Chromatin subunits from baker's yeast: isolation and partial characterization.

Abstract

The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 Before the transcriptional apparatus of yeast can be related with certainty to that of higher cells, similarities in genome organization must be established. Interphase chromatin fibrils of higher eukaryotes have a "beaded string" structure consisting of repeated arrays of globular particles (about 100 A diameter) connected by thin nucleoprotein filaments (9-12). Nucleases cleave these interconnecting regions, producing individual "subunits" and larger oligomeric nucleoprotein units that can be readily isolated (12, 13). Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12,14). Recent neutron diffraction studies (15) have confirmed earlier proposals (10, 11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14,16,17).Yeast chromatin potentially may have a subunit structure since yeast nuclei contain several major basic proteins that electrophorese similarly to histones (18,19). In fact, staphylococcal nuclease digests yeast chromatin at discrete intervals, indicating that proteins are arranged along the DNA in-a periodic manner (18,20,21). However, some caution is warranted in interpreting these results because of reports that yeast lacks histones Hi and H3 (22) and that yeast ribosomal proteins may be coisolated with or otherwise mistaken for histones (23).As described below, we have confirmed the periodic structure of yeast chromatin and have isolated and partially characterized monomeric subunits and oligomer units from yeast that appear to be nearly identical to subunits from higher eukaryotes.MATERIALS AND METHODS Isolation and Digestion of Yeast Nuclei. Saccharomyces cerevisiae strain A8209B was obtained from Dr. Gerald Fink (Cornell University). Yeast cells and protoplasts were prepared as described by deKloet et al. (24). Prior to isolation of nuclei, protoplasts (OD6Wj nm = 1-1.5) were incubated for 45 min at 300 in 20 mM KH2PO4 (pH 6.2)/2 mM MgCl2/2% glucose/ 0.3% casamino acids/20% sorbitol to permit return to a metabolically active state (24). The protoplasts were then rapidly cooled to 40 and washed with cold 20% sorbitol. Nuclei were isolated (at 0-40) by a method modified slightly from that of Blobel and Potter (25). Protoplasts were washed once in TKMC buffer (50 mM Tris-HCI, pH 7.5/25 mM KCI/5 mM MgCI2/1 mM CaCI2) containing 0.25 M sucrose, then twice in the same solution containing 0.5% Triton X-100. The crude nuclear pellet was purified by centrifugation through dense sucrose in TKMC buffer plus 0.1 mM phenylmethylsulfo...

A 368-Base-Pair cis -Acting HWP1 Promoter Region, HCR, of Candida albicans Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p

“…Hta1 and Htb1 have predicted masses of 13,861 Da and 13,988 Da, which matched their observed masses on SDS-PAGE gels. A BLAST analysis also revealed a strong similarity to S. cerevisiae histones Hta1 and Htb1, subunits of the nucleosome (29).…”

Isolation of the yeast nuclear pore complex.

“…Thus by Coomassie staining, nuclear proteins are represented by the four histones ( Fig. 3 A, lane 7, positions marked with dots; Thomas and Furber, 1976;Nelson et al, 1977). A band at around 50 kD was believed to be a cytoplasmic protein, and was found to be a persistent contaminant of yeast fractionation procedures ( Fig.…”

Comparison of the structure and transcriptional capability of growing phase and stationary yeast chromatin: a model for reversible gene activation

“…Based on the a-amanitin sensitivities determined in vitro (24,25) A) growing and stationary oligomeric nucleoprotein, the "oligo" fraction (see Table III Can the kinds of differences noted in stationary vs. growing nuclei be maintained in isolated, fractionated chromatin? To find out, we have fractionated staphylococcal nuclease intranuclear chromatin digests by sedimentation in isokinetic sucrose gradients (8). The kind of resolution we obtain on a sucrose gradient of yeast nucleosomes is shown in Figure 6, Compared to organisms with less active genomes, there are very large amounts of RNA and non-histone proteins in these gradient fractions.…”