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°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.     

Thermal denaturation of DNA in the chromatin fragments released by mild micrococcal nuclease digestion of HTC cell nuclei

In chromatin a minor fraction melts at a temperature lower than deproteinized DNA, which may be assigned to DNA destabilizing proteins. We attempted to localize the destabilized DNA in the various chromatin fragments separated by electrophoresis after a mild micrococcal nuclease digestion. The small released fragments are enriched in coding sequences. About 20% of the DNA extracted from the released nucleosomes are single-stranded, 60% of the DNA in these fragments are digested by nuclease S₁ after incubation at low temperature, which suggests that the DNA destabilizing proteins are present in the released nucleosomes. Hybridization studies have shown that 25% of the DNA in nucleosomes are specific of this class of fragments. DNA destabilizing proteins could be associated with the specific sequences.     

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.     

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.     

Characterization of soluble lignin released from alfalfa by sheep digestion

A faecal soluble lignin fraction (FSL) extracted with 90% dioxane from the faeces of sheep fed on alfalfa hay was characterized by chemical analysis, nitrobenzene oxidation, fourier transform infrared spectroscopy and gel permeation chromatography, and compared with milled wood lignin (MWL) isolated from the alfalfa hay. The amount of FSL in the faeces was low, accounting for only 1% of the lignin present in the alfalfa hay. FSL and MWL consisted mostly of lignin components and contained a small amount of carbohydrate. FSL had a much higher proportion of syringylpropane units than MWL and showed a wide molecular size distribution. The results indicate the selective and limited solubilization of syringyl-rich lignin from alfalfa by sheep digestion.     

Partial characterization of RNA polymerase II complex released by micrococcal nuclease digestion of rat liver nuclei

Two forms of RNA polymerase II were released from rat liver chromatin by micrococcal nuclease digestion of the nuclei. One form behaved like a free RNA polymerase II and the other like a complex with other nuclear components. Both forms of RNA polymerase II activity were recovered in the 0.16 M NaCl-soluble fraction of the nuclear digest, and the complexed form of RNA polymerase II could transcribe its endogenous template under conditions permitting only of elongation of the RNA synthesis. The RNA polymerase II complex was further purified by gel filtration chromatography and column electrophoresis. Analysis of protein and DNA of the partially purified complex suggested that the RNA polymerase II was bound to mono- or dinucleosomes carrying some characteristic nonhistone proteins. Furthermore, in experiments on tissues from starved rats, the two forms of RNA polymerase II were found to originate from different functional states of the chromatin-bound enzyme in vivo.     

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.     

Comparative analysis of proteins selectively released from cell nuclei of different eukaryotic species under conditions of mild hydrolysis by nucleases

Nuclei of cultured Chinese hamster fibroblasts, dechoryonized eggs ofDrosophila melanogaster, calf thymocytes and mouse spleen were digested with micrococcal nuclease or DNAse I under conditions when about 15–20% of nuclear DNA is hydrolyzed. Proteins released from the nuclei with this DNA were analyzed by electrophoresis in polyacrylamide gels in the presence of SDS and the most prominent components were purified in each case. It is shown that these components derived from different species have different electrophoretic mobilities but are very similar in the amino acid composition characterized by high glycine content and a low lysine/arginine ratio. Molecular weights of two of those components, from mouse spleen and thymocytes, were estimated and are shown to be 25,500 and 23,000, respectively. Notwithstanding their different molecular weights they have the same N-terminal amino acid-arginine.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

Background

Adenoviruses force quiescent cells to re-enter the cell cycle to replicate their DNA, and for the most part, this is accomplished after they express the E1A protein immediately after infection. In this context, E1A is believed to inactivate cellular proteins (e.g., p130) that are known to be involved in the silencing of E2F-dependent genes that are required for cell cycle entry. However, the potential perturbation of these types of genes by E1A relative to their functions in regulatory networks and canonical pathways remains poorly understood.

Findings

We have used DNA microarrays analyzed with Bayesian ANOVA for microarray (BAM) to assess changes in gene expression after E1A alone was introduced into quiescent cells from a regulated promoter. Approximately 2,401 genes were significantly modulated by E1A, and of these, 385 and 1033 met the criteria for generating networks and functional and canonical pathway analysis respectively, as determined by using Ingenuity Pathway Analysis software. After focusing on the highest-ranking cellular processes and regulatory networks that were responsive to E1A in quiescent cells, we observed that many of the up-regulated genes were associated with DNA replication, the cell cycle and cellular compromise. We also identified a cadre of up regulated genes with no previous connection to E1A; including genes that encode components of global DNA repair systems and DNA damage checkpoints. Among the down-regulated genes, we found that many were involved in cell signalling, cell movement, and cellular proliferation. Remarkably, a subset of these was also associated with p53-independent apoptosis, and the putative suppression of this pathway may be necessary in the viral life cycle until sufficient progeny have been produced.

Conclusions

These studies have identified for the first time a large number of genes that are relevant to E1A's activities in promoting quiescent cells to re-enter the cell cycle in order to create an optimum environment for adenoviral replication.     

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.     

Characterization of phosphopeptides released during dialysis of EDTA-reacted highly purified DNA prepared from calf thymus nuclei

The treatment of highly purified DNA obtained from calf thymus nuclei (N-DNA) with a chelating agent and subsequent repeated dialyses led to release of phosphopeptides (PPs) into the dialysates. By means of anion exchange column chromatography, the PPs were separated into 9 main fractions. Two of them (P1 and P5) contained the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by deionization with nitrilotriacetate + Chelex. The proportion of phosphoserine was about twice as great in P5 as in P1. Whereas P1 and P5 contained essentially no nucleotide material, the other fractions contained ribonucleotides and deoxynucleotides. The deoxynucleotide content was less than 10% of that of total nucleotides. After a deionizing treatment, the amounts of nucleotides in these fractions were reduced to a level corresponding to 1 nucleotide per peptide of 5-15 amino acids.     

Measurement of growth hormone released by ox anterior-pituitary slices in vitro

1. A method has been developed for the iodination of ox growth hormone by using iodine monochloride, giving a product which is 93+/-4% bindable to antiserum. 2. The (131)I- or (125)I-labelled growth hormone obtained has been used in the development of an immunological assay for growth hormone. 3. The assay could be applied to the measurement of growth-hormone output by pituitary slices in vitro. 4. On incubation ox-pituitary slices liberate 8.1% of their growth-hormone content in the first hour and 4.5% in the second hour. 5. The amount of growth hormone liberated into the medium is greater if the tissue is frequently transferred than if it is incubated in the same medium. 6. The output is stimulated by a hypothalamic extract but inhibited by high concentrations of posterior-lobe powder: a stalk-median-eminence extract had no effect. 7. The output is lowered by temperature reduction, beta-hydroxybutyrate, acetoacetate, and pyruvate, glutamate and fumarate. Palmitoylcarnitine stimulates growth-hormone output. 8. Anoxia and 2,4-dinitrophenol have no effect on output.     

RNA-directed DNA polymerase from particles released by normal goose cells.

Cells from a goose embryo were shown to release particle-associated RNA-directed DNA polymerase and RNase H activities that required the presence of Nonidet P-40 for detection. The particles were not infectious and did not have endogenous DNA synthesis. The goose particle DNA polymerase was related to the DNA polymerase of spleen necrosis virus with respect to size and was inhibited by immunoglobulin G to spleen necrosis virus DNA polymerase. However, goose cells producing DNA polymerase-containing particles did not contain reticuloendotheliosis virus-related nucleotide sequences in their DNA.