FIG. 1.

Monomer extension mapping of nucleosomes positioned on HIS3 in TA-HIS3 chromatin purified from wild-type and gcn4Δ cells. (A) Map of the TRP1ARS1HIS3(TA-HIS3) plasmid (9). HIS3 was inserted at the EcoRI site of the TRP1ARS1 plasmid (37). TA-HIS3 has only 2,435 bp and contains no bacterial sequences. TRP1 is expressed at basal levels because the upstream activation sequence for TRP1 is not present in TRP1ARS1. The unique XbaI site used in the mapping experiments shown here is located in the TRP1 ORF. (B) Schematic diagram of the monomer extension method for mapping the positions of nucleosomes (41). The approach is to obtain DNA from nucleosome core particles (mononucleosomes or monomers) and use this as primer in a primer extension reaction; since both strands of nucleosomal DNA can act as primers, a single-stranded template must be used (otherwise two sets of extension products will be obtained). pGEM-TAHIS3 contains the entire TA-HIS3 sequence as an insert. Note that this method can resolve overlapping positions. (C) High-resolution monomer extension mapping of TA-HIS3 chromatin purified from uninduced (unind.) and induced wild-type cells and from gcn4Δ cells. Nucleosome positions were mapped with respect to the XbaI site in the TRP1 ORF (see panel A). The products of monomer extension reactions using nucleosomal DNA were analyzed with (+) or without (−) XbaI digestion in long sequencing gels. The purpose of monomer extension without XbaI cleavage was to identify bands resulting from premature termination by reverse transcriptase (lanes 4, 6, and 8). In the samples digested with XbaI, each band represents the distance of the downstream border of a nucleosome from the XbaI site. The bands which are quantitatively above background are labeled (nucleosomes D1 to D5 and A4 to A28); some are quantitatively so minor that they have been ignored. Some bands have been grouped (indicated by black bars at right) because they are less than 15 bp apart and so might represent incomplete trimming of the same nucleosome. The HIS3 transcription unit (with transcription start site at nucleotide +1) is shown at right, together with a series of gray ovals indicating the positions of the predominant (D) nucleosomes; these nucleosomes are present in wild-type and gcn4Δ chromatin but are clearly predominant in gcn4Δ chromatin. End-labeled markers: 100 bp ladder (New England Biolabs) (lane 1), a DdeI digest of λ DNA (lane 2), and a HinfI digest of λ DNA (lane 3). A phosphorimage is shown. The position information is summarized in Fig. 2A. (D) Scans of the monomer extension maps shown in panel C. The lanes corresponding to gcn4Δ chromatin (lane 9), induced wild-type chromatin (lane 7), and uninduced wild-type chromatin (lane 5) were scanned using a phosphorimager and normalized to the major peak at the top of the gel.