Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei |
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Authors: | G Veldhuisen M Saloheimo M A Fiers P J Punt R Contreras M Penttil? and C A M J J van den Hondel |
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Institution: | (1) Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Fax: +31-30-6944466; e-mail: P.Punt@voeding.tno.nl, NL;(2) VTT Biotechnology and Food Research, Biologinkuja 1, Espoo, FIN-02044 VTT, Finland, FI;(3) Laboratorium Moleculaire Biologie, Universiteit Gent, B-9000 Gent, Belgium, BE |
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Abstract: | The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway
in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype
in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.
Received: 21 January 1997 / Accepted: 21 June 1997 |
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Keywords: | Gene cloning Protein secretion Filamentous fungi Small GTP binding protein Complementation |
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