Species specificity of informatin

119

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (1O). nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.The periodic structure of chromatin now appears to be well established (for review, see refs. 1 and 2). A repeat unit of chromatin, termed the nucleosome, is made up of an octamer aggregate of histones containing pairs of each of the four main types, H2A, H2B, H3, and H4, which together with histone HI (3) is complexed with DNA of a variable length (150-240 base pairs). The basic structural element of the nucleosome in all cell types seems to be a core particle containing the histone octamer and DNA of about 140 base pairs in length (4, 5). Recent studies of the core particle crystals suggest that its structure is a flat disc of dimensions 110 X 110 X 57 A; about 1.75 turns of DNA in the B form are wound around the protein core (6). The proximity of histones in chromatin has been revealed by histone-histone crosslinking experiments (for review, see ref. 1). Histones H3 and H4 appear to play a central part in nucleosome formation (7) as first suggested by Kornberg (8). In the nucleosome the DNA sites separated by 10-nucleotide intervals are exposed to the action of different nucleases (7, 9). As determined by methylation experiments (10, 11), histones are partly buried in the major groove and leave the minor groove of DNA well exposed.Here we report a high-resolution map for the arrangement of histones along the core DNA determined by a sequencing procedure (11) in which histones were crosslinked to DNA within the core (12). Crosslinking causes the specific scission of one DNA strand at the point of crosslinking and the attachment of only the 5'-terminal DNA fragment to histones. The size of the single-stranded DNA fragments crosslinked to each histone species has been measured to identify the position of histones on one strand of the core DNA from its 5' end. A fairly clear picture of the detailed organization of histones has emerged from this map. Preliminary results of this work have been published elsewhere (11). MATERIALS AND METHODSCrosslinking Histones to DNA. The core particles were prepared from Hl-depleted chromatin of mouse Ehrlich ascites tumor cells as described (3, 11). The core particles (0.5 mg/ml) were methylated (abou...

Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

Mirzabekov¹,

Shick²,

Belyavsky³

et al. 1978

119

“…The DNase I nicking site map determined from digestion of end-labeled core particles does not show a frequently nicked site in the middle of the core particle (11,13,14). However, to satisfactorily simulate different features of the extended ladder pattern of yeast nuclei, of DNase I digestion products.…”

Section: Resultsmentioning

confidence: 99%

Organization of spacer DNA in chromatin.

Lohr¹,

Holde²

1979

Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (lOi + 5) base pairs, where m = 0, 1, 2,2.... This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second DNase I digestion of yeast, chicken erythrocyte, and HeLa nuclei produces "extended ladders" of bands of single-strand DNA fragments extending to well beyond the size of a nucleosomal repeat (1). This shows that the interparticle DNA, so-called linker or spacer DNA, occurs in discrete sizes in at least some fraction of the chromatin of these species. We have used the term "phasing" to denote this characteristic of spacer organization, because a discrete spacer requires that adjacent core particles be in a precise location relative to one another. Phasing, as used here, is not meant to suggest there is a precise location of a nucleosome with respect to a given DNA sequence. We present in this paper further observations on nucleosome phasing that provide at least partial explanation for many of the details of DNase I cleavage patterns in yeast and other eukaryotes.EXPERIMENTAL PROCEDURES HeLa, chicken erythrocyte, and yeast nuclei (the last from growing and stationary cells) were isolated as described (2, 3). DNase I digestion and DNA extraction were performed as in ref. 2. DNA was electrophoresed on either 5.5% or 8% polyacrylamide/98% formamide gels as described (4) or 8, 12, 13, or 15.5% polyacrylamide/urea gels prepared as described (5). Gels were analyzed as in ref. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. the two series overlap. In fact, a plot of band number vs. gel mobility shows precisely this behavior (Fig. 2). Both series are quite regular; however, in the overlap region there is a transition from the "low" (10-z110 b) pattern to the "high" (>140 b) series.We have accurately calibrated the band sizes of the yeast and chicken erythrocyte DNA and of our PM2 Hae III restriction fragments against OX174 Hae III, simian virus 40 Hae III, and pBR322 Hae III restriction fragments, whose sizes are exactly known from DNA sequence determinations (6-8). Results are shown in Table 1. Notice that (i) in the overlap region the two patterns differ by about 4-6 b, quantitatively showing the amount of DNA by which the high and low series of bands are out of phase and (ii) clearly the interval between bands in both the low and high series slightly exceeds 10 b.A graphical representation of the size data (Fig. 3) shows a clear discontinuity in the sizes of the two series of bands obtained by DNase I digestion of yeast nuclei. The lower series can be fi...

“…The filiation of some of the clonal derivatives of H4HEC3 rat hepatoma cells is shown in Fig. 1 (3,23). The differences among the repeat length probability distributions for these clones are negligible (Fig.…”

Section: Methodsmentioning

confidence: 97%

Chromatin repeat length correlates with phenotypic expression in hepatoma cells, their dedifferentiated variants, and somatic hybrids.

Sperling¹,

Weiss²

1980