NEOCALLIMASTIX FRONTALIS ENOLASE GENE, ENOL - FIRST REPORT OF AN INTRON IN AN ANAEROBIC FUNGUS

A DNA clone containing a putative enolase gene was isolated from a genomic DNA library of the anaerobic fungus Neocallimastix frontalis. It was deduced from sequence comparisons that the enolase gene was interrupted by a large 331 bp intron. The enolase gene, termed enol, has an ORF of 1308 bp and encodes a predicted 436 amino acid protein. The deduced amino acid sequence shows high identity (71.5-71%) to those of enolases from the yeasts Saccharomyces cerevisiae and Candida albicans. The G + C content of the enolase coding sequence (43.8 mol%) is considerably higher than the G + C content of the intervening sequence (14.2 mol%) or the 5' and 3' non-translated flanking sequences (15.2 and 4.7 mol%, respectively). The codon usage of the N. frontalis enolase gene was very biased as has been found for the highly expressed genes of yeast and filamentous fungi. The gene has all the canonical features (polyadenylation signal intron splicing boundaries) of genes isolated from aerobic filamentous fungi. Only one enolase gene could be detected in N. frontalis genomic DNA by Southern analysis with a homologous probe. RNA analysis detected a single enolase transcript of about 1.6 kb. When mycelium was grown on glucose, levels of enolase mRNA were markedly increased by comparison with enolase mRNA levels in mycelium grown on cellulose, suggesting that expression of the N. frontalis enolase gene was transcriptionally regulated by the carbon source.