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.     

Nucleosome structure in Aspergillus nidulans

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Subunit structure of simian-virus-40 minichromosome.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 +/- 2 nucleosomes, each containing 190 -- 200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190 - 200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.     

Evidence for a subunit structure of chromatin in mouse myeloma cells

If micrococcal nuclease is allowed to digest chromatin as it exists inside intact nuclei isolated from mouse myeloma tissue culture cells, more than 60% of the DNA can be isolated as a homogeneous fragment on a sucrose gradient. Analytical ultracentrifugation indicates that the protected DNA is native, unnicked, and about 140 +/- 10 base pairs long. After less extensive nuclease digestion, the protected DNA migrates in gels in lengths which are integral multiples of this 140 base pair "monomer" band. A submonomer band, 105 "/- 10 base pairs long, can also be detected. Similar digestion patterns were obtained by two different nuclear isolation procedures and even when intact cells were gently lysed directly in the digestion medium. These results confirm and extend the chromatin digestion studies of previous investigators and provide support for a subunit model for eukaryotic chromatin. The single strand specific S1 nuclease did not digest intranuclear chromatin under the conditions used.     

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."     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Chromatin structure in neuronal and neuroglial cell nuclei as a function of age

Abstract: Nuclei from the cerebral cortices of animals of different ages were separated into neuronal and neuroglial populations. Nuclei from cerebellar neurons were also studied. Using the enzyme micrococcal nuclease as a probe for chromatin structure, we found that the DNA from both neuronal preparations showed a decreased susceptibility to digestion during aging, although the onset of this alteration varies. In addition, both neuronal populations showed dramatic increases in the nucleosome spacing of the chromatin. Cerebral neuronal chromatin has a repeat length (nucleosome core and linker region) of 164 base pairs at 22 days and 11 months, 186 base pairs at 24 months, and 199 base pairs at 30 months. Cerebellar neuronal chromatin has a repeat of 188 base pairs at both 22 days and 11 months, 208 base pairs at 24 months, and 243 base pairs at 30 months. Neuroglial chromatin, on the other hand, showed no change in either accessibility to nuclease or repeat length.     

The measurement of exonuclease activities by atomic force microscopy

We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for measuring nuclease activities. Received: 8 August 1997 / Accepted: 10 September 1997     

Quantitative analysis of the digestion of yeast chromatin by staphylococcal nuclease.

The DNA in intranuclear yeast chromatin is protected from rapid staphylococcal nuclease degradation so as to yield an oligomeric series of DNA sizes. The course of production and disappearance of the various oligomers agrees quantitatively with a theory of random cleavage by the enzyme at uniformly susceptible sites. The sizes of the oligomers are integral repeats of a basic size, about 160 base pairs, and 80-90% of the yeast genome is involved in this repeating structure. Within this repeat there exists a 140 base pair core of more nuclease-resistant DNA. During the course of digestion, the sizes of the oligomers decrease continuously. The widths of the distribution of DNA sizes increase in order: monomer (1 X repeat size, half width = 5-7 base pairs) less than dimer (2 X repeat size, half width = 30 base pairs) less than trimer (3 X repeat size, half width = 40-45 base pairs). The yeast genome thus seems to have variable spacing of the nucleaseresistant cores, to produce the average repeat size of about 160 base pairs. Also, the presence of more than one species of monomer and dimer at certain times of digestion suggests a possible heterogeneity in the subunit structure.     

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.     

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.     

Histone variants and chromatin structure during sea urchin development

At the late blastula stage of sea urchin development a changeover of histone synthesis and chromatin composition takes place. Synthesis of the early histone variants declines while another set, the late histone variants, begins to be detected. During subsequent development the late histones accumulate steadily. In the 9-day larva only late histone variants are detectable. Micrococcal nuclease acts differentially on early and late nuclei. There is a depressed release of acid-soluble DNA when chromatin containing the late histones is digested. Nucleosomal repeat lengths change systematically and in parallel with the changing histone composition. Blastula and preblastula chromatin have a significantly shorter major repeat length than does the chromatin of 9-, 11-, and 16-day larvae. Intermediate stages of development have chromatin with intermediate periodicities. These differences are observed when the determinations are made under denaturing conditions of electrophoresis. Repeat lengths were found to be independent of the extent of digestion at all stages examined except the pluteus, in which there is an increase of the apparent repeat length as digestion proceeds. Pancreatic DNase I digests nuclei from blastulae and 9-day larvae similarly. Changes in the histone composition of chromatin, in nuclease accessibility of chromatin, and in nucleosomal repeat length are all very closely correlated, implying that there are underlying causal relationships.     

Preliminary characterization of chelation-sensitive nucleoprotein particles

Nucleoprotein particles (B2), isolated following digestion of calf thymus chromatin with micrococcal nuclease, are resolved on a non-chelating Bio-Gel A-5m column. B2 protein electrophoresis showed the presence of several H1 species and several nonhistone proteins but was depleted in core histones. DNA electrophoresis demonstrated that native B2 DNA has a length of about 46 base pairs. On DNA sequencing gels, the length distribution of denatured B2 DNA ranged from 12 to 35 bases with a weighted average chain length of about 26 bases. Depletion of a 20 base band in B2 DNA suggested specific protection of internucleosomal DNA sites during the nuclease digestion.     

Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin.

We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.     

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.