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)

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.
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PMID:Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction. 194 68

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.
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PMID:c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts. 219 41

Androgen receptors were quantified in nuclei from human prostate tissue and in nuclear fractions derived by exhaustive digestion with micrococcal nuclease. In nuclei from benign hypertrophic prostate (BPH), the population of androgen receptors solubilized during nucleolysis predominated whereas in carcinoma nuclei the nuclease-resistant population was in excess. This phenomenon was restricted to intranuclear deployment and could not be attributed to recompartmentalization within the cell. Receptor content could not be correlated to the expression of the cellular protooncogenes myc, H-ras, K-ras or sis, in either BPH or carcinoma. However, in both BPH and carcinoma, significant correlation was observed between nuclear androgen receptor content and expression of c-fos. Expression of c-fos was not elevated in carcinoma compared to BPH, whereas expression of c-myc was elevated in carcinoma specimens of all grades of glandular differentiation, and expression of H-ras became increasingly elevated as differentiation was lost.
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PMID:Intranuclear androgen receptor deployment and protooncogene expression in human diseased prostate. 244 4

A procedure for the isolation of transcriptionally active nucleosomes was used to monitor changes in chromatin structure during the activation, repression, and superinduction of the protooncogenes c-fos and c-myc. Nuclei were isolated from murine fibroblasts at successive times after stimulation of quiescent cell cultures with serum or platelet-derived growth factor. The nucleosomes released by a brief micrococcal nuclease digestion were fractionated by HgII-affinity chromatography to separate the unfolded nucleosomes of transcriptionally active genes (in which the sulfhydryl groups of histone H3 are accessible for binding to HgII) from the compactly beaded nucleosomes of transcriptionally inert DNA sequences (in which the H3 sulfhydryl groups are not accessible). The DNA sequence contents of the HgII-bound and unbound nucleosome fractions were compared by slot-blot hybridizations to 32P-labeled cloned probes for c-fos and c-myc. The binding of the c-fos and c-myc nucleosomes to the HgII column accurately reflected both the timing and the degree of their expression, as determined by run-off transcription assays with the isolated nuclei. The superinduction of c-fos and c-myc expression by an inhibitor of protein synthesis (cycloheximide) was reflected in the persistence of the unfolded, transcriptionally active state of their component nucleosomes. These results provide direct evidence that rapid and reversible changes in nucleosome topography accompany the program of oncogene expression, and they suggest a way to monitor aberrant gene activity during malignant transformation.
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PMID:Rapid and reversible changes in nucleosome structure accompany the activation, repression, and superinduction of murine fibroblast protooncogenes c-fos and c-myc. 347 51

Human promyelocytic leukemic (HL-60) cells have amplified c-myc protooncogene sequences which lead to an elevated level of c-myc gene expression. Induction of HL-60 cells by phorbol esters to undergo monocytic differentiation results in the suppression of c-myc, but the activation of c-fos gene transcription. Chromatin structures of c-myc and c-fos were compared by measuring their sequences in nucleosome-associated DNA fragments. These nucleosomal particles were released from chromatin by micrococcal nuclease digestion and subsequently analyzed with two dimensional gel electrophoresis. C-myc related sequences were detected in nucleosomal DNA fragments of differentiated cells only, while the c-fos related sequences were found in nucleosomal DNAs of noninduced HL-60 cells. Since the enzyme preferentially digests relaxed DNAs, these results suggest that nucleosomal subunits of c-myc and c-fos chromatin are relaxed during the state of active transcription, and reassembled once their transcription is repressed.
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PMID:Reassembly of c-myc and relaxation of c-fos nucleosomes during differentiation of human leukemic (HL-60) cells. 354 26

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the post-ribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.
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PMID:Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts. 1093 49

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.
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PMID:The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements. 1127 41

L3MBTL1, a paralogue of Drosophila tumor suppressor lethal(3)malignant brain tumor (l(3)mbt), binds histones in a methylation state-dependent manner and contributes to higher order chromatin structure and transcriptional repression. It is the founding member of a family of MBT domain-containing proteins that has three members in Drosophila and nine in mice and humans. Knockdown experiments in cell lines suggested that L3MBTL1 has non-redundant roles in the suppression of oncogene expression. We generated a mutant mouse strain that lacks exons 13-20 of L3mbtl1. Markedly reduced levels of a mutant mRNA with an out-of-frame fusion of exons 12 and 21 were expressed, but a mutant protein was undetectable by Western blot analysis. L3MBTL1(-/-) mice developed and reproduced normally. The highest expression of L3MBTL1 was detected in the brain, but its disruption did not affect brain development, spontaneous movement, and motor coordination. Despite previous implications of L3mbtl1 in the biology of hematopoietic transcriptional regulators, lack of L3MBTL1 did not result in deficiencies in lymphopoiesis or hematopoiesis. In contrast with its demonstrated biochemical activities, embryonic stem (ES) cells lacking L3MBTL1 displayed no abnormalities in H4 lysine 20 (H4K20) mono-, di-, or trimethylation; had normal global chromatin density as assessed by micrococcal nuclease digests; and expressed normal levels of c-myc. Embryonic fibroblasts lacking L3MBTL1 displayed unaltered cell cycle arrest and down-regulation of cyclin E expression after irradiation. In cohorts of mice followed for more than 2 years, lack of L3MBTL1 did not alter normal lifespan or survival with or without sublethal irradiation.
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PMID:Chromatin protein L3MBTL1 is dispensable for development and tumor suppression in mice. 2059 34

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