Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to [3H]4-OHTAM. The [3H]4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea. The following parameters were determined: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Following mild nuclease digestion, the solubilized [3H]4-OHTAM bound receptor sedimented as an abundant 6-7 S form and a less abundant approximately 12 S species. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000. About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer. Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000. Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000. These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexes with DNA.
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PMID:Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease. 407 72

There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links. "Strong" DNA-matrix interactions proved to be very different. They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide. DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix. A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.
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PMID:[Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method. III. Two types of DNA-nuclear matrix interaction]. 407 22

Exhaustive digestion of chromatin with trypsin leads to the cleavage of only 20-30 amino acids from each of histones III (f3), IV (f2a1), IIb2 (f2b), and IIb1 (f2a1), the remainder of these chains being resistant. This resistance is not altered by removing the histones from the DNA with 2 M NaCl, but is dramatically reduced in 6 M urea. Histones III, IV, IIb2, and possibly IIb1 are cleaved at their N-termini. Histones I and V and the nonhistone proteins are the first to be attacked by trypsin and have no detectable trypsin-resistant fragments. The arginine rich histones, III and IV, are then cleaved as a pair, followed by most of IIb2 and IIb1, also as a pair. This data is consistent with a model in which basic N-terminal "arms" extend from a trypsin-resistant histone complex. The structural arrangement of these arms relative to the trypsin-resistant histone complex may define the spatial coordinates of DNA binding sites and, consequently, the folding of the DNA fiber in the chromosome. Accompanying the tryptic digestion of the N-terminals of histones III, IV, IIb2, and possibly IIb1, is an increased sensitivity of chromatin to staphylococcal nuclease. As analyzed by electrophoresis, untrypsinized chromatin is digested into eight discrete limit-digest fragments by nuclease. Trypsinization results in the nuclease digestion of some, but not all, of these DNA bands. Together with the information on the way trypsin cleaves histones in chromatin, the analysis of the resistant DNA suggests that histone N-terminals are associated with some DNA bands and histone C-terminals with other DNA bands. We propose that histones fold the chromosome by crosslinking the DNA corresponding to these bands.
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PMID:Dissection of chromosome structure with trypsin and nucleases. 453 Mar 1

Cell-specific chromatin antigens have been detected in rat Sertoli cells. Antisera were raised in rabbits to dehistonized chromatin prepared from 5- to 6-day cultures of rat Sertoli cells and immunoreactivity was assessed with microcomplement fixation tests or immunoidentification of antigens separated electrophoretically and transferred to nitrocellulose sheets. Tissue specificity was confirmed further by immunoabsorption. These antisera recognized only components of Sertoli cell chromatin; chromatins prepared from rat liver, kidney, thymus, Novikoff hepatoma, a testes germinal cell fraction, or purified rat DNA showed little or no immunoreactivity. Nitrocellulose transfers of total chromosomal proteins revealed the presence of two high-molecular-weight antigens (greater than 200,000) and a broad range of weaker immunologic species (M tau about 50,000-200,000) in Sertoli cell chromatin but not liver or thymus chromatin. Deproteinization of Sertoli cell chromatin with concentrated salt and urea at pH 6 or 8 produced an increase in complement-fixing activity of the fraction sedimenting with DNA and micrococcal nuclease digestion studies showed these fractions to depend in part on DNA for antigenicity. Individual antigens in the protein-DNA pellets of pH 8 salt and urea fractionated chromatin could not be identified with antigens on nitrocellulose sheets. Collectively, these observations suggest that at least two groups of cell-specific antigens exist in Sertoli cell chromatin; one group is detected after electrophoretic separation and transfer to nitrocellulose and the other group, reactive in complement fixation, cannot be detected through this method, apparently becoming antigenically inactive once the complex of protein and DNA is dissociated.
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PMID:Tissue and cell-specific antigens in chromatin from cultured rat Sertoli cells. 618 1

Micrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-I) DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32P- or 3H-thymidine-labelled HSV-I. Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-I-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-I DNA as well as to 32P-labelled Vero cell DNA.. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with PH 9.3 buffer, pyridine, Sarkosyl or NcCl/urea. Only fragments of heterogeneous size were produced. Thus, HSV-I DNA is found predominantly in structures other than nucleosomes during virus replication.
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PMID:The structure of herpes simplex virus type 1 DNA as probed by micrococcal nuclease digestion. 625 37

The human sperm chromatin was gently decondensed by treating the sperm head sequentially with micrococcal nuclease and 2 M NaCl. All histones, about 10% of DNA, and a small amount of degraded protamines were released into the soluble fraction, leaving mainly nucleoprotamines in the pellet fraction. Transmission and scanning electron microscopic studies revealed that the nucleoprotamine pellet consisted of chromatin cords of two dimensions, viz., 330- to 420-A and 650- to 1200-A thick cords laced together by very fine strands of 60- to 80-A fibers; both types of cord appeared knobby and had zig-zag patterns throughout their length. It appeared that these cords were derived from two types of sperm heads of approximately equal population; one type contained chiefly the thick cords and the other chiefly the thin cords. Further treatment of the pellet nuclease-NaCl with urea and mercaptoethanol resulted in the dissociation of the thick into the thin cords and unravelling of the thin cords into smaller sized fibers; whereas the treatment of the pellet nuclease-NaCl with DNAase I resulted in the disappearance of the 60- to 80-A fibers, and the remaining cords were chiefly of thick type together with the sperm head exoskeletons. From these results the packing order of the chromatin in human sperm was proposed.
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PMID:Transmission and scanning electron microscopic studies of the human sperm chromatin decondensed by micrococcal nuclease and salt. 628 55

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.
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PMID:Hyper(ADP-ribosyl)ation of histone H1. 629 82

Biochemical mapping experiments of the simian rotavirus SA11 genome were performed to determine which double-stranded RNA segment coded for each of the viral polypeptides. Viral RNA transcripts were synthesized in vitro by using the endogenous viral RNA polymerase and fractionated by electrophoresis in acid-urea agarose gels. The fractionated transcripts were translated in two cell-free systems: micrococcal nuclease-treated reticulocyte lysates and wheat germ extracts. The polypeptide products were identified by polyacrylamide gel electrophoresis and partial peptide analysis and compared with polypeptides synthesized in infected cells or found in purified virus. The RNA segment that coded for each transcript was determined by hybridization of the fractionated transcripts to the double-stranded RNA genome and analysis of the hybrids by electrophoresis in polyacrylamide gels. Primary gene products were assigned for 10 of the rotavirus transcripts and 10 of the double-stranded RNA segments. The coding assignments are as follows: the inner-capsid polypeptides, VP1, VP2, and VP6, were assigned to segments 1, 2, and 6, respectively; the major outer-capsid polypeptides, VP3 and VP7, were assigned to segments 4 and 9, respectively; segments 5, 7, and 8 coded for nonstructural polypeptides with molecular weights of 53,000, 34,000, and 35,000, respectively; segment 10 coded for the 20,000-molecular-weight precursor to the 29,000-molecular-weight glycosylated nonstructural polypeptide; and segment 11 coded for a 26,000-molecular-weight polypeptide that may be the precursor to the minor outer-capsid polypeptide VP9. Several methods were used to determine the product of gene segment 3, and the problems associated with the identification of this gene product are discussed.
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PMID:Biochemical mapping of the simian rotavirus SA11 genome. 630 11

Infection of BHK cells with foot-and-mouth disease virus (FMDV) causes a thorough change in the electrophoretic profile of whole nuclear histones. It consists in the disappearance of histone H3 and the appearance of a new polypeptide (Pi) which migrates between histones H2A and H4 on SDS-polyacrylamide gels. Protein Pi is detected at 2 hr postinfection (pi), the time in which viral RNA synthesis begins to increase, and reaches equimolecular amounts with the remaining core histones 1 hr later, when the disappearance of histone H3 is almost complete. Labeling of cells prior to infection demonstrates that Pi is not a novo product but the result of a viral-induced processing of a host precursor synthetized beforehand. Protein Pi comigrates with histone H2A/B in acetic acid/urea polyacrylamide gels and it shares common major peptides with histone H3 under controlled proteolysis with protease V8 or trypsin. The mononucleosomal and nucleosomal DNA pattern analysis after micrococcal nuclease treatment of nuclei from infected and mock-infected cells did not show any significant differences even though after 3 hr (p.i.), protein Pi replaces histone H3 in the nucleosomal structure. It was concluded that FMDV infection is responsible for a specific modification in the nucleus of infected cells which leads, after 3 hr (p.i.), to a complete histone H3 protein Pi transition in the nucleosomes.
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PMID:Histone H3 modification in BHK cells infected with foot-and-mouth disease virus. 633 Sep 87

Antibodies directed against centromeric chromatin characteristically occur in the sera of patients with the CREST variant of scleroderma. We have studied the in situ enzymatic sensitivity and solubility of the centromeric antigen and have isolated an antigenic moiety that reacts with anticentromere antibodies. The centromeric antigen in the human epithelial cell line, HEp-2, was sensitive to DNAase I and micrococcal nuclease but not affected by RNAase A, trypsin or amylase. It was insoluble in 0.15-4 M NaCl but was extracted from the HEp-2 cells by 4 M urea/2 M NaCl. Antigenic activity in a 4 M urea/2 M NaCl extract of rabbit thymus was demonstrated by immunoabsorption. Indirect immunofluorescence of the extract separated by polyacrylamide gel electrophoresis revealed a fluorescent band with a mol. wt of 33,000. Calf thymus and trout testis histone preparations were fractionated by gel electrophoresis and transferred by blotting techniques to diazobenzyloxymethyl cellulose paper for autoradiography. Anticentromere antibodies bound to and were absorbed by trout testis histone 1. We propose that the centromeric antigen may be a variant of histone 1 that is associated with condensed chromatin.
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PMID:Anticentromere antibodies bind to trout testis histone 1 and a low molecular weight protein from rabbit thymus. 638 63


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