INFLUENCE OF ALKYLTRANSFERASE ACTIVITY AND CHROMOSOMAL LOCUS ON MUTATIONAL HOTSPOTS IN CHINESE-HAMSTER OVARY CELLS

High-density mutational spectra have been established for exon 3 of th e gene encoding adenine phosphoribosyltransferase (APRT) of the Chines e hamster ovary (CHO) cell line derivative D422 and closely related an d/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC --> AT transitions yielding a selectable APRT(-) phenotype) was estimated at 31 based on our own a ccumulated data base of 136 sequenced exon 3 mutations and on literatu re reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 ' 'baseline'' sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one compr ising a cluster at nucleotides 1293-1309 and the other at nucleotide 1 365. Collectively, the Latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer(-) as they lack the repair enzyme O-6-methylguanidine methyltransferase (EC 2.1.1.63). In modifi ed repair-proficient CHO cells, the distribution of mutations among al l of the 31 sites was random, with only 3 of the 19 GC --> AT transiti ons in the above hotspots. To determine whether the distribution was l ocus-dependent, two independent lines carrying single copies of transf ected APRT genes were generated from a derivative of D422 carrying a d eletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer(-) cells a re at least in part locus dependent and that some sequences are partic ularly susceptible to EMS mutagenesis and perhaps also to methyltransf erase repair.