Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa.
The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and_expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.
Kapoor
M
M
Department of Biological Sciences, University of Calgary, AB, Canada. mkapoor@ucalgary.ca
Curle
C A
CA
Kalia
S
S
Achari
Y
Y
D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D018274:Electroporation; D004794:Enzyme Repression / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D049452:Green Fluorescent Proteins; D005969:Glutamate Dehydrogenase