NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS Summary 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 α‐d‐N‐acetylneuraminic acid (X‐NeuNAc) and 5‐bromo‐6‐chloro‐3‐indolyl β‐d‐glucuronide (X‐GlucM), respectively. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS

The Plant Journal, Volume 32 (3) – Nov 1, 2002

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Publisher
Wiley
Copyright
Copyright © 2002 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
D.O.I.
10.1046/j.1365-313X.2002.01422.x
Publisher site
See Article on Publisher Site

Abstract

Summary 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 α‐d‐N‐acetylneuraminic acid (X‐NeuNAc) and 5‐bromo‐6‐chloro‐3‐indolyl β‐d‐glucuronide (X‐GlucM), respectively.

Journal

The Plant JournalWiley

Published: Nov 1, 2002

References

  • Cell‐to‐cell and long‐distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues
    Imlau, Imlau; Truernit, Truernit; Sauer, Sauer
  • Use of red fluorescent protein from Discosoma sp. (dsRed) as a reporter for plant gene expression
    Jach, Jach; Binot, Binot; Frings, Frings; Luxa, Luxa; Schell, Schell
  • Initiation of translation in prokaryotes and eukaryotes
    Kozak, Kozak
  • Single‐step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
    Stemmer, Stemmer; Crameri, Crameri; Ha, Ha; Brennan, Brennan; Heyneker, Heyneker

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