Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA clone (designated hamAT101) encoding an arylamine acetyltransferase, AT-1, was isolated from a hamster liver lambda gt11 cDNA library using a specific polyclonal antibody raised against AT-1. The cloned cDNA insert consisted of 1181 nucleotides, including an open reading frame of 870 nucleotides encoding 290 amino acid (Mr 33,503). The isolated cDNA displayed high sequence similarity to those of chicken, rabbit, and human acetyltransferases. In Northern blots, the hamAT101 cDNA probe hybridized to an RNA band of 18S in the livers of both slow and rapid acetylator phenotypes. To confirm that hamAT101 cDNA encodes the monomorphic but not the polymorphic protein, the isolated cDNA was expressed in monkey kidney cells (COS-1 cells) using the vector p91023(B). A protein with a molecular weight similar to that of AT-1 was detected upon Western blotting in the 9000 x g supernatant from the transfected cells. The activity toward four different substrates of the 9000 x g supernatant was also examined. In agreement with the results of purified AT-1, the cDNA-expressed protein exhibited a high capacity for N-acetylation of 4-aminoazobenzene and 2-aminofluorene, and O-acetylation of 2-hydroxyamino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, whereas no activity was found for the N-acetylation of p-aminobenzoic acid. These results, in addition to the RNA blot hybridization, indicate that hamAT101 encodes the hamster acetyltransferase AT-1.
Mol Carcinog 1991
PMID:An arylamine acetyltransferase (AT-I) from Syrian golden hamster liver: cloning, complete nucleotide sequence, and expression in mammalian cells. 200 37

Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.
Mol Endocrinol 1990 Dec
PMID:Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. 208 79

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.
Mol Endocrinol 1990 Dec
PMID:Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase. 208 86

To examine the functional relationship between distinct cis-active elements within the distal enhancer region of the rat PRL gene, we have used deletional and mutational analysis of that region in transient transfection studies in GH3 pituitary tumor cells. Results from these studies demonstrate that the region of the PRL distal enhancer containing the Pit-1-binding sites is critical not only for enhancer activity and the response to cAMP, but also for the response to estradiol. An interaction of the estrogen receptor with factors conferring basal enhancer activity is suggested by studies with a mutant distal enhancer region in which the PRL estrogen response element was converted to a palindromic estrogen response element. To directly examine potential interactions, cotransfection studies using PRL distal enhancer reporter gene constructs and expression vectors for Pit-1 and rat estrogen receptor were performed in two heterologous cell lines. The activity of the reporter gene under the control of the PRL distal enhancer linked to either the thymidine kinase promoter or the PRL proximal promoter was not significantly altered by cotransfection with the Pit-1 expression vector in COS-1 or RAT-1 cells. Coexpression of these reporter constructs and an expression vector for estrogen receptor resulted in only a slight response to estradiol. However, when both Pit-1 and estrogen receptor were cotransfected with the distal enhancer reporter gene, a marked induction was observed in response to estradiol, and this activity was dependent upon the concentration of the Pit-1 expression vector.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Dec
PMID:Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. 208 92

An abnormal human thyroid hormone beta-receptor (hTR beta-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR beta-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR beta-Mf were cotransfected, hTR beta-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR beta-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR beta-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR beta-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state.
Mol Endocrinol 1990 Dec
PMID:Dominant negative transcriptional regulation by a mutant thyroid hormone receptor-beta in a family with generalized resistance to thyroid hormone. 208 93

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.
Mol Biol Rep 1990 Nov
PMID:Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes. 209 3

Based on the sequence of a dopamine D2 receptor cloned from rat brain, we prepared a series of oligodeoxynucleotide probes. A mixture of these probes hybridized with a 2.6-kilobase species of mRNA extracted from several rat tissues including retina and, using in situ hybridization of these probes to cryostat sections of rat retina, they densely label the inner nuclear and outer plexiform layers. Labeling was also observed in the inner plexiform and ganglion cell layers. No hybridization was observed to the photoreceptor layers. A similar pattern of labeling was observed in monkey retina, indicating that the probes also hybridize with a homologous primate mRNA. The probes were used to screen a lambda gt10 library of human retina. A 2.5-kilobase clone was isolated, which encodes a protein that differs from the rat brain protein by 18 amino acids. The 5' and 3' untranslated regions of the human retinal cDNA were also strongly homologous with the rat brain cDNA. The clone was subcloned into the pCD-PS expression vector and transfected into COS-7 cells. The transfected cells bound [3H]-raclopride with a pharmacology expected of dopamine D2 receptors. These data indicate that D2 receptors expressed in the inner retina and outer plexiform layer have genetic identity with those expressed by brain and that the human and rat D2 receptors are derived from highly related genes.
Mol Pharmacol 1990 Jan
PMID:Molecular cloning and expression of a dopamine D2 receptor from human retina. 213 93

The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatographic properties on ion-exchange columns and have higher-molecular-weight forms than the CV-1 cell factors. Major forms of the CV-1 factors migrate between 20 and 24 kilodaltons, while the COS factors migrate between 20 and 28 kilodaltons. The binding sites for the factors from CV-1 and COS cells are similar, covering a rather broad region within the 72-base-pair repeat comprising the AP-1 site and the two-octamer binding protein (OBP100/Oct 1) sites, OBP I and OBP II. Specific binding competition analyses indicate that the two general regions within the binding site (the AP-1-OBP II site and the OBP I site) each retain partial binding ability; however, the factors bind best when the two regions are adjacent in a relatively specific spatial arrangement. The binding site for the Band A factors corresponds very well to sequences necessary for the activation of the late promoter as defined by deletion and base substitution mutagenesis studies (J. M. Keller and J. C. Alwine, Mol. Cell. Biol. 5:1859-1869, 1985; E. May, F. Omilli, M. Emoult-Lange, M. Zenke, and P. Chambon, Nucleic Acids Res. 15:2445-2461, 1987). These data, in combination with the data showing that the Band A factors are modified or induced in the presence of T/t antigen, strongly suggest that T/t antigen mediates its transcriptional activation function, at least in part, through the Band A factors.
 ...
PMID:Activity of simian DNA-binding factors is altered in the presence of simian virus 40 (SV40) early proteins: characterization of factors binding to elements involved in activation of the SV40 late promoter. 215 10

We have recently demonstrated that the alpha 2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for alpha 2-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from alpha 2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known alpha 2-adrenergic receptor subtypes. (b) The ratio of alpha 2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for alpha 2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with alpha 2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.
Mol Pharmacol 1990 Jan
PMID:Nonadrenergic [3H]idazoxan binding sites are physically distinct from alpha 2-adrenergic receptors. 215 10

In this paper we validate a methodology, ligand autoradiographic receptor screening (LARS), for detecting expression of full length receptor cDNAs in COS cells. The method involves transfection of COS cells with receptor cDNAs by spheroplast fusion, production of filter replicas of the cell fragments, ligand binding to the receptors expressed in the membranes, and autoradiographic detection of bound ligand. A beta-adrenergic receptor cDNA cloned into a eukaryotic expression vector reliably induces high levels of beta-adrenergic receptor expression in 2-12% of COS cell colonies transfected with this plasmid after experimental conditions are optimized. Receptor expression is monitored by autoradiographic detection of 125iodocyanopindolol binding to COS cell fragments immobilized on polyester filter replicas. Binding displays appropriate pharmacological properties. The number of high-density binding spots per filter depends on the fraction of the spheroplasts in the fusion mixture that contain the beta-adrenergic receptor cDNA. A 100-plate LARS experiment can assess receptor expression in more than 10(4) transfected colonies. Thus detection of receptor-encoding sequences present in libraries in proportions as low as 1 in 10(4) should be possible. This technique may therefore be useful in detecting expression of other receptor cDNAs for which selective radioligands are available.
Brain Res Mol Brain Res 1990 Apr
PMID:Ligand autoradiographic receptor screening: receptor cDNA expression in replicas of transfected COS cells. 215 85


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