Folylpolyglutamate synthesis in Neurospora crassa: Primary structure of the folylpolyglutamate synthetase gene and elucidation of the met-6 mutation

In Neurospora crassa, the met-6(+) gene encodes folylpoly-gamma-glutamate s ynthetase (FPGS) which catalyzes the formation of polyglutamate forms of fo late. Methionine auxotrophy of the Neurospora crassa met-6 mutant is relate d to a lesion affecting this enzyme. Functional complementation of the muta nt strain was achieved by introducing copies of the wild-type met-6(+) gene into mutant spheroplasts. The complementing sequences were found to be con tained on a 3.5 kb EcoRI-BarnHI restriction fragment. The nucleotide sequen ce of the met-6(+) gene was determined and an open reading frame of 1587 bp was identified, interrupted by two introns. This open reading frame contai ned several AUG codons but translation beginning from either of the first t wo would theoretically produce a protein of appropriate size and with simil arity to five other FPGS proteins. Northern blot analyses of met-6(+) trans cripts revealed a 2.0 kb product. The position of the transcription stop si te and an intron were identified by sequencing partial cDNA clones which we re truncated at the 5' end. DNA sequence analysis of the met-6 mutant allel e revealed a T to C transition which would result in replacement of a highl y conserved serine with a proline. (C) 1998 Elsevier Science Ltd. All right s reserved.