Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Abstract

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping -120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.In the eucaryotic genome, the chromatin loop represents a basic structural unit. The loops are generated by periodic attachment of the chromatin fiber to a nonhistone chromosomal protein scaffolding in the nucleus, which has been referred to as the nuclear matrix (3,8,9,36). During mitosis, part of this matrix probably rearranges to become the metaphase chromosomal scaffold (2,19). Although neither the interphase nuclear matrix nor the mitotic chromosomal scaffold has been fully characterized as yet, topoisomerase II has been identified as an integral component of both (5,14). This finding has led to the suggestion that the nuclear matrix may somehow poise individual chromatin domains for transcription by allowing torsional stress to be introduced into defined regions (7). Nonrandom organization of the chromatin is further suggested by the presence at the base of the loops of specific DNA sequences that associate with the nuclear matrix. In Drosophila cells, specific matrix attachment regions (MARs) have been found in the 5'-flanking regions of several active genes (12,26,33), and these MARs coincide with enhancerlike elements (12). However, in the murine K light-chain gene, an attachment site is found at an intragenic site...

Identifying a property of origins of DNA synthesis required to support plasmids stably in human cells

“…Much data indicate that homing of DNA templates to the operationally defined nuclear matrix correlates with their supporting DNA synthesis (33)(34)(35)(36). Consistent with this data, newly introduced plasmids containing oriP homed rapidly to the nuclear matrix (Fig.…”

Human telomeres are attached to the nuclear matrix.

“…Isolation of LIS-generated halo structures The procedure used is a modification of the protocol of Dijkwel and Hamlin (1988); the LIS technique was originally developed by Mirkovitch et al (1984). All procedures were carried out at 4°C unless indicated otherwise.…”

Studies of an 800-kilobase DNA stretch of the Drosophila X chromosome: comapping of a subclass of scaffold-attached regions with sequences able to replicate autonomously in Saccharomyces cerevisiae.

“…Biol. 190: 255-258, 1986), yeast (3), and vertebrate (7,12,13,15,27,28,33,41,49) genomes. Data have shown that the DNA regions interacting with nonhistone proteins, i.e., the scaffold attached regions (SARs) were nonrandomly distributed with regard to transcriptional units and replicons (20).…”

“…On the other hand, certain data support the idea that SARs may be involved in the replication process. These include the observation of coincidental sizes for replicons and loops in various species (10), the comapping of SARs and either initiation (3,15,16,39,43,51) or termination sites for * Corresponding author.…”