EMS-INDUCED MUTANT FREQUENCY AND SPECTRUM IN BONE-MARROW OF D6-2 TRANSGENIC MICE

EMS-induced mutant frequency and mutation spectrum as well as backgrou nd mutant frequency have been characterized for bone marrow of the D6- 2 transgenic mice. The lad genes carried on pSPORT1 vectors were recov ered from the treated or untreated mouse genomic DNA by excision and c ircularization, and analyzed in vitro for mutations that occurred in t he mouse bone marrow. lacI(-) mutants were positively selected with th e M9/L media. The 6 lacI(-) mutants were identified out of 11 935 vect ors recovered from genomic DNA of the treated mice (mutant frequency w as 50 x 10(-5)), while no mutant was found in 11 649 vectors from untr eated mice (the background mutant frequency was lower than 8.6 x 10(-5 )). Two regions of lad for each mutant, in which the majority of sensi tive sites for inactivation of the lad gene product have been located, were sequenced and 16 mutation events were identified. The predominan t mutations (14/16 or 87.5%) were base substitutions, whereas the rema ining 2 mutations were single base deletions (12.5%). Of these base su bstitutions, transversions made up 9/14 or 64%, and transitions compri sed 5/14 or 36%. These findings were markedly different from the spont aneous spectra characterized by using Big-Blue(R) system, as well as f rom the EMS-induced mutation spectra obtained with in vitro assay syst ems, where the EMS-induced predominant mutations are GC-->AT transitio ns. In addition, 45 % of mutations analyzed occurred at CpG dinucleoti des, which was in accordance with previous studies with other systems. These data show that: (i) the D6-2 transgenic mouse lineage is a suit able model for studying mutagenesis in vivo,; (ii) a fundamental diffe rence in mutagenesis for EMS between in vitro and in vivo, assay syste ms may exist, but more extensive sequence analyses are required to det ermine the possible differences in mutation spectra.