Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica.
Endothiapepsin is an aspartic protease secreted by Cryphonectria parasitica. It has a milk-clotting activity and is used in the cheese industry. The eapA gene encoding endothiapepsin has been cloned and sequenced. An open reading frame of 419 codons, which encodes a precursor differing from mature endothiapepsin by the presence of an 89 aa residue prepro-sequence, was found. The eapA gene is interrupted by three introns. C. parasitica mutant strains deficient in the production of endothiapepsin (eapA-) were constructed using a gene-replacement strategy. Two nonsense mutations were introduced at the beginning of the coding sequence by PCR-induced mutagenesis. The mutated DNA fragment was introduced in C. parasitica by co-transformation with a benomyl-resistant (benR) selection plasmid. Transformants which have the eapA- phenotype were obtained. Protein analysis confirmed that they secreted no detectable amount of endothiapepsin. No ectopic integration of the mutated eapA gene occurred in the eapA- transformants. Moreover, after one conidiation step, eapA- transformants yielded benomyl-sensitive (benS) segregants which were analyzed by Southern blotting experiments. The results revealed no difference with the wild-type strain, suggesting that the eapA-, benS segregants differed from the non-transformed strain only by the presence of the two nonsense mutations in the eapA locus.
Razanamparany
V
V
Unité de Microbiologie, Sanofi Elf Biorecherches, Labège, France.
Jara
P
P
Legoux
R
R
Delmas
P
P
Msayeh
F
F
Kaghad
M
M
Loison
G
G
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000235:genetics*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D014170:Transformation, Genetic / Q000235:genetics
D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D016282:Aspartic Acid Endopeptidases; C042466:Endothia aspartic proteinase