NAD(+)-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping

Abstract

The NAD(+)-specific glutamate dehydrogenase (NAD-GDH) of the filamentous fungus Neurospora crassa is a tetrameric enzyme, regulated by catabolite repression. The amino acid sequence of this enzyme had been published several years ago. With the object of investigating the molecular mechanism of catabolite repression, the nucleotide sequence of genomic clones containing the coding region, along with 5'- and 3'-flanking noncoding segments of the NAD-GDH transcription unit, was obtained. The gdh structural gene was shown to code for a polypeptide of 1047 residues, with a calculated molecular mass of 118,280 daltons. The coding sequence is interrupted by two short introns located close to the N- and C-terminal domains of the polypeptide. Consensus intron boundaries and internal splice sequences resemble closely those of other N. crassa genes. A comparison of the amino acid sequence deduced from the nucleotide sequence with the previously published sequence showed several discrepancies between the two. Nucleotide sequence corresponding to a gap in the amino acid sequence was located in the genomic clone. Genetic mapping by restriction fragment length polymorphism analysis localized the gdh gene close to the loci trp-1 and con-7 on the right arm of linkage group III.