CHARACTERIZATION OF THE PROMOTER REGION OF THE ENOLASE ENCODING GENE ENOL FROM THE ANAEROBIC FUNGUS NEOCALLIMASTIX FRONTALIS - SEQUENCE ANDPROMOTER ANALYSIS

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional sta rt point. The base composition of the enolase upstream sequence reveal ed a very A + T-rich profile (13.5% G + C) leading to many putative ha irpin structures. The functional organization of the N. frontalis enol ase promoter was investigated by heterologous transient-expression ass ays. DNA fragments obtained by the sequential removal of sequences ups tream of the translation start codon were fused to the Escherichia col i lacZ gene and the resulting plasmids were used to transform the asco mycetes Aspergillus nidulans and Penicillium roqueforti and the oomyce te Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating protoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no beta-galactosidase activity was detected in ascomycete protoplasts. DN A hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the tran sformation plasmid which could be rescued in E. coli. Our results indi cate that the transcriptional machinery of the anaerobic chytrid N. fr ontalis may differ significantly from that of ascomycetes but that eno ugh conservation exists within the lower fungi to allow a transient-dr iven expression of a reporter gene in an oomycete fungus.