Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater.
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PMID:Actin in Xenopus oocytes. II. Intracellular distribution and polymerizability. 56 82

A unique cAMP regulatory sequence, -129/-96 base pairs (bp), associated with the gene encoding human cytochrome P450C21 (CYP21B) binds a nuclear protein designated ASP, as described previously (Kagawa, N., and Waterman, M. R. (1991) J. Biol. Chem. 266, 11199-11204). This putative transcription factor required for cAMP-dependent transcription of the human CYP21B gene has been purified from the nuclear extracts of mouse Y1 cells by using sequence-specific DNA-affinity chromatography. The purified ASP is 78 kDa as estimated by SDS-polyacrylamide gel electrophoresis and binds to its specific recognition site, -126/-113-bp CACTCTGTGGGCGG, which has been demonstrated to be the minimum cAMP regulatory sequence of the human CYP21B gene. To characterize ASP more precisely, an antibody was raised against the 78-kDa protein. This antibody led to a supershift of the DNA.ASP complex on gel shift analysis and inhibition of in vitro transcription promoted by the ASP binding sequence, thereby indicating that ASP is a 78-kDa transcription factor. Upon DNase I footprinting experiments, ASP showed a characteristic footprint which very closely resembles but is distinct from that of Sp1 which also occupies a binding site within -129/-96 bp. Furthermore, the addition of purified ASP enhanced the mRNA synthesis promoted by the minimum cAMP regulatory sequence in a cell-free transcription system using HeLa cell extracts, whereas added Sp1 does not. These results indicate that ASP is a primary transcription factor for the cAMP-dependent regulation of the human CYP21B gene.
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PMID:Purification and characterization of a transcription factor which appears to regulate cAMP responsiveness of the human CYP21B gene. 133 85

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E.coli, isolated from E.coli cell extracts, and identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control region (ICR) of the Xenopus 5S ribosomal RNA gene from DNase I digestion. Intact protein bound specifically to the entire ICR (+96 to +43). One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 protected the 5S gene ICR from positions +96 to +63. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR.
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PMID:Zinc finger structure of a ribosomal gene-specific transcription factor. 136 25

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.
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PMID:The perinuclear matrix as a structural element of the mouse sperm nucleus. 151 Oct 98

The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, approximately 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to less than 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to approximately 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S.H. Kaufmann and J.H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These observations reveal that the recovery of pADPRp with a nuclease- and salt-resistant nuclear subfraction is dependent on the source of the nuclei and on the conditions used to fractionate those nuclei. In addition, these observations raise the possibility that there might be different functional classes of pADPRp molecules within the nucleus.
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PMID:Association of poly(ADP-ribose) polymerase with the nuclear matrix: the role of intermolecular disulfide bond formation, RNA retention, and cell type. 170 86

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.
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PMID:Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box. 173 Jul 70

Deoxyribonuclease I (DNase I) was purified about 850,000-fold from human kidney using a rabbit anti-human urine DNase I antibody and sensitive DNase I activity assay. On SDS-PAGE, the purified kidney DNase I gave a single major band, and its molecular mass was estimated to be 38,000 Da. The activity of purified kidney DNase I was dependent on the presence of Mg2+ and Ca2+. G-Actin inhibited the activity, as did the anti-urine DNase I antibody. The properties of the kidney DNase I were the same as those of urine DNase I.
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PMID:Purification and characterization of genetically polymorphic deoxyribonuclease I from human kidney. 176 56

The mammalian DNA repair enzyme beta-polymerase is encoded by a single-copy gene that is expressed in all tissues and cell lines studied to date. A protein fraction with high binding affinity for an ATF/CREB-like binding element, GTGACGTCAC, at -49 to -40 in the core beta-polymerase promoter has been purified to near-homogeneity from a nuclear extract of bovine testes. The major binding activity, as monitored by gel mobility shift assay, is recovered in 20% yield by a procedure involving oligonucleotide affinity chromatography. The purified protein yields DNase I footprinting and gel shift binding patterns indistinguishable from the activity in crude extracts. The final fraction activates transcription in an in vitro transcription reaction. The native molecular weight of the purified binding activity is about 100-120K as measured by gel filtration. SDS-PAGE of the purified fraction revealed that it contains several polypeptides in the molecular weight range of 30-52K, yet two of these peptides (Mr 49K and 52K) are predominant. Specific binding to the palindrome is salt-sensitive and is consistent with the formation of nine ion pairs (from log KA vs log KCl plots) and has a KA at 200 mM KCl of 5.8 X 10(11) M-1. Kinetic studies with synthetic oligonucleotides as binding ligands indicate that the purified protein can bind tighter to or discriminate between the beta-polymerase ATF/CREB element and similar elements derived from somatostatin and chorionic gonadotropin genes.
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PMID:Mammalian beta-polymerase promoter: large-scale purification and properties of ATF/CREB palindrome binding protein from bovine testes. 182 81

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
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PMID:Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation. 184 38


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