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  • 1
    Electronic Resource
    Electronic Resource
    Development of Aspergillus oryzae thiA promoter as a tool for molecular biological studies (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 244 (2005), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In filamentous fungi, the repertoire of promoters available for exogenous gene expression is limited. Here, we report the development and application of the thiamine-regulatable thiA promoter (PthiA) in Aspergillus oryzae as a tool for molecular biological studies. When PthiA was used to express the enhanced green fluorescent protein (EGFP) reporter, the fluorescence in the mycelia was either repressed or induced in the presence or absence of thiamine in the culture media, respectively. In addition, the expression level from the thiA promoter can be controlled by the concentration of external thiamine. Thiamine content in the media did not affect mycelial morphology, making the thiA promoter more useful compared with alcA and amyB promoters that depend on carbon source for regulation. Moreover, as the A. oryzae thiA promoter was also regulated by thiamine in A. nidulans, this promoter can be further applied as an inducible promoter in other Aspergilli.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsle.2005.01.014
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  • 2
    Electronic Resource
    Electronic Resource
    Molecular cloning and functional characterization of avaB, a gene encoding Vam6p/Vps39p-like protein in Aspergillus nidulans (2004)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 232 (2004), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: It has been demonstrated that Saccharomyces cerevisiae Vam6p/Vps39p plays a critical role in the tethering steps of vacuolar membrane fusion by facilitating guanine nucleotide exchange on small guanosine triphosphatase (GTPase) Vam4p/Ypt7p. We report here the identification and characterization of a novel protein in Aspergillus nidulans, AvaB, that exhibits similarity to Vam6p/Vps39p and plays a critical role in vacuolar morphogenesis in A. nidulans. AvaB is comprised of 1058 amino acids with amino-terminal citron homology (CNH) and central clathrin homology (CLH) domains, as observed for other Vam6p/Vps39p family proteins. Disruption of avaB in A. nidulans resulted in the fragmentation of vacuoles and reduced growth rate under various growth conditions, implying its importance in maintaining vacuolar morphology and function. Yeast two-hybrid analysis demonstrated the interaction of AvaB with AvaA, a Vam4p/Ypt7p homolog in A. nidulans, as well as the homooligomer formation of AvaB, suggesting that AvaB performs its function through hetero- or homophilic protein–protein interactions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/S0378-1097(04)00039-4
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  • 3
    Electronic Resource
    Electronic Resource
    Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae (2004)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 239 (2004), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD−sC−). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD−sC−adeA−) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD−sC−ΔargB adeA− or niaD−sC−ΔargB adeB−) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD−sC−ΔargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsle.2004.08.025
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  • 4
    Electronic Resource
    Electronic Resource
    Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae (2001)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 204 (2001), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Δcmp1Δcmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2001.tb10881.x
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  • 5
    Electronic Resource
    Electronic Resource
    Cloning and characterization of vmaA, the gene encoding a 69-kDa catalytic subunit of the vacuolar H+-ATPase during alkaline pH mediated growth of Aspergillus oryzae (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 209 (2002), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar H+-ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (Δvma1) was viable at alkaline pH 8.0 and in the presence of CaCl2 (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11144.x
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  • 6
    Electronic Resource
    Electronic Resource
    τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 60 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser144 and at Ser315. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser144 and nearly one-half of them at Ser315. Phosphorylation at Ser144 and at Thr147 of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb03173.x
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