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Query: UNIPROT:P06889 (Mol)
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Like many eukaryotic genes, the rat albumin promoter contains a CCAAT consensus motif at position -80. In transfected H4II hepatoma cells the strength of this promoter depends to a large extent on the integrity of a hepatic nuclear factor 1 (HNF1) binding site located at position -60 and to a lesser extent on the CCAAT element. However, if the affinity for HNF1 is reduced, the CCAAT-box becomes essential for high, and tissue specific, promoter activity. We wished to determine which, among the different CCAAT binding factors co-existing in eukaryotic cells, was responsible for this co-operativity with HNF1. To this end we prepared a series of mutants of the CCAAT sequence and compared their effects on albumin promoter activity in vivo and on the binding of different CCAAT binding factors in vitro. Our results strongly suggest that a ubiquitous factor NFY (also designated CBF, ACF, CP1) interacts with this CCAAT element in vivo. We propose that during development NFY could facilitate transcription of the albumin gene in hepatocytes when the concentration of HNF1 is limiting. This co-operativity in transcriptional activation is not due to strict co-operativity in DNA binding between the two proteins and is not limited to NFY or a closely related factor, as the CCAAT-box can be replaced by AP1, SP1 or E2 target sites without significantly affecting the final activity.
J Mol Biol 1991 Nov 05
PMID:NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo. 194 67

Forty-eight group A streptococcal strains of different M types were screened for binding of human radiolabeled IgG. Three of the strains bound more than 80% of the added radioactivity and one of them, an M protein type 1 strain designated AP1, was selected for further analysis. Attempts were made to solubilize the IgG binding bacterial molecule, and small amounts of an IgG binding protein with a mol. wt of 40 kDa could be solubilized with mutanolysin, a muramolytic agent. The gene encoding this streptococcal protein was cloned and expressed in E. coli, and the E. coli-produced protein was purified in a single step by affinity chromatography on IgG-Sepharose. When tested with IgGs from different species, the molecule was found to bind human IgG almost exclusively. The N-terminal amino acid sequence was determined and showed no homology with previously isolated Ig binding proteins, and the name protein H (as in human IgG) is suggested for this novel Ig binding bacterial protein. Protein H showed preferential affinity for heavy chains and Fc fragments of human IgG, and did not bind Ig light chains. The affinity constant, determined by Scatchard plots, between protein H and human polyclonal IgG was 1.6 x 10(9). No binding was observed between protein H and IgM, IgA, IgD, or IgE. Finally, when tested against several additional proteins and human plasma, protein H only showed weak binding to alpha 2-macroglobulin, a proteinase inhibitor.
Mol Immunol 1990 Jun
PMID:Protein H--a novel IgG binding bacterial protein. 219 20

We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Mol Cell Biol 1989 Jun
PMID:E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. 247 56

The NGFI-B cDNA was previously isolated by virtue of its induction by nerve growth factor (NGF) in PC12 cells. It encodes a 61-kilodalton protein that has two regions of extensive homology with members of the steroid/thyroid hormone receptor gene family. The rat NGFI-B gene is approximately 7.6 kilobases long and is interrupted by six introns. Although the exon-intron structure of the gene is similar to those of several other members of the steroid/thyroid hormone receptor gene family, there is a novel splice site within the DNA-binding domain which suggests that NGFI-B constitutes yet another evolutionary digression from a postulated common ancestral receptor gene. Primer extension and S1 nuclease protection assays were used to determine the transcription initiation site, which displayed the heterogeneity typical of genes that lack a TATA box. Sequence analysis of the 5' flanking region revealed several GC boxes but no identifiable TATA box. Four potential AP1 binding sites were identified at nucleotides -49, -78, -222, and -242. Neither the serum response element nor the CArG box element, two sequences found in other growth factor-inducible genes, was detected in this region of the growth factor-inducible NGFI-B gene. Nevertheless, results of nuclear runoff experiments demonstrated that the NGFI-B gene was transcriptionally activated by nerve growth factor in PC12 cells. In vivo, a rapid, dramatic increase in NGFI-B mRNA was observed in the cerebral cortex, midbrain, and cerebellum of animals that experienced a convulsant-induced seizure.
Mol Cell Biol 1989 Oct
PMID:The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction. 247 23

We have demonstrated that two sequence elements in the c-fos promoter can mediate the response of the gene to epidermal growth factor and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). The first is the previously described serum response element. The second is a sequence element highly homologous to the consensus binding site for the HeLa cell transcription factor AP1. Although recent reports have shown that fos protein binds to AP1-binding sites through an interaction with AP1 protein and have raised the speculation that fos protein may negatively regulate expression of the c-fos gene via this interaction, we found no role for the AP1 consensus homology in the downregulation of c-fos expression following induction by epidermal growth factor and TPA.
Mol Cell Biol 1989 Mar
PMID:An AP1-binding site in the c-fos gene can mediate induction by epidermal growth factor and 12-O-tetradecanoyl phorbol-13-acetate. 249 46

PEA1 (AP1) motif transcription enhancer activity was stimulated by v-raf and more efficiently by activated c-raf-1 or A-raf than by their normal counterparts, in agreement with a role for PEA1 in transformation by raf. Mutations in the ATP-binding site of v-raf prevented activation, suggesting that phosphorylation is somehow required.
Mol Cell Biol 1989 May
PMID:Expression of raf oncogenes activates the PEA1 transcription factor motif. 250 65

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.
Mol Cell Biol 1989 Nov
PMID:Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer. 260 94

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.
Brain Res Mol Brain Res 1989 Mar
PMID:Preproenkephalin DNA-binding proteins in the rat: 5' flanking region. 271 96

The late promoter of simian virus 40 is transcriptionally activated, in trans, by large T antigen, the primary viral early gene product. Although large T antigen is a well-characterized DNA-binding protein, a variety of data suggest that its trans-activation function does not require direct interaction with DNA. We demonstrate that defined late promoter elements, omega (omega), tau (tau), and delta (delta), necessary for T-antigen-mediated trans-activation, are binding sites for simian cellular factors, not T antigen. Two of the late promoter elements (omega and tau) are shown to bind the same factor or family of factors. These factors bind to a site very similar to that for the HeLa cell factor AP1. We refer to these factors as the simian AP1-sequence recognition proteins (sAP1-SRPs). Compared with normal simian CV-1P cells, the sAP1-SRPs from T-antigen-producing COS cells, or from 14-h simian virus 40-infected CV-1P cells, showed altered binding patterns to both the omega and tau binding sites. In addition, the sAP1-SRPs from T-antigen-containing cells bound to the tau site more stably than did the analogous factors from normal CV-1P cells. The altered pattern of binding and the increased stability of binding correlated with the presence of T antigen in the cell. Additionally, the alteration of the binding pattern within 14 h of infection in CV-1P cells is temporally correct for late promoter activation. Overall, the data show (i) that the late promoter elements necessary for T-antigen-mediated trans-activation contain binding sites for simian cellular DNA-binding proteins; (ii) that the presence of T antigen causes alterations in the binding characteristics of specific simian cellular DNA-binding factors or families of factors; and (iii) that factors which bind to the late promoter elements required for activation have altered and more stable binding characteristics in the presence of T antigen. These points strongly suggest that T antigen mediates trans-activation indirectly through the alteration of binding of at least one specific simian cellular factor, sAP1-SRP, or through the induction of a family of sAP1-SRP factors.
Mol Cell Biol 1988 Apr
PMID:Simian virus 40 T antigen alters the binding characteristics of specific simian DNA-binding factors. 283 51

Malpighian tubules, gut, ovaries and carcasses of the adult female tick Amblyomma hebraeum were incubated in vitro in the presence of 2 microM [3H]ecdysone. Organs and media were separately extracted after 6, 24 and 48 h incubations and the patterns of ecdysone metabolites were analyzed by HPLC. Esterase-susceptible apolar metabolites similar to the AP2 already described in the soft tick Ornithodoros moubata and thus presumably corresponding to the same conjugates (C-22 esters with fatty acids) were rapidly produced in all tissues investigated. They were mainly found within the organs but they were also released into the medium to some extent. By contrast, less apolar metabolites corresponding to the AP1 esters were mainly found in the media. Malpighian tubules and gut were the most active organs regarding the conversion of ecdysone into 20-hydroxyecdysone (20E). However, only low quantities of 20E were formed, reaching respectively 12.5% and 11.6% of the total metabolites after 48 h incubations. In the carcass and in the ovary the formation of 20E was only a minor pathway (1.7% and 3.1% of the total metabolites after 48 h). In ovaries we observed a massive conversion of ecdysone into 3-epiecdysone, which (as in insects) presumably proceeded through the intermediate formation of 3-dehydroecdysone. These two compounds were identified among the metabolites by CI/D mass spectrometry. The 3-epimer was released into the media, in contrast with the AP2 which were essentially stored within ovaries. Epimerization was also realized to some extent by carcasses, and again the epimer was released into the culture media. The different pathways are compared with those found in other tick species and in insects, and the significance of the various metabolites is discussed.
Mol Cell Endocrinol 1986 Oct
PMID:Metabolism of [3H]ecdysone by isolated tissues of the female ixodid tick Amblyomma hebraeum (Ixodoidea; Ixodidae). 375 75


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