Development of a lambda-based complementation assay for the preliminary localization of lacI mutants from the Big Blue mouse: implications for a DNA-sequencing strategy.
The Big Blue transgenic mouse carrying the E. coli lacI gene as a mutational target in a lambda-based shuttle vector has been receiving increasing attention in genotoxicity testing because it offers the potential of studying mutation in a mammalian system in vivo. The system not only provides information on mutant frequency, but it also offers the potential of providing information about mutational specificity. Such data is not only important for studies of mutational mechanisms; it offers a critical advantage for determining the mutational response at levels where significant increases in mutant frequency have not been discerned. The repeated sequencing of the entire 1080-bp lacI target, however, remains a formidable task. Here we report on the adaptation of the "negative complementation" assay for the lacI-d phenotype to accommodate the lambda lacI recovered from the Big Blue transgenic animal. This assay permits the localization of mutations to an approximately 330-bp region to facilitate the production of mutational specificity data. The assay is based upon lysogenization of the lambda containing the lacI mutation into a lacI+ host. Of 107 sequenced lacI mutants recovered from Big Blue mice, 74 were identified as NC+ (lacI-d) using this assay. Of these 74, 49 occurred in the region 32-208 bp, which has traditionally been viewed as the NC+ domain. 33 of these mutations were previously identified as producing the NC+ phenotype while another 7 occurred at sites where NC+ mutants have been recovered, but involved a new base substitution. 9 mutants involved new sites. An additional 25 mutants located downstream of the presumed NC+ region were also found to be NC+ as determined by their blue colour on X-gal plates. Of these, 18 occurred in the 209-360-bp region. In parallel, 54 lacI mutants carrying unknown mutations were examined. 37 of these produced blue colonies in this assay. The sequencing of these mutants revealed that 20 (54%) of the 37 mutants were located in the 32-208-bp region. This complementation assay can potentially reduce the amount of DNA sequencing necessary to produce a mutational spectrum by optimising the choice of sequencing primers, and thus provide a significant saving of the material and time required. Furthermore, evidence indicates that the restriction of the mutational target to the NC+ region extends these savings without reducing the usefulness of the mutational specificity data.
Gu
M
M
Centre for Environmental Health, University of Victoria, BC, Canada.
Ahmed
A
A
Wei
C
C
Gorelick
N
N
Glickman
B W
BW
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D005816:Genetic Complementation Test / Q000379:methods*; D007763:Lac Operon / Q000235:genetics; D008242:Lysogeny; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*
D012097:Repressor Proteins