Effect of gamma rays at the dihydrofolate reductase locus: Deletions and inversions

Abstract

A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of "foreign" DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells.

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

“…Dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44, a double deletion mutant that contains no copies of the hamster dhfr gene (Urlaub et al, 1983(Urlaub et al, , 1986, was used as the host cell line for mAb expression.…”

Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40

“…The SGN-40 variant heavy chain and SGN-40 light chain were each cloned into the expression vector pDEF38 (Running Deer and Allison, 2004) downstream of the CHEF EF-1a promoter and stably expressed in CHO-DG44 (Urlaub et al, 1986) …”

Ionizing radiation-induced mutagenesis

“…The resolution is -100bp. Using this system Chasin's group Urlaub et al, 1986) have analysed the changes in spontaneously arising and y-and UV-irradiation-induced mutants. Of 5 spontaneous mutants none showed deletions.…”

“…Indeed, where both cytogenic and molecular analysis have been performed together, most of the chromosomal changes have been found to be unrelated to the known localization of the genes investigated (Fuscoe et al, 1986;Urlaub et al, 1986;Yandell et al, 1987). There are also locus specific effects.…”