Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations.
A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.
Srienc
F
F
Campbell
J L
JL
Bailey
J E
JE
D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005696:Galactosidases / Q000032:analysis*; D007700:Kinetics; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013194:Staining and Labeling; D001616:beta-Galactosidase / Q000032:analysis*
D005456:Fluorescent Dyes; D005696:Galactosidases; D001616:beta-Galactosidase