NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.
GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.
Kirby
J
J
Department of Genetics, Trinity College, Dublin 2, Ireland.
Kavanagh
T A
TA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D009439:Neuraminidase; D005966:Glucuronidase