Micro-BIA, a colorimetric microtiter assay of lambda prophage induction.
Escherichia coli that are lysogenic for a lambda-lacZ fusion phage produce beta-galactosidase, product of the lacZ gene, upon induction of the prophage by DNA-damaging agents. The miniaturization of a quantitative, colorimetric beta-galactosidase (prophage) induction assay (BIA) is presented. Induction assays are performed in microtiter wells with the aid of multichannel pipetting devices. Results are shown with screening strain BR513 (uvrB delta envA) and a strain, BR339 (uvrB delta lexA3ind-) which exhibits enhanced induction. A method developed for strain BR339 utilizes bacteria stored frozen in log phase, permeabilized in vitro, and used immediately; with this method, 2 consecutive assays may be completed in 1 working day. Mutagens utilized for the model studies included 4NQO, ENNG, daunorubicin, bleomycin, acetoxy-AAF, B[a]P, DMBA, and DEN (the last three in the presence of liver S9). Induced levels of beta-galactosidase were monitored using a vertical light path photometer that measured color absorbance in each microtiter well. Alternatively, color intensity could be determined by using a color chart prepared for this assay. These values were then plotted to generate dose-response curves. Considerable savings in labor and materials are achieved with the method described, one which may be used as a screen for DNA-damaging chemicals. Automated equipment and computers may be used to advantage with this assay, but they are not required.
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D010582:Bacteriophage lambda / Q000187:drug effects; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D051379:Mice; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D008862:Microsomes, Liver / Q000378:metabolism; D009152:Mutagenicity Tests / Q000295:instrumentation; D009153:Mutagens / Q000494:pharmacology; D014775:Virus Activation / Q000187:drug effects*; D001616:beta-Galactosidase / Q000032:analysis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D009153:Mutagens; D001616:beta-Galactosidase