Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

Abstract

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

“…There is one copy of DHFR gene per amplisome, and no large repetitive structure is detected. This is in sharp contrast with the observed structure and organization of the other cytogenetic anomalies such as DMs, episomes, or homogeneous staining regions that are also associated with amplified genes (Cowell 1982;Carroll et al 1987;Looney et al 1988;Ma et al 1988). Many groups have studied the size of the amplification units and the organization of the amplified genes on DMs, episomes, and HSRs.…”

“…Many groups have studied the size of the amplification units and the organization of the amplified genes on DMs, episomes, and HSRs. The structure of the amplified DNA sequences in different loci has been shown to be organized as head-to-tail tandem arrays Ma et al 1988) or as inverted duplications (Carroll et al 1987). The structure and organization of the amplified DNA sequences in DMs have also been shown to be highly heterogeneous, even in a single cell or cell line.…”

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

“…4}. It is also interesting to note that several examples of inverted duplications have been observed in the smaller amplicons that characterize cell lines with high amplicon copy numbers (e.g., see Ford and Fried 1986;Looney and Hamlin 1987;Ma et al 1988;Hyrien et al 1989). In each of these examples, however, the palindromic junctions are amplified, and they are relatively close to the selected gene.…”

“…Although some information is available concerning the structure and disposition of amplicons late in the amplification process (see Ardeshir et al 1983;Federspiel et al 1984;Debatisse et al 1986;Guilotto et al 1986;Looney and Hamlin 1987;Looney et al 1988;Ma et al 1988), very little is known about the molecular mechanisms that initiate and perpetuate the amplification process in any mammalian system. The following models have been proposed (for review, see Schimke 1982;Wahl 1984i Hamlin et al 1991): (1) unequal sister chromatid exchange (USCE) could result in the juxtaposition of both copies of a locus on one chromosome arm, presumably followed by additional rounds of USCE (Fig.…”

“…6Corresponding author. Debatisse et al 1986;Ma et al 1988]; (3} the multiple amplicons are arrayed in tandem, either in the body of chromosomes as abnormally banding regions (ABRs; Biedler and Spengler 1975) or as acentromeric, autonomously replicating episomal elements (termed double minutes; Kaufman et al 1979;Barker 1982).…”