We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less expensive and toxic reagents, requires only inexpensive laboratory equipment and is more readily adapted to high-throughput DNA extraction. This protocol yields approximately 5-30 microg of total DNA from 200 mg of tissue fresh weight, depending on plant species and tissue source. It can be completed in as little as 5-6 h.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric P-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

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...

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric 0-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold.In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than 4 0 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increase expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.