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...

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.

hydrophobic except for a few relatively hydrophilic Pro residues that are highly conserved. The central domain is a unique feature of the oleosins because it is Over 70 amino acids long, which is the longest con~nuous hydrophobic stretch of any known protein.It is of interest to study the ontogeny of oil bodies because Of the organelle's importante to TAG accumulation and storage. Techniques that might be expected to illustrate the mechanisms of oil body assembly have instead rendered confusing and inconclusive results. The processes involved in oil body formation, especially the con&ibution of the ER, are still controversial (see Murphy, 1990, and H~~~~ 1992, for recent reviews). One hypothesis suggests that newly synthesized TAG accumulate in the hydrophobic interior of the ER membrane between the two phospholipid layers.Soybean (GlYCine max) seed oil is stored as droplets of Eventually, a droplet of TAG surrounded by a phospholipid TAG, which acCumuhte in the CytoPlasm as discrete organmonolayer disconnects and is released into the cytosol (Freyelks tenned Oil bodies.Oil or is involved in an indirect manner. may prevent coalescence of the oil bodies.In the present study, we have focused on learning more Oleosin cDNAs and genomic clones have been character- Murphy et al., 1991; Cummins and Murphy, 1992). A comparison of the predicted amino acid sequence of these clones has demonstrated that oleosin proteins share a common secondary structure, which consists of three domains. The amino-and carboxy-terminal domains have very little sequence identity or similarity; however, the hydopathic nature of these domains has been conserved. The a-o teenus foms a hyhophdic a-helix, whereas the carboxy terminus foms an amphipathic a -h e h . In contrast, there is an extremely high degee of sequence identity in the central domain of the protein. 24-kD oleosin is cotranslationally integrated into microsomal membranes. We demonstrate that oleosin is integrated into a bilayer membrane in preference to the oil body monolayer membrane, indicating that oleosin is synthesized on the endoplasmic reticulum (ER). A new model of oil body assembly involving a conformational change through initial association with the ER