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At least 595 records · Page 33Linked to original sources
PubMed1997

Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer.

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.

3T3 Cells↗
PubMed1979

Cations and the accessibility of chromatin to nucleases.

When rat liver nuclei prepared with polyamines as stabilising cations are digested with DNAase II, release of both inactive chromatin and Mg-soluble, active chromatin is greatly reduced, in comparison to digestion of liver nuclei prepared with Mg2+ as stabilising cation. Chromatin release from polyamine stabilised nuclei is also inhibited relative to Mg-stabilised nuclei following digestion with micrococcal nuclease under two very different cation conditions. Nuclei prepared with polyamines and monovalent ions as stabilising cations exhibit properties intermediate between these two extremes with both nucleases. These effects are due to residual binding of polyamines to chromatin, which is thus maintained in a condensed state, inaccessible to nucleases. Since polyamine binding is not easily reversed, concentrations of polyamines and other cations must be rigidly controlled in experiments on chromatin structure if artefacts are to be avoided. The significance of these findings to the nature and properties of active chromatin within the intact nucleus is considered.

Animals↗
PubMed1980

Differential nuclease sensitivity of the ovalbumin and beta-globin chromatin regions in erythrocytes and oviduct cells of laying hen.

We have monitored the differential nuclease sensitivity of defined regions of the chicken genome in different cells using a method which combines restriction enzyme digestion and blotting to diazobenzyloxymethyl (DBM)-paper (see Ref. 11). By using different specific probes and by scanning the bands on the autoradiograms, it is possible to compare on the same blot the digestion patterns of similar-sized fragments from different regions of the genome corresponding to "active" and reference "inactive" genes. We have demonstrated the preferential sensitivity to DNaseI and micrococcal nuclease digestion of the ovalbumin gene region in hen oviduct chromatin. The beta-globin gene region (containing both an adult and an embryonic gene) is also preferentially digested by DNaseI in hen mature erythrocyte nuclei, but at a lower rate than the ovalbumin gene region in oviduct. These observations raise the possibility that there may be several types of preferential nuclease sensitivities, all characterized by increased rates of digestion but to different levels, the highest corresponding to the very actively transcribing genes.

Animals↗
PubMed1980

The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease.

Brief digestion of HeLa nuclei with mirococcal nuclease releases monomer hnRNP particles as well as monomer and polynucleosomes. Sucrose gradient analysis of the nuclease released material reveals a series of small A260 peaks overlapping a more predominant peak in the 40S region of the gradient. Analysis of the proteins, DNa, and RNA in successive gradient fractions has confirmed that the smaller peaks are monomer and polynucleosomes, and that the larger peak is 40S hnRNP. Like 40S particles isolated by low salt extraction or by sonication, the nuclease released particles are composed of rapidly labeled RNA associated with a group of non-histone proteins the most predominant of which are the 32,000-44,000 MW proteins previously identified as core hnRNP proteins. These results provide further evidence that 40S hnRNP particles exist as discrete structural components of larger in vivo ribonucleoprotein complexes.

Cell Nucleus↗
PubMed1984

Nuclease sensitivity of adenovirus type 2 chromatin in lytic infection.

We have investigated the sensitivity of adenovirus type 2 naked DNA and chromatin at 5 h and 20 h after infection to digestion by DNase I, micrococcal nuclease and endogenous nuclease between map coordinates 11.3 and 18.0 (SmaI-F fragment) using a terminal labelling method. Infected cell nuclei were gently digested with nucleases, DNA was extracted and digested to completion with SmaI and the fragments shorter than the SmaI-F fragment mapped by hybridization with a 708 base pair probe co-terminal with the SmaI-F fragment. Early chromatin contained hypersensitive sites at 16.0 and 14.3. These sites became minor cleavage sites in late chromatin and new hypersensitive sites appeared at 13.5 and 13.0. The change in the location of the hypersensitive sites in the course of infection correlated with the early to late switch in the transcription pattern in this region and the early to late change in the overall structure of adenovirus chromatin.

Adenoviruses, Human↗
PubMed1990

Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei.

Flow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA-specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed from. In contrast, when nuclei were digested in a buffer lacking both the mono- and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.

Cell Nucleus↗
PubMed1975

Reassessment of the coagulase and thermostable nuclease tests as means of identifying Staphylococcus aureus.

A total of 91 enterotoxigenic strains of Staphylococcus auerus isolated from foods and tested for production of coagulase and thermostable nuclease and the ability to ferment glucose and mannitol showed, with the exception of four strains, a complete correlation among these properties. A similar correlation was observed with 103 cultures of S. aureus isolated from clinical material. In all instances, the coagulase reactions were sufficiently strong to be scored at either the 3+ or 4+ levels. Presumptive staphylococcal cultures isolated during routine examination of foods and yielding 2+ coagulase reactions or lower were invariably negative for thermostable nuclease production. It is suggested that the thermostable nuclease test be performed on cultures with doubtful coagulase reactions before classifying them as S. aureus.

Aerobiosis↗
PubMed1981

Simian virus 40 maturation: chromatin modifications increase the accessibility of viral DNA to nuclease and RNA polymerase.

The accessibility of extracellular and nuclear simian virus 40 (SV40-M and SV40-I, respectively) virion chromatin DNAs to micrococcal nuclease, DNase I, BglI, EcoRI, and RNA polymerase was examined. Our results support the following conclusions: (i) the intranucleosomal DNA of SV40-I chromatin, similar to the precursor 75S chromatin complex, is resistant to enzymatic activity; and (ii) SV40-M virion chromatin is modified in a manner which increases the accessibility of viral DNA to enzymes, and the distinction between nucleosomal DNA and linker DNA is absent. Micrococcal nuclease digestion of SV40-I virion chromatin gave a typical nucleosomal DNA ladder pattern with a repeat unit of 205 base pairs of DNA. SV40-I chromatin was sensitive to cleavage with endonuclease BglI, but not with EcoRI. When SV40-I virion chromatin was used as a template, the rate of incorporation of ribonucleoside triphosphates into RNA was 5% of that obtained with naked form SV40 form I DNA. Micrococcal nuclease digestion of SV40-M virion chromatin resulted in submonomeric DNA fragments of approximately 55 base pairs, but no larger repeating unit of DNA was observed. SV40-M virion chromatin was sensitive to cleavage with either BglI or EcoRI and was approximately 20% more susceptible to digestion with DNase I than was SV40-I virion chromatin. The transcriptional efficiency of the extracellular virion chromatin was almost equivalent to that of naked SV40 form I DNA and was 16-fold higher than the rate observed with nuclear virion chromatin. The increased transcriptional activity was dependent upon the presence of nonhistone viral protein VP1 or VP2 or both.

Chromatin↗
PubMed2001

Therapeutic effect of a Gag-nuclease fusion protein against retroviral infection in vivo.

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.

Animals↗
PubMed1988

DNase I- and micrococcal nuclease-hypersensitive sites in the human apolipoprotein B gene are tissue specific.

We have mapped the DNase I- and micrococcal nuclease-hypersensitive sites present in the 5' end of the human apolipoprotein B (apo-B) gene in nuclei from cells expressing or not expressing the gene. Four DNase I-hypersensitive sites were found in nuclei from liver-derived HepG2 cells and intestine-derived CaCo-2 cells, which express the apo-B gene, but not in HeLa cells, which do not. These sites are located near positions -120, -440, -700, and +760 base pairs relative to the transcriptional start site. Undifferentiated CaCo-2 cells exhibited another site, near position -540. Six micrococcal nuclease-hypersensitive sites were found in nuclei from HepG2 and CaCo-2 cells, but not in HeLa cells or free DNA. These sites are located near positions -120, -390, -530, -700, -850, and +210. HepG2 cells exhibited another site, near position +460. Comparison of the DNA sequence of the 5' flanking regions of the human and mouse apo-B genes revealed a high degree of evolutionary conservation of short stretches of sequences in the immediate vicinity of each of the DNase I- and most of the micrococcal nuclease-hypersensitive sites.

Apolipoproteins B↗
PubMed1976

Effect of magnesium and some nutrients on the growth and nuclease formation of a moderate halophile, Micrococcus varians var. halophilus.

Production of halophilic nuclease by a moderate halophile, Micrococcus varians, ATCC 21971, was maximal at 2.5 to 3.5 M NaCl concentration in a complex medium (CM) composed of 1% casamino acids, 1% yeast extract, and NaCl. The addition of 81 mM MgSO4 to CM inhibited nuclease production in spite of good growth. Microscopic observation showed that this inhibition was accompanied by complete clumping of the cells. The Sehgal and Gibbons complex medium (SGC) which contained 0.75% vitamin-free casamino acids, 1% yeast extract, and NaCl, however, supported good production of the nuclease in spite of the presence of 81 mM MgSO4. It seemed that both magnesium sulfate and some substances present in CM might be responsible for this inhibition and clumping. A synthetic medium optimal for enzyme production was developed consisting of 16 amino acids, 4 vitamins, 0.73 mM KH2PO4, 2.7 mM KCl, 20 mM MgSO4, and 2.5 M NaCl. The organism required biotin as an essential growth factor, and thiamine, riboflavin, and choline as stimulating factors. Omission of isoleucine from the medium reduced markedly the growth rate. Glutamic acid, proline, and arginine were consumed completely during cultivation in the synthetic medium.

Amino Acids↗
PubMed1977

Identification of specific phosphoproteins in nuclease-digested chromatin subunits.

Rat liver chromatin subunits (nucleosomes), which were isolated from nuclease-digested chromatin either treated or not treated with 0.5% sodium deoxycholate and 5% Triton X-100, were found to be similar with regard to sedimentation at 11 S and appeared as spherical particles with a diameter of about 100 A (1 A = 0.1 nm) in electron micrographs. The ratio of histone to nonhistone chromatin proteins (NHCP) in these subunits was 1:0.1 in the absence of detergents and 1:0.25 in the presence of detergents. After ultracentrifugation of the nuclease-digested chromatin in a 5--30% sucrose density gradient two groups of phenol-soluble NHCP were identified, one being released in the top of the gradient (fA) and the other still bound to the chromatin subunits after the nuclease digestion (fB). Two specific phosphoproteins were found in fA, one (A4) with molecular weight (MW) 39 000, pI 5.3--6.0, and the other (A5) with MW 31 000, pI 5.1--5.8. These proteins were not present in fB. Another protein in fB (B2) was highly phosphorylated, with MW 68 000, PI 6.5--8.2, and this was not found in fA. These phosphoproteins were further characterized and found to contain phosphoserine. The presence of specific phosphorylated protein(s) in fB suggests that the interaction of phosphoproteins with DNA in the eukaryotic genome is more than a random process.

Animals↗
PubMed1980

3,5,3'-triiodothyronine receptor-containing chromatin fragments: production by nuclease digestion.

Nuclear thyroid hormone (T3) receptors are nonhistone proteins which are tightly bound to rat liver chromatin. The solubilization of the T3 receptors by micrococcal nuclease was studied using an assay which allows the delection of in vitro hormone binding and which is independent of the state of solubility of the chromatin. Nuclease digestion produces a receptor containing moiety which sediments at a rate of 5--6S. This form of the receptor is different than that released from chromatin at high ionic strength (3.8S) and potentially represents the stable association of the receptor which other elements of chromatin. Partial release of chromatin compaction by the use of dilute buffer solutions increases the rate of nuclease digestion, facilitates the release of the (5--6S) T3-receptor complex, and allows the isolation of sucrose gradient fractions which are enriched with receptor.

Animals↗
PubMed1984

Increased micrococcal nuclease sensitivity and/or solubility in nuclei from Graves' disease thyroid tissue.

The sensitivity of nuclei to micrococcal nuclease was compared in thyroid tissue obtained from euthyroid patients with solitary cold nodules and from patients with Graves' disease. A significant increase in the solubility and/or the sensitivity of nuclei to the nuclease was found in thyroid tissue from patients with Graves' disease. Electrophoretic analysis of DNA in chromatin solubilized by the nuclease revealed that the amount of oligonucleosomal DNA was increased, and that of polynucleosomal DNA was even more increased, in nuclei from Graves' thyroids than in those from normal thyroids. Polyacrylamide gel electrophoretic analysis in Triton acid-urea showed that the extent of histone acetylation in nuclei from Graves' thyroids was almost the same as in those from normal thyroids. These findings suggest that the state of chromatin organization in Graves' thyroid nuclei is different from that in normal thyroid nuclei and is independent of the extent of histone acetylation.

Acetylation↗
PubMedDate not supplied

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease.

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.

Adenosine Triphosphate↗
PubMed1998

Conformational features of a peptide model Ac-DTVKLMYKGQPMTFR-NH2, corresponding to an early folding beta hairpin region of staphylococcal nuclease.

Recent H-D exchange 1H NMR studies of the refolding of Staphylococcal nuclease (P117G) variant suggest that, a region of the protein corresponding to a beta hairpin in the native structure folded early in the refolding process. In order to investigate whether the formation of beta hairpin is an early folding event, we investigated the conformational features of the beta hairpin peptide model Ac-DTVKLMYKGQPMTFR-NH2 from Staphylococcal nuclease with 1H NMR techniques. It appears that the peptide aggregates even at a low concentration. However, based on the observation of weak dnn(i, i + 1) NOEs between K8-G9, G9-Q10, an upfield shift of Gly9 NH and a low temperature coefficient (-d delta/dT) for Gly9 NH, we suggest that the sequence YKGQP as part of the beta hairpin peptide model samples conformational forms with reduced conformational entropy and turn potential. The presence of aggregation could be restricting the population of folded conformational forms and formation of beta hairpin at detectable concentrations. We suggest that, formation of beta hairpin could be an early event in the folding of Staphylococcal nuclease and this observation correlates with H-D exchange 1H NMR results and also with the prediction of a protein folding model proposed in literature.

Amino Acid Sequence↗
PubMed1990

A 21 KDa protein resides in the nucleosomes of micrococcal nuclease sensitive liver chromatin of Atlantic salmon treated with estradiol.

Atlantic salmon was treated with 17-beta-estradiol to induce the process of vitellogenesis in liver. When the isolated liver nuclei were incubated with micrococcal nuclease at increasing enzyme/DNA ratios a 21 KDa protein appeared in the nuclease sensitive chromatin, the S-fraction. After HPLC gel filtration the 21 KDa protein resided with the oligo- and mononucleosomes. The S-fraction contained vitellogenin gene sequences at low nuclease/DNA ratios. The sequences were detectable also in the mononucleosomes derived from the S-fraction. After hormone treatment the vitellogenin gene exposed a higher sensitivity to micrococcal nuclease than the hormone-untreated controls. The results indicate that the 21 KDa protein took part in the hormone-mediated changes in gene expression by modulating the structure of estradiol responsive chromatin domains including the vitellogenin gene.

Animals↗
PubMed1984

Undermethylation of DNA in mononucleosomes solubilized by micrococcal nuclease digestion of HeLa cell nuclei.

To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with [3H-methyl]methionine and [14C] thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease. When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes. Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA. This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides. Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles.

Cell Nucleus↗