Analysis of DNA of isolated chromatin subunits.

Abstract

Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleoprotein complexes consisting of one or more v bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from v bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in v body DNA. These results indicate that v body formation may be random with respect to DNA sequence and suggest that the mere presence of v bodies over a specific regioni of DNA is not sufficient to restrict its transcription.Over the past year an experimentally consistent model of chromatin structure has emerged which postulates the presence of regularly repeating nucleoprotein subunits joined by short segments of DNA. Biochemical evidence for a periodic protein-DNA complex was initially introduced by Hewish and Burgoyne (1) and subsequently confirmed in several laboratories (2-6). These data demonstrate that mild nuclease digestion of nuclei results in the liberation of a series of nucleoprotein particles, v bodies, containing DNA fragments whose molecular weights are all multiples of a unit fragment 150-200 base pairs in length. Subsequent studies (4,5,7,8) have revealed that the v body represents a transient intermediate in the digestion process and that cleavage of DNA within the P body results in the generation of a true limit digest reflecting the internal structure of the monomeric subunit.Electron microscopic observations (9, 10) are in accord with the biochemical data and reveal the existence of spherical nucleoprotein particles which are joined by a short filament of DNA. These particles can be observed closely apposed to one another in gently disrupted nuclei, providing convincing evidence that these nucleoprotein particles reflect a level of structure characteristic of the chromatin fiber in vio.The elucidation of a subunit structure in chromatin immediately poses the question as to the possible role of this structure in those biological processes in which chromatin participates. In this report we describe procedures for the isolation and characterization of highly purified v bodies.We find that most if not all of the DNA sequences in the genome are involved in the formation of these particles. No specific frequency class of DNA is restricted from particle formation. Further, virtually all sequences express...

Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study

“…Extended nonbeaded states of chromatin with the full nucleosomal histone complement have so far been described only in high urea concentrations (for references, see Olins et al ., 1977). The findings in various laboratories that transcriptional complexes and/or sequences complementary to transcriptional products can be recovered in the nucleosomal fraction obtained by nuclease digestions (for example, Lacy and Axel , 1975;Gottesfeld et al ., 1975;Garel and Axel, 1976;Mathis and Gorovsky , 1976;Kuo et aI. , 1976;Piper et aI., 1976;Reeves and Jones, 1976;Reeves, 1976Reeves, , 1977Brown et al ., 1977) are not contrad ictory to the conclusion that nucleosomal beads are absent in chromatin regions engaged in transcription.…”

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

“…Biophysical and electron microscopic methods have indicated that high salt causes chromatin to become less compact (25,28). Assuming that the viral transcription complex contains nucleosomes, as appears to be the case for template active cellular chromatin (18)(19)(20), a salt-mediated conformational change such as the conversion from an octamer to two tetramers may facilitate a maximal rate of RNA chain elongation in nucleosomal DNA, at least in vitro.…”

“…Nucleosomes are comprised of about 200 base pairs of DNA wound around a complex containing two each of the four major histones (11)(12)(13)(14)(15)(16)(17). Transcriptionally active and inactive regions of cellular chromatin are both organized in this basic manner (18)(19)(20), although there is some structural difference between them, as evidenced by a selective deoxyribonuclease I sensitivity of active genes (21,22).…”

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

“…A distinctive eucaryotic feature is that the genomic DNA is organized into nucleosomes by intimate association with histones and other proteins to form chromatin. Treatment of chromatin with micrococcal nuclease results in a characteristic nucleosome repeat structure (16). Moreover, chromatin can exist in both transcriptionally active and inactive forms.…”