Embryogenesis is the fundamental process of differentiation of all tissues from a fertilized egg. This process involves establishment of distinct stem cells that will later differentiate to all cell ...types. Recently, lines of embryonic stem cells have been established in culture from blastocysts. The cells are pluripotent and can differentiate in vivo to all lineages. Interestingly, differentiation of these cells can also be induced in vitro. Morphological and molecular events that are characteristic of the development of the embryo can be mimicked in vitro by growing the cells under controlled conditions. Furthermore, the process of early development can now be studied and manipulated in vitro and specific lineages of stem cells can be obtained and grown in culture.
The sensitivity to DNase I digestion of the gene encoding rat phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was assessed during development and prior to the onset of expression. This gene is ...resistant to DNase I digestion in nuclei isolated from livers of 19-day rat fetuses. Gradual acquisition of sensitivity of the phosphoenolpyruvate carboxykinase gene, which starts later than the 19th day of gestation and is completed by the 21st day, occurs before initiation of gene expression. As transcription of the phosphoenolpyruvate carboxykinase gene is not detected until birth, the events observed may represent a shift from a dormant to an active gene. Injection of N6,O2-dibutyryladenosine 3′,5′-cyclic monophosphate into fetuses on the 19th day of gestation induces gene expression and sensitivity to DNase I digestion within 3 hr of treatment. While this short treatment does not affect the methylation pattern of the gene, longer treatment of fetuses (2 days) with dibutyryl-cAMP results in premature hypomethylation of the gene. A hierarchy of modifications of the phosphoenolpyruvate carboxykinase gene during development is discussed.
The cytosolic phosphoenolpyruvate carboxykinase PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32 gene was isolated from a rat genomic library, and a map of the methylatable ...sites C-C-G-G and G-C-G-C has been constructed. The extent of methylation of 18 sites in the PEPCK gene in adult liver, kidney, spleen, and heart muscle and in fetal liver has been analyzed using the 5-methylcytosine sensitive enzymes Hpa II and Hha I. This analysis revealed extensive undermethylation of the PEPCK gene in the adult liver and kidney (PEPCK-expressing tissue), whereas the gene in adult spleen and heart muscle as well as in fetal liver (PEPCK-nonexpressing tissues) was heavily methylated. However, unlike the gene in the adult nonexpressing tissues, a region in the middle of the gene was found to be partially hypomethylated in fetal liver. This hypomethylation correlates with the competence of the fetal liver gene to be expressed. Treatment of fetuses by in utero injection of 5-azacytidine causes a hypomethylation-associated activation of the PEPCK gene. Taken together, the present findings suggest a sequential loss of methyl groups during development. When related to PEPCK gene expression, the sequential loss of methyl groups demonstrates an early stage prior to transcription characterized by hypomethylation of discrete sites and a later developmental hypomethylation of all sites associated with the mature active PEPCK gene around the time of birth.
Fibroblast growth factors (FGF) are expressed at high levels in the central nervous system (CNS), however their function in the CNS is not well understood. The immortalized neuronal cell line (BK1), ...derived from a transgenic mouse central nervous system tumor, expresses high levels of FGF receptor 1 (FGFR1) and demonstrates both morphologic and biochemical changes when treated with basic FGF (FGF-2). We have derived subclones of BK1 cells with varying degrees of FGF responsiveness by transfecting either a wild type (FRW) or a truncated (FRX) form of FGFR1. Cells expressing high levels of FGFR1 rapidly and uniformly respond to FGF, while cells expressing FRX fail to respond to FGF, either morphologically or by the expression of molecular markers. These BK1 subclones will prove useful to study FGFR mediated signal transduction and FGFR responsive genes in a CNS derived cell. These studies also demonstrate that a dominant negative FGF receptor can be used as a tool to elucidate the function of FGF in the central nervous system.
The PCK gene, encoding cytosolic phosphoenolpyruvate carboxykinase, is specifically expressed in gluconeogenic tissues, liver and kidney. Hence it serves as a model of a class of single-copy genes ...whose transcription is restricted to a few tissues, rather than a unique tissue. To begin delineating the mechanisms that govern this pattern of expression, cis-regulatory elements of PCK were examined using transient transfection assays in PCK-expressing kidney and hepatoma cell lines. The analyses enabled us to identify a proximal element, between nucleotide (nt) positions -121 and -98, relative to the transcription start point that is sufficient for specific expression in kidney cells, but is just one of the elements required for expression in hepatoma cells. A distal element (between nt -487 and -417), which is essential for hepatoma-specific expression, is not needed in kidney cells. We suggest that the differential regulation of PCK expression in the liver and kidney results from an interplay between different cis-regulatory elements and trans-acting factors.
The gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32, a key enzyme in gluconeogenesis and glyceroneogenesis, ...is expressed in tissues that arise from different embryonal origins: the gluconeogenic liver arises from endoderm, whereas the gluconeogenic kidney cortex and glyceroneogenic adipose tissue arise from the mesoderm. To identify the cis-regulatory elements conferring the differential gene expression, PEPCK chimeric genes were transfected into two rat hepatoma cell lines (H4IIEC3 and HTC-M1.1) and mouse adipocytes (3T3F442A), which express the endogenous gene, and into myoblasts and preadipocytes, which do not express it. The results demonstrate that 597 base pairs of the 5′ flanking region of the PEPCK gene are sufficient to confer cell-specific gene expression in the PEPCK-expressing hepatoma cells and adipocytes. However, different elements within this 597-base-pair region enhance the gene expression in the hepatoma cells (endoderm) and adipocytes (mesoderm). In the hepatocytes, expression is conferred by two elements--one 5′ of position -362 and the other 3′ of position -98 with respect to the transcription start site. The region in between these two elements (from -362 to -98), which seems to inhibit the gene expression in the hepatocytes, confers enhanced expression in the adipocytes. Moreover, the distal positive regulatory element of the hepatocytes seems to be orientation and PEPCK promoter dependent. In contrast, the positive regulatory element of the adipocytes seems to act as a more typical enhancer. These results suggest that separate cis-regulatory elements confer cell-specific expression of the PEPCK gene.
Upon birth, the liver acquires new functions as a result of the initiation of expression of key enzymes. One example is the initiation of gluconeogenesis which depends on the induced appearance of ...phosphoenolpyruvate carboxykinase (P‐pyruvate‐CK) at birth. To characterize other genes that undergo such regulation, a differential screening was performed on a cDNA library from well‐differentiated hepatoma cells. The pattern of tissue‐specific and developmental‐specific expression was determined for seven genes. Three clones, out of which two encode for the known genes alcohol dehydrogenase class I (ADH) and phenylalanine 4‐monooxygenase (PAH) and a new gene (clone 116‐3), exhibited a pattern of expression similar to that of the P‐pyruvate‐CK gene, i.e. their expression was liver and kidney specific and induced in the liver upon birth. Determination of the sequence of clone 116‐3 revealed that it belonged to the UDP‐glucuronosyltransferases type 2 (UGT2) family and thus was named UGT2B‐rH4. To examine whether expression of the various genes could be prematurely induced by hormones in the fetal liver, either high levels of CAMP or low levels of insulin were induced in utero. The results demonstrated that cAMP induced a marked expression only of the genes for P‐pyruvate‐CK and ADH but not of those for PAH or UGT2B‐rH4, while insulin deficiency induced premature expression of all four genes. We suggest that a set of genes whose expression is specifically induced in the liver upon birth can be prematurely induced by the hormones in utero.
Differentiating tissue is characterized by a specific repertoire of proteins out of which some are developmentally controlled. This review describes modifications in the structure of genes which ...encode developmentally regulated proteins. Evidence is provided for changes in chromatin conformation and DNA methylation of specific genes-either change can be observed in various stages of some vertebrates. The involvement of hormones in regulating DNA modifications is suggested, and interrelationships between DNA modifications and gene expression are discussed.
The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by ...dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.
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