The rat alpha 1-fetoprotein gene: characterization of the 5'-flanking region and tandem organization with the albumin gene.
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Biomedical subjects
Publications and source records attributed to M Guertin.
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Three major regions of DNase I hypersensitivity (DH) were found in alpha 1-fetoprotein (AFP) chromatin of rat liver. DH site I is located at the transcription initiation site and associated with ongoing AFP transcription. DH site II is located 2.5 kb upstream from the cap site: it is developmental stage-dependent but dissociable from ongoing AFP transcription. DH site III, 3.7 kb upstream from the cap site, behaves as hepatocyte-constitutive. DH sites are present in similar regions of liver albumin chromatin. Dexamethasone-induced AFP gene repression is accompanied by the selective loss of AFP DH site I, a likely result of glucocorticoid receptors binding to a DNA recognition sequence located 5'-adjacent to DH site I. Sl nuclease-hypersensitive sites were found on naked superhelical AFP and albumin DNA, but do not appear to contribute DH sites in liver chromatin. The extent of hypomethylation of HpaII sites at the 5'-end of the AFP gene correlates positively with the level of potential and actual expression of the gene. We conclude that developmental and hormonal regulation of the AFP gene is confined within congruent to 4 kb of 5'-flanking DNA, and we discuss possible hierarchical interactions among DH sites, in relation to DNA methylation and replication.
This article deals with the notion of supportive psychotherapy. This overall approach is often discredited even if, in the mental health network, it is widely used. Therefore, it appears important to analyze this approach and to define the idea of support in psychotherapy; this article reviews secondly when it is opportune to offer such an approach, what are the techniques related to this approach and finally, what are the results we can hope for? This article wants to initiate a reflexion while integrating some readings on the matter and using a clinical experience evolved from a practice in sector psychiatry.
Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were used in liver nuclear run-off assays. Under standard assay conditions, transcription signals detected with 5' probes were systematically stronger than with 3' probes. Heparin eliminated this phenomenon, which suggests that nuclear run-off assays are subject to in vitro reinitiation occurring preferentially in promoter gene regions. Transcription in the presence of heparin indicates that very few polymerases are engaged on the AFP gene in adult rat liver. Dexamethasone treatment of developing rat liver results in the loss of transcribing polymerases from all regions of the AFP gene. Albumin gene transcription is unaffected. Inhibition of liver protein synthesis with cycloheximide does not modify the AFP gene suppressive action of dexamethasone. Glucocorticoid hormone receptors may thus directly interact with the AFP locus, blocking polymerase initiation. We also report the sequence analysis of rat AFP mRNA, which reveals the existence of two potential initiation codons on this molecule.
The administration of glucocorticosteroid hormones to newborn rats interrupts selectively (and reversibly, if the hormone is withdrawn) the hepatic production of alpha 1-fetoprotein (AFP). This results from a decreased concentration of AFP mRNA in the liver [Bélanger, L., Frain, M., Baril, P., Gingras, M.C., Bartkowiak, J., & Sala-Trepat, J.M. (1981) Biochemistry 20, 6665]. We have delineated further the mechanism and time course of this hormonal action in 4-day-old rats treated with dexamethasone (DEX). DNA from a recombinant plasmid containing a 578-bp insert of rat AFP cDNA was used to develop a cell-free nuclear run-off system and directly assess AFP gene transcription activity. Five minutes after DEX injection, AFP gene transcription activity is unchanged, but after 30 min, it drops to 25% that of the control; this correlates with the time required for translocation of DEX receptors to the nucleus. Dose-response curves also show that the degree of AFP gene suppression is closely correlated with the amount of DEX receptor translocated to the nucleus. The nuclear concentration of AFP mRNA, monitored by dot-blot hybridization, decreases to undetectable levels within 48 h, whereas that of albumin mRNA increases slightly, which indicates the selectivity of DEX action. These results show that DEX suppresses AFP gene expression at the transcriptional level and suggest a direct negative action of DEX-receptor complexes on the AFP chromatin transcription unit.
The main features of the oncodevelopmental biology of alpha 1-fetoprotein (AFP) are reviewed. Progress made in the molecular biology of AFP gene regulation is discussed and we present our recent data on the mechanisms of AFP suppression by glucocorticoid hormones. The relationship between AFP gene transcription and cell replication is examined, and it is suggested that the degree of methylation of the AFP gene (or of co-methylated regulatory DNA sequences) conditions its response to hormones.
Chlamydomonas reinhardtii cells treated with toluene at 0 degrees C and 25 degrees C incorporate ribonucleoside triphosphates (NTPs) into chloroplast RNA at 25 degrees C and also at 35 degrees C. The incorporation requires all four NTPs and Mg2+, and is completely inhibited by DNase, RNase, actinomycin D (40 microgram/ml) and rifampicin (350 microgram/ml). However, the incorporation is almost totally insensitive to both alpha-amanitin and streptolydigin at 200 microgram/ml.
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RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis. Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length. In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.
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