DNA associated with nucleosomes in plants.

50 to 55% of tobacco and barley nuclear DNA is accessible to micrococcal endonuclease digestion. The DNA fragments resulting from a mild endonuclease treatment are multiples of a basic unit of 194 +/- 6 base pairs in tobacco and 195 +/- 6 base pairs in barley. After extensive digestion, a DNA fragment of approximately 140 base pairs is predominant. Hence the "extra-core" or "linker"-DNA is 55 base pairs long. Other fragments having 158 and less than 140 base pairs are present as well. Treatment with DNase I results in multiples of 10 bases when analysed under denaturating conditions. These results show that the general organization of the DNA within the nucleosomes is about the same in higher plants as in other higher eukaryotes.     

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.     

Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease.

Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.     

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.     

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.     

Subunit organization of Euglena chromatin

Digestion of $\text{Euglena}$ nuclei or extracted chromatin with micrococcal nuclease results in the identification of a repeating structure. The DNA repeat size, analyzed on agarose and polyacrylamide gels, is found to be 225±13 base pairs. DNase I digestion produces a serie of fragments multiples of roughly 10 bases. Eventhough pressure shearing is necessary to disrupt the though pellicule of the phytoflagellate, we confirm that, in $\text{Euglena}$, chromatin organization is similar to that of other eukaryotes.     

The same amount of DNA is organized in in vitro-assembled nucleosomes irrespective of the origin of the histones.

The four histones H2A, H2B, H3 and H4 from calf thymus, CHO and sea urchin gastrula cells were associated by stepwise dialysis from 2 M NaCl with SV40 DNA Form I. The in vitro-assembled chromatins were visualized by electron microscopy and the size of the DNA fragments generated by digestion with DNase II was determined. Irrespective of the origin of the histones, the size of the smallest DNA band generated at early times of digestion was about 190 base pairs, whereas oligomeric DNA bands were multiples of 140 bp. These results support our previous proposal that the four histones H2A, H2B, H3 and H4 are able to organize more than 140 bp of DNA, but do not provide any evidence that the variability of histones H2A and H2B plays a role in the variability of the DNA repeat length of native chromatins.     

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.     

Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

Structure of the joint region and the termini of the DNA of herpes simplex virus type 1.

Analysis of restriction endonuclease cleavage sites within the inverted, repeated sequences in the joint region of the DNA of herpes simplex virus type 1 strain KOS revealed the presence of two types of sequence heterogeneity. The first was an insertion of 280 base pairs or multiples of 280 base pairs which was found in approximately half of all DNA molecules from every plaque-purified stock of virus. These insertions seemed to be tandem duplications of sequences which were present at the joint and correspond closely to the inverted terminal redunancy. The second type of heterogeneity was due to variable insertions and deletions which were present in some, but not all, plaque-purified virus stocks. Comparison of restriction fragments from the joint region with fragments from the termini indicated that in the simplest observed molecules of herpes simplex virus type 1 DNA, only one copy of the inverted terminal redundancy was present at the joint. A map of restriction endonuclease cleavage sites in the joint region is presented.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Internal structure of the chromatin subunit

The digestion of chromatin in situ with DNase I reveals, after denaturation, a regular series of single stranded DNA fragments the lengths of which represent multiples of 10 bases. These experiments are compatible with the DNA being on the outside of the chromatin subunit and suggest that the subunit structure itself contains repetitive structural elements. Possible models are discussed.     

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.     

Chemical evidence that chromatin DNA exists as 160 base pair beads interspersed with 40 base pair bridges.

Digestion of rat liver nuclei by an endogenous endonuclease generates double-stranded DNA fragments which are initially about 205 base pairs long, as reported previously by Hewish and Burgoyne. As digestion proceeds, the average size of these fragments is reduced to about 160 base pairs. Electrophoresis under denaturing conditions shows that these DNA fragments contain single strand nicks at ten base intervals. Fifteen bands, 10-150 bases, are clearly resolvable. DNA Fragments of 160 to 200 nucleotides are not resolved as distinct species. The results suggest that the chromosomal subunit contains both a 160 base-pair DNA segment, in a conformation susceptible to single strand nicking at ten base intervals, and a forty base-pair DNA segment in a conformation more uniformly susceptible to endogenous endonuclease activity. This chemical evidence agrees with morphological observations suggesting that chromatin has a "bead and bridge" structure.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Preparative separation of the complementary strands of DNA restriction fragments by alkaline RPC-5 chromatography.

High pressure reversed phase chromatography (RPC-5) at pH 12 was used for preparative separation of the complementary strands of the smaller DNA fragments which are generated by the Hae III restriction endonuclease digestion of Col El DNA. A single high pressure RPC-5 chromatographic step at neutral pH served to purify duplex fragments 70, 172, 250 and 440 base pairs long; each of these yielded two elution peaks upon chromatography under alkaline denaturing conditions.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

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.