Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.

Abstract

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoffet al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhifr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.

“…6) (14) have shown that the order of three genes (LARS, CHR, and EMT) has been conserved on human chromosome 5 and the long arm of hamster chromosome 2. As shown here, the human DHFR gene has been assigned also to chromosome 5; however, the hamster DHFR gene has been mapped previously to the short arm of chromosome 2 (19,46). Thus, our results, taken together with those cited above, provide evidence for homology between both the short and long arms of hamster chromosome 2 and human chromosome 5.…”

“…The deletion in chromosome 2a seems to include bands p25 and p26, according to the standard nomenclature proposed by Ray and Mohandas (38). Since the DHFR gene has been mapped to the short arm (p) of chromosome 2 (19,46), it was possible that deletion of region p25-÷p26 resulted in loss of the DHFR gene from chromosome 2a.…”

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

“…Deletion cell line 20A1 appears to have been cloned from a cell that lost the acentric arm; in deletion clone 20A2, the acentric arm appears to have rejoined with the centric Z2 fragment to generate a Z2 chromosome with an interstitial deletion. The translocation of the acentric arm to chromosomes other than Z2 as a result of DHFR gene amplification has been observed previously (Biedler 1982;Flintoff et al 1984). The reasons why terminal sequences of the centric arm in 20A1 are preserved are unknown but could involve telomere addition (see Zakian 1989) or extremely slow resection, as observed in Drosophila (Levis 1989).…”

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…Hence, the LC transgene was most probably integrated in the same sites as the dhfr transgene, whereas the HC transgene was likely to be integrated further away from the dhfr gene and outside the amplification unit. During the gene amplification process, DNA rearrangements such as homologous recombination and translocation occur in high frequency (Bacsi and Wejksnora, 1986;Flintoff et al, 1984;Kim et al, 1998a;Rolig et al, 1997), resulting in the gene-amplified cells becoming heterogeneous in regard to transgene copy numbers although being derived from a single clone. In course of these DNA rearrangements, the distance between the HC transgene and the dhfr gene might become too wide so that the HC transgene is not within the amplification unit any more and, as a result of that, will no longer be coamplified in the following rounds of gene amplification.…”