Manipulation of beta-glucuronidase for use as a reporter in vacuolar targeting studies.
It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, beta-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of beta-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.
Farrell
L B
LB
Washington University, Department of Biology, St. Louis, MO 63130.
Beachy
R N
RN
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016368:Frameshift Mutation; D005821:Genetic Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic; D014617:Vacuoles
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase