Nuclear scaffolds and scaffold-attachment regions in higher plants.

Abstract

DNA in the nuclei of eukaryotic organisms undergoes a hierarchy offolding to be packaged into interphase and metaphase chromosomes. The first level ofpackaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber. Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs). Support for the ubiquitous nature of such higher order packaging of DNA is presented here by. our work with plants. We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold. We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes. This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold. Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs. The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA faments derived from the 3'-flanking regions ofthe root-specific genes are confirmed to contain SARs by this exogenous assay.The structure of the nucleosome core, a complex of eight histones and 146 base pairs (bp) of DNA wrapped around the outside, is now fairly well understood (1). Much less well understood is the structure of the 30-nm chromatin fiber and how these fibers are coiled and folded to form interphase and metaphase chromosomes (2). Central to many models of this "higher order" chromatin structure is the concept ofdomains formed by loops of the 30-nm fibers attached at their bases to a proteinaceous nuclear or chromosome scaffold. Early evidence for such a model came from electron micrographs of histone-depleted chromosomes and nuclei showing loops of DNA spilling out to form a halo (3-7). Mirkovitch et al. (8) showed that the loops were not randomly attached to the nuclear scaffold but that specific DNA regions were involved. These regions, which have been called scaffold attachment regions (SARs), have been partially characterized. The binding sites have been mapped to regions generally ranging from 300 to 1000 bp, which are generally A+T-rich.The domains formed by SAR-bounded loops may have functional, as well as structural, significance. It has long been realized that regions of DNase I-sensitive chromatin, which contain transcriptionally poised genes, are not confined to the genes themselves but rather extend over much larger domains (9-11). These DNase I-sensitive domains have been shown to correspond to SAR-bounded loop domains (12-14). Moreover, inconsistencies in the levels of expression of genes in transgenic...

Presence of an SAR‐like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

“…In the ARS1 sample approximately 60% and 40% of the total radioactivity (T), respectively, were detected in the pellet (P) and supernatant (S) fractions. These ®ndings are very similar to those of a previous report (Hall et al, 1991). In contrast, almost all radioactivity was recovered in the S fraction of the pUC19 sample, only a trace amount being present in the P fraction.…”

“…DNA was isolated without fractionation from the pellet and supernatant fractions, as well as from the DNA/nuclear scaffold mixture, by incubation with a lysis buffer (1% SDS/500 mg ml ±1 Proteinase K/ 20 mM EDTA/20 mM Tris±HCl, pH 8.0) for 16 h at 37°C followed by phenol extraction. The DNAs were separated by electrophoresis as reported previously (Hall et al, 1991) and autoradiographed.…”

Characterization of Randomly-Obtained Matrix Attachment Regions (MARs) from Higher Plants,

“…Nuclei were isolated from a Nicotiana tabacum cell line (NT-1) as previously described (35,41). Nuclear matrixes were prepared from the isolated nuclei by a modification (35,69) of the method of Mirkovitch et al (37), which employs lithium diiodosalicylate (LIS).…”

“…The binding of the cloned fragments to isolated nuclear matrixes was performed by the exogenous binding assay as previously described (13,35,41,69). To generate radioactively labeled probes for the binding assays, plasmids were cut with either EcoRI or HindIII to release the insert.…”

“…In many cases, the large region of genomic DNA assayed for MARs was chosen because it harbors a gene or genes of particular interest. Examples of this approach include a 10.5 kb region containing Drosophila heat shock genes and a 5 kb region containing Drosophila histone gene repeats (37), a 19 kb region containing the mouse kappa immunoglobulin gene (6), a 36 kb region containing the human interferon-gene (9), a 35 kb region containing the Chinese hamster dihydrofolate reductase gene (38), a 90 kb region containing a human globin gene complex (39), a 19 kb region containing the chicken lysozyme gene (40), a 23 kb region containing a root-specific gene from tobacco (41), a 3.6 kb region containing the -phaseolin gene of bean (42), a 7 kb region containing a tomato heat shock cognate gene (43), a 17 kb region containing a seed-specific lectin gene from soybean (26), and a 16 kb region containing the Arabidopsis plastocyanin gene (44).…”