Organization of spacer DNA in chromatin.

Abstract

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...

Nucleosomal arrangement affects single-molecule transcription dynamics

“…Finally, we note that earlier work has shown that yeast exhibits a very tight nucleosomal organization, with adjacent nucleosomes placed on opposite sides of the DNA helix due to the offset of one-half a helical turn (8)(9)(10)(11). Our results therefore suggest that this type of nucleosomal arrangement might be critical for efficient chromatin transcription by RNA polymerase II.…”

“…A central outstanding question is to what extent the nucleosomal arrangement affects Pol II transcription behavior. This arrangement might be particularly important in budding yeast, an organism with a compact genome with short internucleosomal distances (8)(9)(10)(11).…”

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning

“…Considering a 1-to 2-bp overstretching effect of nucleosome DNA (33,36), the S. pombe genome displays a preferential linker length form of ∼10n + 4/5 bp. Because linker length is closely related to the high-order chromatin structure (37,38), this implies that these two genomes may share some similar aspects in high-order chromatin structure, regardless of the evolutionary divergence and distinctions in nucleosome formation between the species.…”

Organization of internucleosomal DNA in rat liver chromatin.

“…Gel electrophoresis of the DNA in denaturing conditions shows a ladder of discrete fragments extending up to or into dimer-size DNA. This observation was made for yeast, HeLa cells and chicken erythrocyte chromatin digested with DNase I (Lohr et al, 1977; Holde, 1979); rat thymus chromatin digested with Serratia marcescens nuclease (Pospelov et al, 1979); Drosophila chromatin digested with micrococcal nuclease (Karpov et al, 1982); and chicken erythrocyte (Riley and Weintraub, 1978), rat liver 1Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. *To whom reprint requests should be sent.…”