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Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori
R M Berka1, M Ward, L J Wilson
1Genencor, Inc., South San Francisco, CA 94080.
Abstract:
We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.
Insights
Researchers cloned the pepA gene from Aspergillus awamori, revealing its exon-intron structure and identifying a signal peptide and propeptide. Gene replacement successfully created PEPA-deficient mutants, confirming the gene
Area of Science:
- Molecular Biology
- Enzymology
- Mycology
Background:
- Aspergillus awamori produces aspartic proteinase aspergillopepsin A (PEPA).
- Understanding the pepA gene structure and its regulation is crucial for protein production.
- Aspartic proteinases play significant roles in various biological processes.
Purpose of the Study:
- To clone and characterize the genomic pepA gene from Aspergillus awamori.
- To investigate the structure of the pepA gene, including exons, introns, and regulatory regions.
- To develop a method for generating PEPA-deficient mutants for further study.
Main Methods:
- Cloning of the pepA gene using synthetic oligodeoxyribonucleotide probes.
- Nucleotide sequencing to determine gene structure (exons, introns).
- Northern blot analysis to study mRNA transcription.
- Gene replacement strategy using a plasmid with a deleted coding region and an argB marker.
- Immunoassay and Southern hybridization for mutant screening and confirmation.
Main Results:
- The pepA gene consists of four exons and three introns.
- A 69-amino acid sequence, likely a signal peptide and propeptide, precedes the mature PEPA.
- A single 1.4-kb mRNA transcript for pepA was detected.
- Gene replacement successfully generated PEPA-deficient mutants in 16-40% of transformants.
Conclusions:
- The genomic structure of the Aspergillus awamori pepA gene has been elucidated.
- A functional gene replacement system was established for creating PEPA-deficient mutants.
- These findings provide a foundation for further research into PEPA function and its genetic manipulation.