Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells.
To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection.
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D005959:Glucosidases / Q000235:genetics*; D006801:Humans; D007122:Immunoelectrophoresis; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology; D051379:Mice; D014170:Transformation, Genetic; D000520:alpha-Glucosidases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; C034251:4-nitrophenyl-alpha-glucosidase; D005959:Glucosidases; D000520:alpha-Glucosidases