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Query: UNIPROT:P06889 (Mol)
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The effect of secondary stimulation with estrogen on synthesis of nuclear and nucleolar proteins is studied in chick oviduct. Isolated nuclei and nucleoli have a protein/DNA ratio of 5.2 and 5.6, respectively. 35% of nuclear and nucleolar protein is recovered in the histone fraction after hydroxylapatite chromatography. Gel electrophoretic separations of nuclear and nucleolar nonhistones are largely similar as to visible bands and distribution of radioactivity. Nucleoli bind 1.4 times more [3H] estradiol as compared to whole nuclei. Nucleolar histones are labelled slightly more actively with [3H] leucine than nuclear histones; nucleolar nonhistones are labelled about 3 times more actively than nuclear nonhistones. An 18 hour secondary stimulation with estrogen increases the radioactivity of histones by 6-fold and that of nonhistones by 2.5-fold in whole nuclei as well as in nucleoli. Stimulation appears to increase preferentially radioactivity of nonhistones at 50 000 daltons. As this change is observed in whole nuclei and nucleoli and is not reduced with hydroxyurea, it is suggested that this may be related to a gross structural reorganisation of chromatin induced by the hormone.
Mol Cell Biochem 1976 Mar 26
PMID:Labelling of oviduct nuclear and nucleolar proteins during estrogen induced differentiation. 127 54

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.
Cell Mol Biol 1992 Sep
PMID:Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork. 130 7

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.
Mol Cell Biol 1992 Jan
PMID:Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus. 130 93

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.
Mol Cell Biol 1992 May
PMID:Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements. 131 53

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.
J Mol Biol 1992 Jun 20
PMID:Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication. 131 98

In order to analyze the transcriptional regulation of the muscle-specific subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes composed of the upstream region of the PGAM-M gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured cardiac muscle cells, the segment between nucleotides -165 and +41 relative to the transcription initiation site was sufficient to confer maximal CAT activity. This region contains two E boxes and one MEF-2 motif. Deletion and substitution mutation analysis showed that a single MEF-2 motif but not the E boxes had a substantial effect on skeletal and cardiac muscle-specific enhancer activity and that the cardiac muscle-specific negative regulatory region was located between nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected with MyoD into C3H10T1/2, the profile of CAT activity was similar to that observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M MEF-2 oligomer. These observations define the PGAM-M enhancer as the only cardiac- and skeletal-muscle-specific enhancer characterized thus far that is mainly activated through MEF-2.
Mol Cell Biol 1992 Oct
PMID:A single MEF-2 site is a major positive regulatory element required for transcription of the muscle-specific subunit of the human phosphoglycerate mutase gene in skeletal and cardiac muscle cells. 132 54

We previously studied the transcriptional mechanisms involved in expression of the murine nerve growth factor (NGF) gene. To investigate the regulation of transcription of the human NGF gene, the promoter region was cloned. The nucleotide sequences of the human and mouse genes are greater than 90% similar near their promoters. The cloned human promoter was transcriptionally active in mouse L929 fibroblasts. 5' Deletion analyses indicated that the -85 to -45 region stimulates basal transcription 6-fold. This segment is greater than 80% identical in human and mouse genes except for an AP-1 consensus sequence found only in the human gene. A second AP-1 consensus sequence at +34, previously shown to function as a regulatory element in the mouse gene, is identical in both genes. Gel shift analyses of L929 cell extracts revealed binding of protein to oligonucleotide probes spanning each of the two AP-1 consensus sequences of the human gene. The gel shift patterns differed, suggesting interaction of different proteins with the two probes. Our results demonstrate that the human NGF gene promoter is transcriptionally active in mouse fibroblasts, and implicate an upstream region in basal transcription.
Brain Res Mol Brain Res 1992 Sep
PMID:The human nerve growth factor gene: structure of the promoter region and expression in L929 fibroblasts. 133 71

The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1992 Oct
PMID:Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter. 133 54

The cauliflower mosaic virus 35S promoter confers strong gene expression in plants, animals and fission yeast, but not in budding yeast. On investigating this paradox, we found that in budding yeast the promoter acts through two domains. Whereas the upstream domain acts as a silencer, the downstream domain couples expression to the nutritional state of the cells via the RAS/cAMP pathway. Point mutations indicate that two boxes with similarity to the cAMP regulated element (CRE) of mammalian cells mediate this response. Gel retardation assays show that, in both yeast and plant protein extracts, factors bind to this promoter element. Therefore, transcriptional activation appears to be highly conserved at the level of transcription factors and specific DNA target elements in eukaryotes. This offers new ways to investigate gene regulation mechanisms of higher eukaryotes, which are not as amenable to genetic analysis as yeast.
Mol Gen Genet 1992 Nov
PMID:The cauliflower mosaic virus 35S promoter is regulated by cAMP in Saccharomyces cerevisiae. 133 31

The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.
Mol Endocrinol 1992 Dec
PMID:Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc. 133 41


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