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)

A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) [1]. The RNP sediments in sucrose gradients with s-values of 70-110S. Formaldehyde-fixed preparations band at Q = 1.40 in isopycnic CsCl gradients. The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with Mr 74000 and 72000. It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophoresis. Treatment of rats with alpha-amanitin leads to a significant decrease in the amount of recovered RNP. In the presence of 0.7 M NaCl the s-value of the complex changes from 70-110S to 40-80S. The RNP structure is stable to mild RNase A or micrococcal nuclease digestion. Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300-360 A. The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al. [29].
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PMID:Isolation and characterization of nuclear particles containing rapidly labelled hnRNA and snRNA in combination with a distinct set of polypeptides of Mr 74000 and 72000. 257 70

HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.
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PMID:Solid-phase processing of U2 snRNA precursors. 294 22

The U1 small nuclear ribonucleoprotein (snRNP) particle, a cofactor in mRNA splicing, contains nine proteins, six of which are also present in other U snRNPs and three of which are specific to the U1 snRNP. Here we have used a reconstituted human U1 snRNP together with snRNP monoclonal antibodies to define the RNA binding sites of one of the U1 snRNP-specific proteins. When Sm monoclonal antibody (specific for the B', B, and D proteins of U snRNPs) was bound to U1 snRNPs prior to micrococcal nuclease digestion, the same approximately equal to 24 nucleotide fragment of U1 RNA (corresponding to nucleotides 120-143 and termed the "Sm domain") was protected as when no antibody was bound prior to digestion. In contrast, when RNP monoclonal antibody, which reacts with the U1 snRNP-specific Mr 70,000 protein, was bound, additional U1 RNA regions were protected against nuclease digestion. This phenomenon, which we term "antibody-mediated nuclease protection," was exploited to map the position of the Mr 70,000 protein to stem-loop I of U1 RNA. However, there were also sites of Mr 70,000 protein interaction with more 3'-ward regions of U1 RNA, particularly the Sm domain. This indicates that in the three-dimensional structure of the U1 snRNP, the RNP and Sm antigens are in contact with each other. The proximity of the Mr 70,000 protein's RNA binding site (stem-loop I) to the functionally important 5' end of U1 RNA suggests that this protein may be involved in the recognition of, or stabilization of base pairing with, pre-mRNA 5' splice sites.
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PMID:The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins. 296 36

Model transcripts containing mammalian pre-rRNA sequences were incubated with a HeLa cell extract, digested with T1 RNase, and immunoprecipitated with anti-(U3)RNP or control antibodies. Two overlapping fragments derived from the 3' domain of human 28S rRNA were specifically immunoprecipitated although transcripts which spanned the transcription initiation site, the ETS processing site, the 5' end of 18S, and both termini of 5.8S yielded no protected fragments. The sequence of these fragments was determined using a novel technique in which the [32P]-labeled fragment was co-finger-printed with [3H]-labeled total transcript serving as an internal marker. The fragments immunoprecipitated derive from nucleotides 4570-4590 and 4575-4590 of human 28S and are adjacent to the alpha-sarcin site. Protection most likely involves the U3 RNA since it is sensitive to pretreatment of the extract with micrococcal nuclease. Complementarity between U3 and this rRNA region is phylogenetically conserved in species ranging from human to S. cerevisiae. The possible significance of this finding is discussed.
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PMID:An in vitro interaction between the human U3 snRNP and 28S rRNA sequences near the alpha-sarcin site. 297 35

A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP) fractions, which showed binding activities for both 5' and 3' splice site RNAs, were studied for the sequence specificity of their binding. Results indicate that the U1-rich snRNP fraction can recognize both 5' and 3' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5' splice site sequences, but not to a consensus 3' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5' splice site, but not a 3' splice site. The binding activity for the 5' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60 degrees C. The binding activity to a 3' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.
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PMID:Recognition of 5' and 3' splice site sequences in pre-mRNA studied with a filter binding technique. 304 Jul 11

Monoclonal antibody 3C5 recognizes a family of protein antigens present predominantly within the nucleus of interphase cells. We have shown previously that the epitope recognized by 3C5 is phosphorylated and have concluded that the proteins defined by this antibody share a common phosphorylation site. Using a combination of immunofluorescence microscopy and immunogold labelling in conjunction with electron microscopy, we have studied the distribution of 3C5-reactive material within interphase and mitotic cells. Antibody 3C5 was found to label specific structures within the interphase nucleus that, on the basis of their characteristic granulofibrillar morphology and strong staining with bismuth, have been identified as clusters of interchromatin granules (IG clusters). Double-labelling experiments with 3C5 and monoclonal antibodies to DNA have shown that these structures contain no detectable DNA. However, by indirect immunofluorescence, we have shown that 3C5-reactive nuclear structures do label with human autoantibodies to the Sm antigen, a component of small nuclear RNP particles (snRNPs). Granulofibrillar structures that stained strongly with bismuth, and were morphologically identical to nuclear IG clusters, were observed in the cytoplasm of mitotic cells. These structures also labelled with 3C5 but not with anti-Sm antibodies. Our results suggest that IG clusters remain essentially intact through mitosis though some snRNP components are apparently lost. In situ extraction of cultured cells with Triton X-100, micrococcal nuclease and 1-2M-NaCl failed to deplete 3C5-reactive material in either interphase or mitotic cells, though some redistribution was evident. In addition, 3C5-reactive proteins were identified in nuclear matrices prepared from rat liver by high-salt extraction procedures. However, the recovery of such proteins was strongly influenced by the preparation technique employed. Our results suggest that 3C5-reactive proteins and IG clusters are anchored to, but not integral components of, salt-resistant structural elements of the interphase nucleus and the mitotic cytoplasm, presumably the nuclear matrix and the cytoskeleton, respectively.
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PMID:Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells. 365 87

An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.
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PMID:Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex. 384 Apr 35

The sera of patients with mixed connective tissue disease (MCTD) have high titers of antibodies directed against nuclear U1-ribonucleoprotein (U1-RNP). This antigen is easily extracted from nuclear preparations with physiologic saline and from tissue sections with 0.1 HCl, leaving the nucleic acids and nuclear matrix behind. When U1-RNP is extracted from HEp-2 cells with 0.1 N HCl, the sera of 32/32 patients with MCTD react with another antigen that is exposed by the extraction procedure. This antigen is not destroyed by trypsin and deoxyribonuclease 1 treatment but is sensitive to both purified ribonuclease A and purified micrococcal nuclease. Absorption studies showed that the antibody reacting with this antigen cannot be absorbed by sheep red blood cells coated with extracts of rabbit thymus that contain U1-RNP. Radioimmunoassay showed that the reaction of the unadsorbed antibody was with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) and not with transfer RNA or ribosomal RNA. The hnRNP/RNA antigen is demonstrated as discrete particles in the internucleolar chromatin of interphase cells, but in metaphase cells the antigen is diffusely dispersed. The distribution, solubility, and biochemical characteristics suggest that the antigenic moiety is part of the nuclear matrix. Therefore, MCTD sera contain antibodies that react with at least two species of nuclear RNP: small nuclear RNP (snRNP), as described by others, and a high m.w. hnRNP/RNA bound to the nuclear matrix.
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PMID:Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix. 619 84

Ribonucleoprotein complexes (hnRNP) sedimenting at 30-40 S and containing fragments of heterogeneous nuclear RNA (hnRNA) have been extracted from HeLa cell nuclei. Besides hnRNA fragments (8-12 S), the complexes contain eight mostly basic core proteins of Mr 31 000-41 000 as shown by two-dimensional gel electrophoresis. Other proteins (mostly of higher molecular weight) seem to be peripherally associated since they are lost after pelleting and recentrifugation of the hnRNP complexes. The particle dissociates into its protein components after digestion of the endogenous hnRNA fragments by micrococcal nuclease. After inactivation of the nuclease and addition of a wide variety of exogenous RNAs [MS2 phage RNA, poly(U), poly(C), poly(A), and poly(A,U)], a RNP particle is re-formed which resembles the native hnRNP complex according to its sedimentation value (35 S), its appearance in the electron microscope, its density in metrizamide, and its protein composition. No particles are formed on double-stranded RNA [poly(A) . poly(U)] or native DNA whereas denatured DNA allows complex formation. On MS2 RNA (3569 nucleotides), the formation of tri- and tetrameric complexes is observed. This indicates the presence of 900-1200 nucleotides per particle. In vivo, 40S hnRNP particles are a unit component of larger RNP structures. Hence, we conclude from our results that the hnRNP core proteins have the intrinsic capability to associate with nascent single-stranded hnRNA regions to form these RNP complexes. Because of the lack of any sequence specificity, the complexes may function in packaging of the hnRNA and in connection with other nuclear components may provide a scaffold for subsequent processing reactions.
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PMID:In vitro reconstitution of 35S ribonucleoprotein complexes. 662 17

A method is described for preparation of a fraction of chromatin enriched in transcribing regions from nuclei of mouse GR cells. This fraction, released by mild staphylococcal nuclease digestion of isolated nuclei, contains 2 to 10% of the DNA as polynucleosomal chromatin together with 50-70% of pulse-labelled RNA and about 90% of all template-engaged RNA polymerase B molecules, titrated with (3H)-alpha-amanitin. Hybridisation of DNA from this chromatin fraction to total nuclear RNA in excess shows that it is enriched in frequently-transcribed DNA sequences. A modification of the Miller technique, allowing the spreading of the active chromatin fraction for electron microscopy, has been developed. Examination of the spreads reveals that this chromatin fraction contains 20-100 nucleosome-long polynucleosomal chains bearing lateral RNP fibrils interpreted as nascent transcripts. The average length of the DNA fragments in the fraction is greater than that of average transcribed regions, suggesting that the transcribed regions are linked to flanking segments whose chromatin conformation probably contributes to the selective release of transcribing chromatin.
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PMID:Isolation and characterisation of a transcribing polynucleosomal chromatin fraction. 667 92


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