A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.
We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.
Datla
R S
RS
Molecular Genetics Section, NRC Canada, Saskatoon.
Hammerlindl
J K
JK
Pelcher
L E
LE
Crosby
W L
WL
Selvaraj
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005819:Genetic Markers; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D015151:Immunoblotting; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D004251:DNA Transposable Elements; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D005966:Glucuronidase