Transformation by integration in Aspergillus nidulans

Tilburn, Joan; Scazzocchio, Claudio; Taylor, Graham G.; Zabicky-Zissman, J H; Lockington, Robin; Davies, R. Wayne

doi:10.1016/0378-1119(83)90191-9

Cited by 646 publications

(347 citation statements)

References 21 publications

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“…Transformation of A. fumigatus D141 was performed according to a protocol that has been used for Aspergillus nidulans and A. fumigatus (Tilburn et al, 1983;Paris, 1994) with 10 7 protoplasts and 5 mg of plasmid. After overnight expression of the hygromycin B phosphotransferase gene (HPH), the transformants were incubated on agar based on minimal medium (Cove, 1966) containing 200 mg hygromycin ml 21 (Sigma), and selected after 5 days incubation at 20 uC followed by an overnight incubation at 42 uC.…”

Section: Methodsmentioning

confidence: 99%

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

Léchenne

Reichard²,

Zaugg

et al. 2007

Microbiology

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Dermatophytes and other filamentous fungi excrete sulphite as a reducing agent during keratin degradation. In the presence of sulphite, cystine in keratin is directly cleaved to cysteine and Ssulphocysteine, and thereby, reduced proteins become accessible to hydrolysis by a variety of secreted endo-and exoproteases. A gene encoding a sulphite transporter in Aspergillus fumigatus (AfuSSU1), and orthologues in the dermatophytes Trichophyton rubrum and Arthroderma benhamiae (TruSSU1 and AbeSSU1, respectively), were identified by functional expression in Saccharomyces cerevisiae. Like the S. cerevisiae sulphite efflux pump Ssu1p, AfuSsu1p, TruSsu1p and AbeSsu1p belong to the tellurite-resistance/dicarboxylate transporter (TDT) family which includes the Escherichia coli tellurite transporter TehAp and the Schizosaccharomyces pombe malate transporter Mae1p. Seven genes in the A. fumigatus genome encode transporters of the TDT family. However, gene disruption of AfuSSU1 and of the two more closely related paralogues revealed that only AfuSSU1 encodes a sulphite efflux pump. TruSsulp and AbeSsulp are believed to be the first members of the TDT family identified in dermatophytes. The relatively high expression of TruSSU1 and AbeSSU1 in dermatophytes compared to that of AfuSSU1 in A. fumigatus likely reflects a property of dermatophytes which renders these fungi pathogenic. Sulphite transporters could be a new target for antifungal drugs in dermatology, since proteolytic digestion of hard keratin would not be possible without prior reduction of disulphide bridges.

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Section: Methodsmentioning

confidence: 99%

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

Léchenne

Reichard²,

Zaugg

et al. 2007

Microbiology

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“…Transformation protocols basically followed the procedures published by Tilburn and colleagues (42). Transformants were selected according to the selection marker used (Tables 1 and 2).…”

Section: Vol 74 2008mentioning

confidence: 99%

N-Glycan Modification in Aspergillus Species

Kainz

Gallmetzer

Hatzl

et al. 2008

Appl Environ Microbiol

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The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional ␣-1,2-mannosidase producing increased amounts of N-glycans of the Man 5 GlcNAc 2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan 5 GlcNac 2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man 3 GlcNAc 2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

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“…This strain carries a deletion removing Ϸ0.5 kb of the 5Ј region of prnD, 1 kb of the 5Ј end of prnB and all of the cis-acting control region (R. Gonzalez and C. Scazzocchio, unpublished) and therefore was unable to use proline as the carbon or nitrogen source. Fungal transformation was performed using the method of Tilburn et al (1983). Transformants were selected on media containing L-proline as the sole nitrogen source and analysed by Southern blot (Sambrook et al, 1989) and DNA-sequencing techniques (Sanger et al, 1977).…”

Section: Rna Extraction and Northern Blot Analysismentioning

confidence: 99%

The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation

Tazebay

Sophianopoulou

Scazzocchio

et al. 1997

Molecular Microbiology

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SummaryIn Aspergillus nidulans a highly specific L-proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism. In mycelia, the expression of the prn structural genes is finely co-regulated in response to proline induction and nitrogen/carbon catabolite repression. In this study we establish that prnB expression is also activated during germination of conidiospores. This activation persists until the development of 6 h-old mycelia and it is independent of proline induction mediated by the pathway-specific prnA gene product. We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation. This process has two components: a prnA-dependent and a prnA-independent component. A cis-acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation. We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium. This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.

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Transformation by integration in Aspergillus nidulans

Cited by 646 publications

References 21 publications

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

N-Glycan Modification in Aspergillus Species

The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation

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