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  • 1
    ISSN: 1432-0983
    Keywords: Key wordsAspergillus niger ; Heterologous expression ; Protein disulphide isomerase ; Protein folding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Current strategies to improve the secretion of heterologous proteins from Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). Here we report the isolation of a gene, pdiA, encoding a putative protein disulphide isomerase (PDI) from A. niger using the Saccharomyces cerevisiae PDI gene as a probe. Sequencing of a genomic clone and RT-PCR products predict a 515-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (Mr = 54.3 kDa). The predicted protein also contains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal. Three introns were identified within the pdiA gene and Southern- and dot-blot analysis indicates that the gene is present in a single copy. Northern-blot analysis shows a transcript of the predicted size. Sequence homology to a motif associated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter. Transcription of pdiA is induced 3–4-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. The kinetics of induction suggest that pdiA expression is not part of the primary stress response.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Key wordsTrichoderma reesei ; Autonomously replicating sequence ; Transformation ; Matrix attachment region ; Nuclear matrix
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed.
    Type of Medium: Electronic Resource
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