenous PEPCK gene in the liver. The potential of this method for targeting genes to the liver is discussed.

Article de synthese sur le metabolisme de l'azote et la synthese d'uree chez les mammiferes. Degradation des proteines. Transport des acides-amines entre les organes et a travers la membrane plasmique des hepatocytes. Cycle de l'ornithine et distribution des enzymes de ce cycle dans le foie. Ontogenese de la synthese d'uree. Pathophysiologie de l'hyperammonemie

In recent years, the rat genes encoding glutamate dehydrogenase (GLUD; Das et al. 1993), glutamine synthetase (glutamateammonia ligase, GLUL; van de Zande et al. 1990) and carbamoylphosphate synthetase 1 (CPS; Van den Hoff et al. 1995) have been isolated. These enzymes have important functions in ammonia metabolism, and each of them is encoded by a single gene. GLUD (E.C. 1.4.1.3) catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, using NAD+ or NADP+ as cofactor. GLUL (E.C. 6.3.1.2) catalyzes the synthesis of glutamine from glutamate, thereby hydrolyzing ATP to ADP. CPS (E.C. 6.3.4.16) is the first and rate-determining enzyme of the ornithine cycle and catalyzes the production of carbamoylphosphate from ammonia, bicarbonate and ATP. In the present communication, we mapped the position of these three genes in the rat by FISH and by somatic cell hybrids. In addition, we determined the chromosomal location of the human GLUL gene and of the mouse, Cps1 gene by FISH. For the FISH mapping of the human glutamine synthetase gene (also called glutamate-ammonia ligase, approved human gene symbol GLUL), a human cDNA (total 2738 bp in pBluescript; Van den Hoff et al. 1991) was used. As shown in Fig.1a, the gene could be unequivocally mapped to Chromosome (Chr) (HSA) 1, band q25. The three genes were mapped in the rat both with a rat-mouse somatic cell hybrid panel (Szpirer et al. 1984; Klinga Levan et al. 1993) and with FISH. For the mappings with the cell hybrid panel, cDNA probes of approximately 1000 bp from the 38 ends of the genes were used (Glul 1111 bp, van de Zande et al. 1990; Glud1 957 bp, Das et al. 1989; Cps1 883 bp, De Groot et al. 1986). The probes were labeled with radioactivity by use of a-P-CTP and the random priming method. They were subsequently hybridized to filters containing 15 mg of genomic DNA from each hybrid restricted with EcoRI or BamHI. In each case the rat hybridizing fragments could be distinguished from the mouse bands, and the genes were assigned as follows: Glud1 to rat Chr (RNO) 16, Cps1 to RNO9, and Glul to RNO13. In the rat, the FISH results corroborated and refined the findings from the somatic cell hybrid panel. Longer probes are preferred in FISH analysis, and for the regional mapping of the Glul gene with FISH, two genomic clones were used (pgGS2, 5000 bp including exons 2–6, and pgGS4, 4500 bp including exon 1; van de Zande et al. 1990). Both probes gave very clear signals at the same chromosomal location, and rat Glul could be sublocalized to RNO13q22 (Fig. 1b). The rat G-band nomenclature is according to Levan (1974); for an updated recent version of the idiogram, see RATMAP database (URL http://ratmap.gen.gu.se/ratmap/WWW Nomen/RNOIdiogrRev96new.GIF). For the mapping of rat Glud1, two clones of genomic DNA (pgGDH6d, 6700 bp containing exon 1, and pgGDH2u, 5800 bp containing exons 8–12; Das et al. 1993) were used. The results from each of the probes were the same, and Glud1 could be sublocalized to RNO16p16 (Fig. 1c). Since RNO16 is a metacentric chromosome in which both chromosome arms have very similar stainability and banding pattern except in optimal metaphases, we wanted to check our conclusion with respect to which chromosome arm carried the Glud1 gene. Sasaki and associates (1994) have published excellent pictures of FISH mapping of the Atp7b gene (Wilson Disease gene homolog), and convincingly assigned this gene to RNO16q12.3. We used the same probe (designated pWD4) in simultaneous hybridizations with the Glud1 probe and could show that the two genes were located on opposite chromosome arms (Fig. 1d), thus verifying our conclusion that Glud1 is at RNO16p16. For the FISH mapping of the rat carbamoyl-phosphate synthetase gene (Cps1), a full-length cDNA probe (cCPSf.1, 5500 bp; De Groot et al. 1986) was used. The findings corroborated the previous hybrid panel mapping, and the Cps1 gene could be sublocalized to 9q34 (Fig. 1e). Since the Cps1 gene had not been mapped in the mouse, we attempted to map it by FISH with the rat cDNA probe. This worked out well, and the mouse Cps1 gene could be assigned to mouse Chr (MMU) 1, band C3 (Fig. 1f). Comparative mapping shows that the human GLUL gene is comprised in a region spanning bands HSA 1q22–1q42 and containing 11 human genes for which there are homologous rat genes on RNO13 (Table 1). Only seven of these genes have been mapped also in the mouse, but they are all situated distally in MMU1 (spanning about 26 cM from C4bp at map position 68 to Atp1a2 at map position 94; mouse data from Mouse Genome Database, MGD). The human homolog of the rat Glud1 gene is located at HSA 10q23.3, and the mouse homolog is on MMU14. Glud1 is included in a group of three genes (also comprising Rbp3 and Sftp1) that is conserved on these chromosomes. In contrast, the human and mouse genes homologous to the Atp7b gene on the long arm of RNO16 are on HSA13 and MMU8, respectively, as is the Atp4b gene, which is also located on RNO16. Thus, it looks as if RNO16 resulted from the fusion of two chromosome segments that are on separate chromosomes in both humans and mice, and, therefore, occurred after the separation of the rat lineage from human and mouse lineages. The human homolog of Cps1 is on HSA2q33–36. In total, there are nine genes on HSA2q (spanning the segment 2q32–2q37) that have their homologs on RNO9 (Table 1). A corresponding segment in the mouse is located on MMU1 (spanning 31 cM from Slc9a2 at map position 21 to Ugt1a1 and Akp3 at position 52). We have pointed out earlier that Correspondence to: G. Levan Mammalian Genome 8, 362–364 (1997).

Perennial ryegrass (Lolium perenne L.) is the most important temperate forage grass species. Unfortunately, the nutritional value of perennial ryegrass declines as maturity progresses, mainly because of a high concentration of poorly digestible compounds in inflorescences. Therefore, the development of forage-type ryegrass varieties with extended vegetative growth is of interest for agriculture. To delay floral transition in perennial ryegrass the Arabidopsis ATH1 gene driven by the maize ubiquitin promoter, the rice actin promoter or the rice OSH1 promoter, respectively was introduced. In ATH1-expressing plants heading was delayed, and in a number of cases the plants never flowered at all. Such non- or late-heading was accompanied by the outgrowth of normally quiescent lateral meristems into extra leaves, resulting in a leafy growth habit. When eventually heading, these plants generally produced a reduced number of inflorescences. These observations suggest that ATH1-mediated delay of heading may be useful to improve fodder quality of perennial ryegrass.

Background: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two ‘knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. Results: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. Conclusions: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45+/− mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45−/− embryos could be caused by defective TGFβ signalling, as the amount of TGF β1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45−/− embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45−/− embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).

This study was undertaken to investigate the effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion. Seven healthy subjects participated in two experiments performed in random order during normoglycemia and hyperglycemic clamping at 15 mM. Duodenal outputs of bilirubin, trypsin, amylase, and bicarbonate were measured by aspiration with a recovery marker under basal conditions for 60 min and during continuous infusion of bombesin (1 ng/kg x min) for 60 min. Plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels were determined at regular intervals. Compared to normoglycemia, during hyperglycemia basal outputs of bilirubin (17 +/- 3 vs. 0.9 +/- 0.4 micromol/60 min), trypsin (24 +/- 4 vs. 4 +/- 1 U/60 min), amylase (12 +/- 1 vs. 3 +/- 1 kU/60 min), and bicarbonate (2.9 +/- 0.5 vs. 1.2 +/- 0.2 mmol/60 min) were significantly p < 0.05) reduced. Bombesin significantly (p < 0.05) increased pancreaticobiliary output during both normo- and hyperglycemia. During hyperglycemia bombesin-stimulated 60-min outputs of bilirubin, trypsin, amylase, and bicarbonate were not significantly different compared to those during normoglycemia. Basal and bombesin-stimulated plasma PP concentrations were significantly (p < 0.05) reduced during hyperglycemia, but plasma CCK levels were not significantly different. It is concluded that acute hyperglycemia reduces basal but does not affect bombesin-induced pancreaticobiliary secretion.

A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.

Abstract The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at ≈7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.

Changes in protein and mRNAs for enzymes of glutamine metabolism were determined in rat kidney cortex at different times after induction of NH4Cl acidosis. After NH4Cl, phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 16-fold by 10 h (P < 0.05) and then returned to control levels by 30 h. In situ hybridization (ISH) showed that PEPCK mRNA was confined to medullary rays; after NH4Cl, expression of PEPCK expanded throughout the cortex, reaching a maximal intensity at 10 h. Phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH) mRNAs increased 8- and 2.6-fold, respectively (both P < 0.05), by 10 h before decreasing; the increased expression was confirmed by ISH. Immunohistochemistry showed that increased PEPCK, PDG, and GDH protein occurred at variable times after the rise in mRNAs. The increase was confined to proximal tubules and was sustained, a finding noted also by Western blot analysis. In contrast, glutamine synthase protein and mRNA, confined to deep cortex and outer medullar, did not change after NH4Cl. These studies reveal striking changes in PEPCK and PDG mRNAs in rat renal cortex during acidosis. The ISH pattern suggested that increased amounts of PEPCK were synthesized in recruited cells which contained little enzyme under physiological conditions. mRNA levels for PEPCK, PDG, and GDH peaked at 10 h before returning to control levels. Despite the decrease in mRNAs, a sustained increase in proteins was noted.

The developmental appearance and spatial distribution pattern of gap junctions were studied in prenatal and adult rat hearts. Gap junctions were visualized immunohistochemically with an antibody raised against a unique cytoplasmic epitope of connexin43, and the spatial distribution pattern was determined by three-dimensional reconstruction. The results demonstrate that from embryonic day 13 onward, connexin43 becomes detectable immunohistochemically in the myocardium of atria and ventricles. No expression is initially detectable in the myocardium of the sinus venosus, the sinoatrial node, the posterior wall of the atrium and pulmonary veins, the interatrial septum, the atrioventricular canal, including atrioventricular node and bundle, the interventricular septum, and the outflow tract. The developmental increase in the density of gap junctions in atria and ventricles of prenatal hearts correlates well with the reported developmental increase in conduction velocity. Whereas connexin43 becomes expressed in the derivatives of the sinus venosus (except for the sinoatrial node) and in the subepicardial layer of the ventricular free wall shortly before birth, it remains undetectable in the atrioventricular node and bundle and the proximal part of the ventricular conduction tissue, even in the adult heart. The apparent absence of an abundant expression of connexin43 at a location with a supposedly high conduction velocity (i.e., the atrioventricular bundle and bundle branches) is unexpected. These observations were confirmed in studies of the adult mouse heart, which showed, in addition, that connexin32 is not expressed in any part of the heart.

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.

The effects of starvation, glucose refeeding, dibutyryl cAMP, and dexamethasone on expression of the gene for phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from rat liver cytosol was studied by using a cloned cDNA probe. The rate of transcription of the gene for phosphoenolpyruvate carboxykinase in hepatic nuclei isolated from starved rats decreased rapidly after refeeding with glucose. Administration of dibutyryl cAMP to glucose-refed animals increased the rate of phosphoenolpyruvate carboxykinase gene transcription seven-fold within 20 min. Phosphoenolpyruvate carboxykinase mRNA in the cytosol is 2.8 kilobases long whereas liver nuclei contain four precursor RNA species that are up to 6.5 kilobases long. Feeding glucose to starved rats rapidly decreased the sequence abundance of enzyme mRNA in both nuclei and cytosol. However, the decrease in cytosolic phosphoenolpyruvate carboxykinase mRNA was preceded by a transient increase in enzyme mRNA over the first 20 min after glucose refeeding. Administration of dibutyryl cAMP to glucose-refed starved animals increased the concentration of the nuclear RNA precursors of phosphoenolpyruvate carboxykinase five- to eight-fold within 30 min and induced the mRNA for the cytosolic enzyme over a period of 60 min. We conclude that cAMP induces phosphoenolpyruvate carboxykinase mRNA by increasing the rate of gene transcription.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and β-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of “primary” myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.