Analysis of protein localization by use of gene fusions with complementary properties.
This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.
Manoil
C
C
Department of Genetics, University of Washington, Seattle 98195.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005798:Genes, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D005696:Galactosidases; D001616:beta-Galactosidase