CHROMATIN STRUCTURE OF THE YEAST URA3 GENE AT HIGH-RESOLUTION PROVIDES INSIGHT INTO STRUCTURE AND POSITIONING OF NUCLEOSOMES IN THE CHROMOSOMAL CONTEXT

To characterize nucleosome structure and positioning in the chromosoma l context, the chromatin structure of the whole URA3 gene was studied in the genome and in a minichromosome by testing the accessibility of DNA to micrococcal nuclease and DNase I. The cutting patterns and henc e the chromatin structures were almost indistinguishable in the genome and in the minichromosomes. The only notable exception was enhanced c utting between nucleosomes U3/U4 and U4/U5 in the minichromomes. The r esults demonstrate that there is no severe constraint acting from outs ide the URA3 gene in chromosomes and minichromosomes. While low-resolu tion mapping showed six regions with a positioned nucleosome (U1 to U6 ), each region resolved in a complex pattern consistent with multiple overlapping positions. Some regions (U1, U4, U5 and U6) showed multipl e positions with a dominant rotational setting (DNase I pattern), whil e U2 showed positioning within 10 bp but with no defined rotational se tting, demonstrating that nucleosome positions were not in phase and n ot coordinately regulated. Reduced DNase I cutting from about 50 bp fo rm the 5' end towards 3' end was common to all nucleosome regions. Thi s polarity has been observed on isolated core particles. The results d emonstrate that the DNase I pattern observed in vitro indeed reflects a structural property of nucleosomes in the chromosomal context. It is emphasized that despite the local heterogeneity revealed by high-reso lution mapping, the low-resolution map is a reasonably accurate repres entation of the chromatin structure. (C) 1996 Academic Press Limited