Minimal promoter for the NAD+-specific glutamate dehydrogenase gene ofNeurospora crassa
| Autor: | C A Curle, S Kalia, Y Achari, M. Kapoor |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Recombinant Fusion Proteins
Blotting Western Green Fluorescent Proteins Molecular Sequence Data Catabolite repression Electrophoretic Mobility Shift Assay Polymerase Chain Reaction Biochemistry Neurospora crassa Transformation Genetic Glutamate Dehydrogenase DNA Fungal Promoter Regions Genetic Molecular Biology Gene Binding Sites Base Sequence biology Chemistry Glutamate dehydrogenase fungi Cell Biology NAD biology.organism_classification Minimal promoter Blotting Southern Luminescent Proteins Electroporation Protein Biosynthesis NAD+ kinase Enzyme Repression Function (biology) Protein Binding |
| Zdroj: | Biochemistry and Cell Biology. 80:177-188 |
| ISSN: | 1208-6002 0829-8211 |
| DOI: | 10.1139/o01-229 |
| Popis: | 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.Key words: Neurospora crassa, repression, induction, GFP fusion, electroporation, microconidia. |
| Databáze: | OpenAIRE |
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