Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

Abstract

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies -240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.It has been known for several years that there is a temporal order of replication of defined genomic sequences in the DNA synthesis period of higher eucaryotic cells (11,22,29,32,54,56). It is also clear that early-and latereplicating sequences can be interspersed on the same chromosome (11) and that a given sequence can be replicated at different intervals of the S period in different cell types of the same organism (22,29,32). In fiber autoradiographic studies, mammalian chromosomal DNA has been shown to be synthesized through the agency of thousands of tandemly arranged replication units (replicons; 31). DNA synthesis within each replicon usually initiates at a centered origin from which the forks proceed outward bidirectionally. There is also evidence that groups of adjacent origins can fire at the same time in the S period (31). Presumably, control over the temporal order of replication is exerted at the level of initiation at each origin of replication.It is not known whether initiation sites in the individual replicons of mammalian cells are fixed genetic elements analogous to the origins of microorganisms, but different lines of evidence suggest that they may be. For example, several laboratories have used in vivo labeling protocols to examine the pattern of replication fork movement through large, defined chromosomal loci (1,5,8,30,32,35). Within the limits of resolution of these studies, forks appear to originate from preferred zones or sites. There have also been a few recent reports of small clone...

Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription

“…This was unexpected, since only a few later-firing origins have been identified to date (e.g., Ma et al 1990;Larner et al 1999;Lin et al 2003). Furthermore, many of these fragments appear to be intimately admixed with early firing fragments (logearly; Fig.…”

Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.

“…High-molecular-weight genomic DNA from CHO-K 1 cells was prepared as described previously (Ma et al 1990) and was digested partially with Sau3A1. The digest was separated on a NaC1 gradient, the 30-50 kb fractions were pooled, and the fragments were cloned into the BamHI site of dephosphorylated PWE-16 (Wahl et al 1987).…”

Densely methylated DNA islands in mammalian chromosomal replication origins.

“…3 was repeated by amplifying both strands of chromosomal DNA with primers P1 to P4 (Table 1) and by sequence determinations on "60 DNA clones, using both the M13 forward and reverse sequencing primers. Also, DNA replication was reversibly arrested by depriving cells of a required amino acid (proline) and restored in medium containing 0.5% serum by transformation with the papovavirus simian virus 40 (1,6,18,20,42,43). ori-1 has been mapped biochemically by analysis of imbalanced DNA replication (7,15), nascent DNA strand lengths (42), and Okazaki fragment distribution (6) to a short segment of cloned DNA whose complete base sequence was determined (8).…”

“…OBRs: orS14 and ori-1 downstream of the Chinese hamster dhfr (dihydrofolic acid reductase) locus (1,6,18,20,42,43 (Table 1). Oligonucleotides used to amplify sodium bisulfite-treated DNAs (see below) were designed on the assumption either that none of the dC residues within the priming sequences were methylated and therefore were converted to dTs through bisulfite modification or that all dCs were methylated and therefore remained dCs.…”