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At least 919 records · Page 51Linked to original sources
PubMed1991

A pH-dependent conformational change of staphylococcal nuclease: hydrogen-1 two-dimensional nuclear Overhauser spectroscopy studies.

Two-dimensional nuclear magnetic resonance (2d-NMR) dipolar correlation spectroscopy (NOESY) for the calcium binding protein, staphylococcal nuclease, was used to study the conformations in solution as a function of pH. A series of 2d-NOESY spectra of the nuclease H124L-Ca(++)-thymidine 3'5'-bisphosphate ternary complex demonstrated significant pH dependent changes: NOE cross peaks between 46His delta H and an aliphatic proton at 2.66 ppm appeared at pH 10.2, but not at pH 5.5. Some other NOE cross peaks between aromatic and aliphatic protons displayed chemical shift changes in omega 2 or both dimensions (omega 1, omega 2). These results indicated that a local rearrangement accompanies the ionization of the functional groups and has a role in enzyme function.

Hydrogen↗
PubMed1992

Nuclease-hypersensitive sites define a region with enhancer activity in the third intron of the human apolipoprotein B gene.

The positions of several DNase I-hypersensitive (DH) sites have been mapped in the second and third introns of the human apolipoprotein B gene. Two such DH sites, I and V, are present both in human hepatoma (HepG2) and colon carcinoma (CaCo-2) cells that express the gene but absent from HeLa cells that do not express the gene. These DH sites map near sequence elements that have been highly conserved between the human and mouse genes. A PvuII-EcoRI fragment (+1064 to +2977) from the hypersensitive region exhibited enhancer activity, which was further localized by means of deletion experiments to a 155-base pair segment located entirely within the third intron and flanked by two DH sites. Three DNase I footprints were observed within this core enhancer, one of which contains putative binding sites for three liver specific nuclear proteins. Experiments are presented that suggest that this enhancer operates by a similar mechanism as that described previously for the strong second intron enhancer, involving an interaction with the basal transcriptional machinery. Digestions with low levels of micrococcal nuclease were performed to ascertain whether nucleosomes were present in the DNase I sensitive enhancer region. Nine different micrococcal nuclease-hypersensitive (MH) sites were detected in HepG2 cells but not in HeLa cells; one MH site was common to both cell types, and HeLa cells exhibited three unique MH sites. The first six MH sites (I-VI) are spaced approximately 200 base pairs apart, suggesting the presence of positioned nucleosomes in that region. MH sites VI-X are more closely spaced, suggesting either additional cutting sites within the core particle or the absence of one or two nucleosomes in this segment of the third intron enhancer.

Apolipoproteins B↗
PubMed1991

The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.

Animals↗
PubMed1990

Structural features of the amplified N-myc oncogene detected by micrococcal nuclease digestion of neuroblastoma cell nuclei.

The structure of chromatin containing amplified N-myc in neuroblastoma and retinoblastoma cells was investigated using micrococcal nuclease digestion of isolated nuclei. The size distribution of DNA fragments containing N-myc, produced by micrococcal nuclease digestion of nuclei, was determined and compared to that of DNA containing the structural gene for dihydrofolate reductase. A perturbation of the native structure of chromatin containing N-myc was evident from the association of N-myc with more extensively digested DNA when compared with chromatin containing dihydrofolate reductase.

Cell Nucleus↗
PubMed1985

Studies on nuclease digestion of chromatin phosphorylated in vivo.

We have previously shown that, by culturing cells in hypertonic media, histone 2A becomes hyperphosphorylated (Pantazis, P., West, M. H. P., and Bonner, W. M. (1984) Mol. Cell. Biol. 4, 1186-1188). In the present study we have probed the effect of this histone modification on the overall chromatin structure by micrococcal nuclease and DNase I digestion. Although no significant quantitative differences in the extent of hydrolysis were observed between control and hyperphosphorylated chromatin by micrococcal nuclease, DNase I digested hyperphosphorylated chromatin at a 3- to 4-fold higher rate than unmodified chromatin.

Animals↗
PubMed1988

Crystallization and preliminary X-ray analysis of a quadruple mutant of staphylococcal nuclease.

A quadruple mutant of staphylococcal nuclease, nuclease (V66L/G79S/G88V/L108V), has been crystallized in a form well suited to moderate-to-high resolution x-ray diffraction analysis. This mutant is highly unstable; only about 20% of the protein in solution at room temperature is in its folded form. Under the crystallization conditions, the protein exhibits circular dichroism properties similar to, but not identical with, those of native wild type protein. The crystals belong to the space group P6(1)22 or P6(5)22 with unit cell dimensions of a = b = 61.1 A, c = 170.1 A and diffract to at least 2.5 A resolution. A data set complete to 3.7 A resolution has been collected and processed; attempts to determine the structure using molecular replacement techniques are under way.

Circular Dichroism↗
PubMed1988

[Stability of DNA-protein interactions in chromatin fractions with different sensitivity to nucleases].

It was revealed by means of nucleoprotein-celite-chromatography that DNA-protein interactions in the chromatin fraction sensitive to micrococcal nuclease and DNase II are weaker that in the resistant one. The micrococcal nuclease destroys the DNA-matrix bond resistant to salt-urea, while DNase II does not change the DNA-matrix integrity. Tightness of the DNA-protein interactions is weakened by the increasing chromatin fragmentation, but does not depend on the size of chromatin particles.

Animals↗
PubMed1987

[Internucleosome interactions: detecting the dinucleosome fragmentation of chromatin using micrococcal nuclease. Heterogeneity of nucleosomes and localization of histone H1].

The content of histone H1 (H1/H4 ratio) in dinucleosomes with the DNA of various length liberated from L-cell nuclear chromatin by micrococcal nuclease was analyzed. It was found that the histone H1 content in the dichromatosome is two times as low as that in the largest dinucleosome and in the complete mononucleosome. The set of chromatin fragments liberated from the Triton X-100 pretreated nuclei differs considerably from that of chromatin sites devoid of histone H1 (the de novo replicating chromatin and the chromatin formed on the undermethylated DNA). A scheme for asymmetric distribution of histone H1 with molecules oriented along the nucleosomal fibril, which reflects the peculiarities of chromatin fragmentation by micrococcal nuclease with predominant liberation of the dichromatosome, is proposed.

Animals↗
PubMed1985

Drug action and chromatin structure. I. Adriamycin binding to core particle of nucleosomes and subsequent enhanced DNA fragmentation induced by micrococcal nuclease.

The relevance of the subunit structure of chromatin to the mode of action of Adriamycin in Novikoff hepatoma had been investigated. To elucidate whether drug binding takes place at random along the chromatin fiber or not Novikoff hepatoma nuclei treated with Adriamycin in vivo were digested with Micrococcal nuclease and were subsequently separated into three fractions, S1, S2 and P2. In these chromatin fraction Adriamycin and the DNA content was determined. DNA were isolated from the above fractions and were analyzed by polyacrylamide gel electrophoresis, furthermore the S2 fraction was sedimented on sucrose density gradient. The results indicate that binding of Adriamycin takes place preferentially on the core particle inducing structural alterations. Adriamycin binding not only enhanced DNA nuclease digestion but altered the size of the fragments.

Animals↗
PubMedDate not supplied

[Accumulation of one of the histone H1 subfractions and increase in chromatin resistance to the nuclease effect in the course of carp and grass carp spermatogenesis].

Chromatin of immature testes and sperma of grass carp and carp was hydrolyzed by micrococcal nuclease and DNA I. The hydrolysis kinetics was studied. Chromatin of sperm maintains the nucleosomal repeat typical to somatic cells and is more resistant to the nuclease effect than chromatin of testes. Histones H1 and H2B of grass carp differ from the respective histones of the calf thymus. The core histones of sperm testes and liver chromatin in grass carp and carp have similar electrophoretic mobility. However the amount of slowly moving subfraction of histone H1 increases in the course of spermatogenesis. The amino acid composition of histone H1 from the carp sperm is characterized by a high lysine (34.6%) and low glycine (4.5%) content. A histone H1 molecule contains one tyrosine. The lysine/arginine ratio is 21.6 which is considerably higher than in H1-like histones of sperma in other species (for instance of Echinodermata).

Amino Acids↗
PubMed1983

Histone deacetylase. Association with a nuclease resistant, high molecular weight fraction of HeLa cell chromatin.

The chromatin-bound histone deacetylase of HeLa cells has been studied using endogenous [3H]acetyl-labeled polynucleosomes containing the enzyme, prepared in the presence of 40 mM butyrate. Histone deacetylase was assayed upon removal of the butyrate, and it was found that active enzyme is found only in association with a high molecular weight complex. This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease. No activity is found on mononucleosomes or oligonucleosomes. Up to 90% of labeled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core histones. Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins. Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities. Taken together, the results on the chromatographic behavior, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex.

Amidohydrolases↗
PubMed1980

Isolation and susceptibility to nucleases of transcriptionally active and inactive chromatin fractions from Physarum polycephalum.

Transcriptionally active and inactive chromatin fractions were isolated from Physarum polycephalum after depolymerization of chromatin with DNAase II or micrococcal nuclease, followed by fractionation in 5 mM-MgCl2. The active fraction of chromatin comprised up to 21% of nuclear DNA and was enriched 22-fold in the labelled nascent RNA. Both chromatin fractions were shown to have the nucleosomal structure. DNA of the active fraction of chromatin was degraded much faster with DNAase I and micrococcal nuclease than the DNA of the inactive fraction.

Chromatin↗
PubMed1981

Removal of histone H1 from intact nuclei alters the digestion of nucleosome core DNA by staphylococcal nuclease.

We have examined the gel profiles of staphylococcal nuclease digests of intact nuclei following different extents of removal of histone H1 by low pH. It was found that the submonomer fragment pattern (i.e. fragments less than 140 base pairs (bp) changed dramatically following removal of H1. The most striking feature of this change was a marked increase in the relative intensity of a band migrating at 102 +/- 4 bp when about 20-50% of the nuclear DNA is rendered acid soluble. All other submonomer bands decreased in relative intensity. There was no evidence for an approximately 100-bp repeat pattern accompanying the enhanced generation of the 102-bp fragment following H1 removal. This result, along with the comparisons of gel profiles for different extents of digestion, suggests that removal of histone H1 from nuclei results in an increased susceptibility of the DNA to staphylococcal nuclease at one or both ends of many of the core particles and that a strong block to further digestion occurs within these core particles resulting in the formation of a relatively stable 102-bp fragment.

Cell Line↗
PubMed1978

Comparison of susceptibility to staphylococcal nuclease and behaviour in metrizamide gradients of normal and 5-bromodeoxyuridine-substituted chromatin from Physarum polycephalum.

Replacement of 20--30% of thymine by 5-bromodeoxyuridine in chromatin DNA of Physarum polycephalum does not cause any visible change in a typical, regular pattern of DNA products obtained upon digestion of chromatin with staphylococcal nuclease. The time course of digestion is similar for normal and substituted chromatin even under conditions when the nuclease cleaves preferentially the dAT regions in DNA. 5-Bromodeoxyuridine label does not significantly affect the DNA/protein ratio in chromatin; this is reflected by similar behaviour of normal and substituted chromatin in metrizamide-density gradients.

Bromodeoxyuridine↗
PubMed1993

DNase I and micrococcal nuclease analysis of the tomato proteinase inhibitor I gene in chromatin.

The chromatin structure of the wound-inducible proteinase inhibitor I gene was investigated in nuclei from leaves of wounded and unwounded tomato plants. DNase I digestion of intact nuclei revealed that the inhibitor I chromatin structure was highly sensitive to the enzyme compared to the inactive ribosomal chromatin. This sensitivity was independent of wounding. Digestion of tomato nuclei with micrococcal nuclease, which produces a nucleosomal ladder from bulk chromatin in agarose gels, supported these observations. Micrococcal nuclease produced only a faint nucleosomal repeat superimposed on a smear for the coding region of the inhibitor I gene, whereas digestion of inactive ribosomal chromatin produced a well defined nucleosomal ladder. Two DNase I-hypersensitive sites were found in the promoter region of the inhibitor I gene. Both were present before and after wound induction. These two constitutive DNase I-hypersensitive sites may correspond to DNA regulatory regions of the gene. The combined results indicate the existence of an open chromatin conformation in the inhibitor I gene DNA region, present before gene induction by wounding.

Cell Fractionation↗
PubMed1997

Studies of the unfolding of an unstable mutant of staphylococcal nuclease: evidence for low temperature unfolding and compactness of the high temperature unfolded state.

Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, deltaC(p), between the unfolded and native states. This analysis gives a deltaC(p) of 2.2 kcal/mol/ x K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These deltaC(p) values are consistent with significant population of the cold unfolded state at approximately 0 degrees C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0 degrees C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements).

Chromatography, Gel↗
PubMed1998

Equilibrium unfolding of partially folded staphylococcal nuclease A2- and A3-forms is accompanied by the formation of an intermediate state.

Structural properties of an equilibrium intermediate formed upon urea-induced unfolding of more ordered staphylococcal nuclease A-forms (A2 and A3) are studied. The effect of association on the structural properties and conformational stability of this unfolding intermediate is also considered. A close structural similarity (including tendency for association) is shown between this intermediate and the least ordered A1-form, induced in the acid-unfolded nuclease by moderate sulfate or chloride concentrations.

Micrococcal Nuclease↗
PubMed1990

Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing.

We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric focusing. Protein separation takes place entirely in solution, with shear-stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE-Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially-purified protein (250 mg, 6700 U/mg) was focused in pH 3-4 carrier ampholytes. 67% of the activity was recovered in pooled peak fractions with a specific activity of 54,000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four-fold increase in specific activity (from 4000 U/mg protein to 15,000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.

Ammonium Sulfate↗