Ribosomal gene clusters are uniquely proportioned between open and closed chromatin structures in both tomato leaf cells and exponentially growing suspension cultures.

Abstract

The accessibility of regulatory molecules to specific DNA sequences and chromatin regions in the nucleus is crucial to gene expression. In this study, we examined the chromatin structure in tomato leaf cells and in exponentially growing tomato cell suspension cultures. The structure of ribosomal chromatin was investigated by micrococcal nuclease and psoralen photocrosslinking. We showed that ribosomal genes in tomato are folded into two While the structure of inactive bulk chromatin is fairly well understood (for reviews, see refs. 1 and 2), there is still no agreement on the organization of the active chromatin in transcribing genes and replicating DNA. With DNase I, it has been found that the active chromatin is degraded more rapidly than the bulk inactive chromatin and therefore is considered to be in a less condensed, or more open, conformation (for review, see ref.3).Particular interest in the chromatin structure of ribosomal genes is based on the peculiar organization of the rRNAencoding DNA (rDNA); active rRNA genes are transcribed at a maximal polymerase density [-1 polymerase per 100 base pairs (bp) or two polymerases per nucleosomal DNA] with a high elongation rate (-30 nucleotides per sec) (for review, see ref. 4). In spite ofthe high transcriptional activity, >100 rRNA gene copies are required in dividing cells to produce the needed amount of rRNA. Essentially all eukaryotic cells contain between 100 and 5000 ribosomal genes per haploid genome. The rDNA transcription rate is regulated as a function of the cellular growth rate, being down-regulated in stationary cells and up-regulated in exponentially growing cells (5, 6).The chromatin structure of transcribing nucleolar genes is still controversial. Davis et al. (7) showed that the ribosomal sequences in a mouse cell line could be fractionated into a nucleosome-containing chromatin component and a component where the ribosomal genes did not show a repeating structure. Mapping the sites of topoisomerase I interaction in ribosomal chromatin of Xenopus oocytes, Culotta and Sollner-Webb (8) found that these sites were spaced with a nucleosomal periodicity, a result suggesting that active ribosomal chromatin is in a nucleosomal array. Ribosomal chromatin was also found in modified nucleosomes in Physarum polycephalum (9,10). In contrast to these results, studies with micrococcal nuclease, DNase I, and psoralen crosslinking showed that the majority of the ribosomal genes in Physarum and Dictyostelium discoideum are not organized in the repeating structure (11)(12)(13) tTo whom reprint requests should be addressed. 5256The 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.

Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster

“…2 to 4). The most sensitive assay for detecting the level of active rDNA units is psoralen cross-linking (13,15,16,47). However, our psoralen crosslinking study did not reveal active units, suggesting such units represent less than 10% of the units in any of the bands we detected on Southern blots (Fig.…”

“…Sogo and colleagues have demonstrated that transcriptionally active rDNA units are more accessible to psoralen and after UV cross-linking can be separated from the inactive units by their slower migration during electrophoresis (14). This psoralen accessibility has been used to detect active rDNA units in many organisms (13,15,16,47). Isolated nuclei from 1-16-h embryos were incubated with psoralen and UV crosslinked (15,47,58).…”

“…Most organisms appear have more than the minimum number of rDNA units needed for full viability. In eukaryotes as diverse as yeast, plants, and mammals, less than half of the units appear to be active at a given time (13)(14)(15). The active rDNA units are more accessible to nucleases and cross-linking reagents than the inactive units.…”

Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

“…Data are the mean Ϯ 1 SD of three independent experiments. bracket) correspond to incomplete digestion of some rDNA sequences, because of the presence of psoralen crosslinks close to the restriction sites (27,38).…”

Repair-Independent Chromatin Assembly onto Active Ribosomal Genes in Yeast after UV Irradiation

“…The ribosomal genes are present in multiple copies (ϳ150 in yeast) that are organized in long tandem repeats (55). In most organisms only a portion of rDNA is transcriptionally active (reviewed in references 20 and 32), and the fraction of active rDNA varies markedly among organisms and cell types (e.g., from ϳ20% to ϳ70%) (8)(9)(10)(11). Furthermore, in yeast, the active rDNA population varies according to growth rate, with the highest proportion of active genes (about 50%) present in exponentially growing cells (10,13).…”