MOLECULAR-CLONING AND SEQUENCING OF THE GENE ENCODING AN EXTRACELLULAR ASPARTIC PROTEINASE FROM ASPERGILLUS-FUMIGATUS

Oligonucleotide primers based on conserved regions of the aspergillope psins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergi llus fumigatus. The segment was used as a probe for isolating and sequ encing the gene from a genomic library of the fungus. Likewise the cDN A was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequ ence of the mature enzyme consisting of 325 aa with a calculated molec ular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of P EP exactly. The nucleotide and the aa sequences of the pre-proenzyme w ere 70% and 71% homologous to the corresponding sequences of the asper gillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the prese nce of a single copy of the pep gene.