RESUMO
We have cloned and determined the nucleotide sequence of a genomic DNA segment from Aspergillus oryzae which contains pepO, the gene encoding the aspartic proteinase, aspergillopepsin O (PEPO). The organization of pepO is strikingly similar to that of pepA from A. niger var. awamori (previously called A. awamori) in that both are composed of four exons and three introns with virtually identical lengths, and the positions of the introns are exactly conserved. From the deduced amino acid (aa) sequence, it appears that PEPO, like other fungal aspartic proteinases, is synthesized as a zymogen containing a putative N-terminal prepro-region of 77 aa followed by a mature protein of 327 aa. Southern blotting experiments suggest that a single copy of pepO exists in the A. oryzae genome.
Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus oryzae/genética , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Aspergillus oryzae/enzimologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Fúngico , Genes Fúngicos , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycin-resistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5' untranslated sequences with those from other fungi.
Assuntos
Aspergillus niger/genética , Genes Fúngicos , Marcadores Genéticos , Oligomicinas , ATPases Translocadoras de Prótons/genética , Transformação Genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Resistência Microbiana a Medicamentos/genética , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.