The variation with age of nuclear phosphoproteins released during micrococcal-nuclease digestion and nucleosomal phosphoproteins in three cell types from rat liver.

The nucleosomal non-histone phosphoproteins, and phosphoproteins released during the digestion of nuclei by micrococcal nuclease, were studied in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2- and 4-month-old rats with increasing proportions of parenchymal tetraploid nuclei. Qualitative and quantitative differences in nucleosomal phosphoprotein band patterns were found among different types of nuclei and ages. More phosphoprotein bands were found in nucleosomes derived from parenchymal than stromal nuclei. The number of phosphoproteins released during micrococcal-nuclease digestion increased with age for parenchymal nuclei. The significance of these results, considered in conjunction with the increase of DNA repeat length and decrease of nuclease accessibility with age, is discussed.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.     

Altered chromatin structure of cerebral nuclei in experimental diabetes mellitus.

To determine whether diabetes alters chromatin structure in vivo, micrococcal nuclease digestion kinetics were analyzed in cerebral cortical and hepatic nuclei of streptozotocin-induced diabetic rats. Cerebral nuclei of diabetic rats maintained for 6 weeks were less susceptible to micrococcal nuclease digestion compared with control rats. Insulin treatment reversed diabetes-related changes in nuclease digestion kinetics. There were no changes in the kinetics of digestion in hepatic nuclei. The reduced digestibility of cerebral DNA in diabetes could not be attributed to altered DNA fluorescence spectra, or altered distribution of most abundant chromatin proteins that were either solubilized or that remained insoluble immediately following nuclease digestion. It is concluded that chronic, uncontrolled hyperglycemia can alter chromatin structure of some tissues in vivo, and this change is probably related to subtle alterations in DNA-protein interactions.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.     

Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons

We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a “core particle” containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.     

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.     

A Low Repeat Length in Oligodendrocyte Chromatin

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."     

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.     

Persistence of the ten-nucleotide repeat in chromatin unfolded in urea, as revealed by digestion with deoxyribonuclease i.

It is shown by enzymatic digestion of chromatin from rat liver or Guerin ascites tumour (GAT) that treatments, which abolish the 180 base pair repeat, as revealed by digestion with micrococcal nuclease (shearing in salt solutions of medium ionic strength, sonication, fixation with formaldehyde in the presence of 5 M urea), have little effect on the 10 nucleotide repeat, observed in deoxyribonuclease I digests.     

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.     

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.     

DNA repeat lengths of erythrocyte chromatins differing in content of histones H1 and H5

Among the erythrocytes of chicken, trout, carp, and sucker, the relative proportion of the lysine-rich histone H5 varied from 20 to 0% of the total histones. Following digestion of nuclear chromatin with micrococcal nuclease, each of them displayed a longer DNA repeat length and greater repeat length heterogeneity than found in liver chromatin. Fish erythrocytes possessed similar repeat lengths of 207-209 base pairs which was 10-12 base pairs shorter than in chicken erythrocyte chromatin and approximately 10 base pairs longer than in liver chromatin. No correlation existed between the DNA repeat length or repeat length heterogeneity and the relative proportion of H5.     

Tissue-specific and periodic changes in the nuclease sensitivity of the fibroin gene chromatin in the silkworm Bombyx mori

Nuclei of substantial purity were isolated from the middle or posterior silk glands of the silkworm Bombyx mori larvae. Both the fibroin H- and L-chain gene sequences in the isolated nuclei from the posterior silk glands of the fifth instar larvae, where the genes are transcribed actively, are extremely sensitive to the digestion with DNaseI; on the other hand, these sequences in the middle silk gland nuclei from the same larvae, where the genes are not expressed, are markedly resistant to the digestion. The H-chain gene sequences in the posterior silk gland nuclei from the fifth instar larvae are also highly susceptible to the digestion with micrococcal nuclease, HinfI, and HhaI. The digestion products with micrococcal nuclease show a continuous size distribution. The H-chain gene sequences in the middle silk gland nuclei or the posterior silk gland nuclei from the fourth molting stage are cleaved partially into nucleosome dimer to oligomer sizes upon digestion with higher concentrations of micrococcal nuclease, suggesting that the inactive forms of the H-chain gene chromatin are constructed by folding of the chromatin fiber containing a regular array of nucleosomes. Hypersensitive sites to micrococcal nuclease are present near both ends of the second exon, a major body of the fibroin H-chain gene, in both the active and inactive forms of the chromatin. The DNaseI or micrococcal nuclease sensitivity of the H-chain gene chromatin in the posterior silk gland nuclei shows periodical changes corresponding to the intermolt-molt-intermolt cycle.     

Expansion of chicken erythrocyte nuclei upon limited micrococcal nuclease digestion. Correlation with higher order chromatin structure

A sensitive method for measuring nuclear volumes with a Coulter counter is described. It has been applied to the digestion of chicken erythrocyte nuclei by micrococcal nuclease and DNase I. Early in digestion, micrococcal nuclease induced a 20% increase in the effective spherical volume of the nuclei, followed by a gradual reduction. At the peak of nuclear swelling, about 17% of the chromatin was soluble after lysis and its average chain length was about 18 kilobase pairs (kb). DNase I digestion did not give rise to a corresponding expansion of the nuclei. Several preparation conditions, including the treatment of nuclei with 0.2% Triton X-100, led to a loss of the expansion effect upon subsequent micrococcal nuclease digestion. The results support the domain theory of higher order chromatin structure. In the context of this model, the observed maximum nuclear expansion correlates with an average of one nuclease scission per domain.