NAD-specific glutamate dehydrogenase of Neurospora crassa. cDNA cloning and gene expression during derepression

The catabolic NAD-specific glutamate dehydrogenase of Neurospora crassa is one of the many enzymes regulated by carbon catabolite repression. To achieve an understanding of its regulation, cDNA and genomic clones were isolated. Total poly(A+) RNA from derepressed cells was used for the construction of a cDNA library in the expression vector, lambda gt11. By screening this library with a polyclonal antiserum against NAD-specific glutamate dehydrogenase, a positive clone with a 0.9-kilobase insert was isolated and the insert DNA sequenced. The insert was shown to code for approximately one-third of the known amino acid sequence, close to the carboxyl terminus. Using this truncated cDNA as a probe, the structural gene was shown to be transcriptionally activated approximately 60-fold during derepression, producing an approximately 4.7-kilobase mRNA transcript. N. crassa genomic clones, hybridizing to this cDNA probe, were isolated and the structural gene (on two BamHI fragments) was subcloned in pUC13.