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At least 829 records · Page 46Linked to original sources
PubMed1996

A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus.

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization. Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome. Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse alpha-satellite sequences. NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix. Salt-fractionation experiments showed that NRC sequences are matrix associated. These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation.

Animals↗
PubMed1991

Chromatosomes are not produced from Tetrahymena chromatin by micrococcal nuclease digestion.

The subnucleosomal organization of Tetrahymena chromatin, which has an unusual H1 histone, was investigated by NaCl extraction and micrococcal nuclease digestion of nuclei. It was found that Tetrahymena histone H1 is extracted with 0.35 M NaCl, whereas bovine thyroid H1 is not. Micrococcal nuclease digestion of Tetrahymena nuclei did not yield chromatosomes as a stable intermediate, whereas digestion of bovine thyroid nuclei did.

Animals↗
PubMed2002

Multi-copy repression of Serratia marcescens nuclease expression by dinI.

The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced as the cells entered stationary phase, through inhibition of basal SOS expression.

Amino Acid Sequence↗
PubMed1997

DNA binding and nuclease protection by the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus.

The DNA-binding and nuclease-protection properties of the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus have been shown to be consistent with the formation of nucleosome-like structures (NLS). These proteins bind to DNA molecules as short as 20 bp and form complexes that protect DNA fragments from micrococcal nuclease (MNase) digestion that are 30 bp, approximately 60 bp and multiples of approximately 60 bp in length. The sequences of 49 of the approximately 60-bp DNA fragments protected from MNase digestion by HMfA have been determined and their intrinsic curvatures calculated. A circular permutation gel mobility-shift assay was used to determine directly the curvatures for five of these sequences. HMfA bound to intrinsically curved and noncurved DNAs, but exhibited a slight preference for the model curved DNA in binding competitions with a model noncurved DNA. The results obtained are consistent with the concept that the archaeal NLS is analogous, and possibly homologous, to the central core of the eukaryal nucleosome formed by a histone (H3 + H4)2 tetramer.

Base Sequence↗
PubMed1984

Two human colon tumor cell lines with similar nuclease sensitivities have different ethidium bromide binding characteristics.

Chromatin from two human colon adenocarcinoma cell lines (HT-29 and LoVo) showed similar digestion kinetics when sensitivities to DNase I and micrococcal nuclease were examined. Chromatin conformations were probed by examining the binding of ethidium bromide. A Scatchard plot revealed that both chromatins bound the same amount of ethidium bromide per mole of DNA, but the DNA from LoVo cells was more accessible to the intercalator. The results indicate that differences in chromatin conformation are not necessarily accompanied by different nuclease sensitivities.

Adenocarcinoma↗
PubMed1985

Formation of single-stranded regions in the course of digestion of DNA with DNAase II and micrococcal nuclease.

In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy. Most of the ss stretches are located internally in the DNA molecules. The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.

Animals↗
PubMed1976

Enzymic activity at interfaces. II. Enzymic activity of microsomal nuclease and bovine pancreatic ribonuclease at the air/water interface.

The adsorption isotherms and the spreading tendency of microsomal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) and bovine pancreatic ribonuclease (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) in the presence of isopropyl alcohol as spreading agent have been determined using enzymes radioactively labelled by acetylation. In parallel, the concentration-surface pressure relations have been established. The enzymic activity of microsomal nuclease spread from isopropyl alcohol containing aqueous solutions was only a few percent of its activity in bulk, while the activity of the adsorbed enzyme was only slightly reduced. Adsorbed monolayers of RNAase were almost inactivated, while the spread monolayers in the presence of isopropyl alcohol became reactivated after exposure to the substrate for several hours. The exposure time for the reactivation decreases with increasing surface concentration.

Air↗
PubMed1978

A study of an endogenous nucleolytic reaction and of the action micrococcal nuclease and DNAase I on a salt-soluble, compact form of chromatin.

The endogenous nucleolytic reaction occurring in rabbit thymus nuclear lysates has been studied at extended incubation times (up to 4 h). Production of nucleosomal polymers containing multiples of 205 base pairs of DNA was observed. The stability of the bands and the low release (1%) of acid-soluble nucleotides indicated there was only a small fraction of sensitive DNA between the subunits. The salt-soluble chromatin formed in the endogenous reaction at short incubation times (14--24 min) and purified over Sephadex G-200 has been treated with micrococcal nuclease and DNAase I. With micrococcal nuclease, nucleosomal polymers containing multiples of 201 base pairs of DNA were formed. Extensive digestion reaveled a core subunit containing 145 base pairs of DNA. With DNAase I only random degradation was observed and nucleosomal complexes were not produced.

Animals↗
PubMed1985

Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength.

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.

Animals↗
PubMed1986

Temperature-dependent cleavage of chromatin by micrococcal nuclease near the nucleosome center.

Digestion of nuclei at 4 degrees C with micrococcal nuclease results in significant intranucleosomal cleavage compared to digestion conducted at 37 degrees C. Employing nucleoprotein gel electrophoresis in one dimension followed by DNA electrophoresis in a second dimension, we demonstrate that such temperature-sensitive, internal cleavage predominantly occurs about 20 bp from the nucleosome center. We suggest that lower temperatures reduce the stability of hydrophobic interactions within the histone octamer and lead to a conformational alteration in nucleosomes that is detected by micrococcal nuclease.

Cells, Cultured↗
PubMed1991

The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding-folding kinetics.

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro117 is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.

Bacterial Proteins↗
PubMed1988

The upstream limit of nuclease-sensitive chromatin in Dictyostelium rRNA genes neighbors a topoisomerase I-like cluster.

In the chromatin of Dictyostelium ribosomal RNA (rRNA) genes, the coding and upstream flanking regions are sensitive to endonucleases. This sensitivity stops about 2.3 x 10(3) bases upstream from the transcription start, at a point we call the structural boundary. Upstream from the boundary an 850 base-pair region is strongly protected against micrococcal nuclease cleavage, particularly in rapidly transcribing vegetative cells, and upstream from this the pattern of nuclease protection suggests that positioned nucleosomes are present. On the gene side of the structural boundary nucleosomes are known to be absent in vegetative cells but present in differentiating slug cells where the rRNA synthesis rate is lower. We show that in slugs these nucleosomes are randomly distributed, in contrast to those upstream from the boundary. Close to the gene side of the boundary is a duplication of the putative promoter located 29 base-pairs distant from four clustered topoisomerase I recognition sequences, which are cleaved by endogenous topoisomerase I-like activity. An additional topoisomerase I recognition sequence found upstream from the structural boundary is not cleaved in chromatin. The possible significance of these sequences and structures in transcription is discussed.

Binding Sites↗
PubMed1983

Fractionation by micrococcal nuclease digestion of Drosophila embryo chromatin: isolation of a fraction enriched in two major nonhistone proteins.

Limited digestion of Drosophila melanogaster embryo nuclei with mitochondrial nuclease, followed by selective solubilization in 0.1 M NaCl, yields a soluble nucleoprotein fraction (S3) enriched in two dominant protein bands of apparent molecular weight of 44000 and 48000. The analysis of the nucleosome monomer and multimer peaks, separated on sucrose gradients after slight digestion with micrococcal nuclease, shows that these proteins are associated with chromatin subunits, and that they are principally found in the subnucleosome region of fraction S3. This doublet is tentatively identified as a member of the noncanonical HMG of Drosophila. The thermal denaturation and the circular dichroism spectra of the fraction soluble in 0.1 M NaCl (S3) and insoluble fraction (P3) show that both fractions are also structurally different.

Animals↗
PubMedDate not supplied

The effects of histones, in vitro age, and culture state on the digestion of DNA by micrococcal nuclease and deoxyribonuclease I.

Nuclei from confluent and mitotically arrested populations of human diploid fibroblast-like cells were subjected to digestion by micrococcal nuclease and deoxyribonuclease (DNase I) following the removal of various histone components by salt extraction. There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion. There was an age related inhibition of DNA digestion by DNase I in nuclei from older confluent cells before and after the removal of H1 histone but not after the removal of core particle histones. This inhibition was not detected in older arrested populations. These results indicate that an age-related masking by nucleosome core histones may limit the accessibility of DNA to enzymatic activities in older confluent cells. Since this inhibition was absent in older arrested populations, the importance of limited DNA accessibility as a primary cause of cellular senescence is questionable.

Cell Survival↗
PubMed1986

Nuclease digestion of DNA and RNA in nuclei from young adult and senescent Caenorhabditis elegans (Nematoda).

Nuclei prepared from young adult and senescent Caenorhabditis elegans (Nematoda) were subjected to digestion by micrococcal nuclease and DNaseI. The kinetics of digestion of nuclei by micrococcal nuclease showed no change with age. There was, however, an age-related increase of acid-soluble deoxyribonucleotides released by DNaseI, suggesting that subtle alterations of chromatin conformation occur in aged nematodes. The ratio of nuclear RNA to DNA decreased and the nuclear RNA became more susceptible to enzymatic degradation as the worms grew old. These findings appear to indicate that nuclear RNA is less protected by protein in old nematodes. The decline of the nuclear RNA/DNA ratio with age is in good agreement with the generally accepted idea that there is a reduced level of RNA and protein synthesis in old animals.

Aging↗
PubMed1996

An improved method to distinguish micrococcal nuclease sensitivity of chromatin.

The resolution of the standard micrococcal nuclease assay for sensitivity of active chromatin has been enhanced by the inclusion of an additional step of digesting nuclease-digested DNA with a suitable restriction enzyme prior to Southern hybridization. The improved assay has been used to analyze the chromatin structure of the lamin A, albumin and alpha-fetoprotein genes during rat liver development.

Animals↗
PubMed1984

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells. Stimulation by initiation factor eIF-2B.

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.

Animals↗
PubMed1984

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion.

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0 degrees C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock , J.M., Jr., and Rill , R.L. (1979) Biochemistry 18, 3739-3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1-2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.

Animals↗