Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, β-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of β-glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.
- Bradford M: A rapid and sensitive method for the quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976). CrossRef
- Horsch R, Klee H: Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process. Proc Natl Acad Sci USA 83: 4428–4432 (1986).
- Iturriaga G, Jefferson RA, Bevan MW: Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco. Plant Cell 1: 381–390 (1989). CrossRef
- Jefferson RA: Assaying chimeric genes in plants. The GUS fusion system. Plant Mol Biol 1: 387–405 (1987).
- Jefferson RA, Burgess SM, Hirsh D: β-glucuronidase from Escherichia coli as a gene fusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).
- Kavanagh TA, Jefferson RA, Bevan MW: Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein. Mol Gen Genet 215: 38–45 (1988). CrossRef
- Kunkel TA, Roberts JD, Zakour RA: Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch repair system of E. coli. Meth Enzymol 153: 367–382 (1987).
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA (1989).
- Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
- Schmitz UK, Lonsdale DM: A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell 1: 783–791 (1989). CrossRef
- Vieira J, Messing J: Production of single-stranded plasmid DNA. Meth Enzymol 153: 3–11 (1987).
- Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies
Plant Molecular Biology
Volume 15, Issue 6 , pp 821-825
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers
- Additional Links
- protein targeting
- reporter gene
- site-directed mutagenesis
- Industry Sectors