In higher eukaryotes, the DNA composition of centromeres displays a high degree of variation, even between chromosomes of a single species. However, the long-range organization of centromeric DNA apparently follows similar structural rules. In our study, a comparative analysis of the DNA at centromeric regions of Beta species, including cultivated and wild beets, was performed using a set of repetitive DNA sequences. Our results show that these regions in Beta genomes have a complex structure and consist of variable repetitive sequences, including satellite DNA, Ty3-gypsy-like retrotransposons, and microsatellites. Based on their molecular characterization and chromosomal distribution determined by fluorescent in situ hybridization (FISH), centromeric repeated DNA sequences were grouped into three classes. By high-resolution multicolor-FISH on pachytene chromosomes and extended DNA fibers we analyzed the long-range organization of centromeric DNA sequences, leading to a structural model of a centromeric region of the wild beet species Beta procumbens. The chromosomal mutants PRO1 and PAT2 contain a single wild beet minichromosome with centromere activity and provide, together with cloned centromeric DNA sequences, an experimental system toward the molecular isolation of individual plant centromeres. In particular, FISH to extended DNA fibers of the PRO1 minichromosome and pulsed-field gel electrophoresis of large restriction fragments enabled estimations of the array size, interspersion patterns, and higher order organization of these centromere-associated satellite families. Regarding the overall structure, Beta centromeric regions show similarities to their counterparts in the few animal and plant species in which centromeres have been analyzed in detail.
The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.
Recently, it has been suggested that nuclear processes, such as replication, transcription, and splicing, are spatially organized and associated with a nuclear framework called the nuclear matrix, a structure of unknown molecular composition. It has been shown that chromatin is attached to the nuclear matrix via specific DNA fragments called matrix attachment regions (MARs). We have begun to dissect the plant nuclear matrix by isolating a DNA binding protein with specific affinity for MARs. Here, it is shown that MAR binding filament-like protein 1 (MFP1) is associated with specklelike structures at the nuclear periphery that are part of isolated nuclei and the nuclear matrix. A predicted N-terminal transmembrane domain is necessary for the specific targeting of MFP1 to the speckles, indicating an association with the nuclear envelope-endoplasmic reticulum continuum. In addition, it is shown that a marker protein for plant microtubule organizing centers, which has been shown to be localized on the outside of the plant nuclear envelope, is also part of the nuclear matrix. These findings indicate a close and previously undescribed connection in plants between the nuclear envelope and the internal nuclear matrix, and they suggest a function for MFP1 in attaching chromatin to specific sites at the nuclear periphery. INTRODUCTIONThe nuclear matrix hypothesis proposes a structural framework for the eukaryotic nucleus that is similar to the cytoskeleton. Biochemically, the nuclear matrix is defined as the insoluble material that remains after extraction of nuclei with high-salt solutions (Berezney and Coffey, 1974) or with the chaotropic agent lithium diiodosalicylate (Mirkovitch et al., 1984) and treatment with DNases. Electron microscopy has shown a network of fibers of ف 10 nm in diameter. These fibers resemble the intermediate filaments of the cytoskeleton. Because they can be detected only under certain preparation conditions (He et al., 1990), their in vivo existence has been somewhat controversial.The isolated nuclear matrix binds specifically to certain DNA elements called matrix attachment regions (MARs). MARs are generally AT-rich DNA sequences that are several hundred base pairs long and are localized in the noncoding regions of the DNA, often flanking genes (Gasser and Laemmli, 1987). MARs have a positive effect on gene expression (Allen et al., 1993(Allen et al., , 1996Mlynárová et al., 1996) that is believed to be due to the establishment of independent and transcriptionally active chromatin domains between two MARs (Spiker and Thompson, 1996). Although the nuclear matrix was identified more than 20 years ago (Berezney and Coffey, 1974), none of its structural components has been isolated and molecularly characterized from any organism. One strategy to identify such components has been to isolate proteins that specifically bind to MARs. A small number of MAR binding proteins have been purified and cloned from animals, and they subsequently have been shown to be localized in the nuclear matrix (von Kri...
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