Transformation ofAspergillussp. andTrichoderma reeseiUsing the Pyrithiamine Resistance Gene (ptrA) ofAspergillus oryzae

Kubodera, Takafumi; Yamashita, Nobuo; Nishimura, Akira

doi:10.1271/bbb.66.404

Cited by 111 publications

(82 citation statements)

References 8 publications

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“…We generated both an episomally replicating plasmid and an integrative vector for delivery of reporter genes. The episomal vector used was derived from pPTRII (25) with the AMA1 origin of replication and was modified to contain a hygromycin resistance cassette for selection in A. fumigatus (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…DNA manipulations were done according to the method of Sambrook et al (48) or according to reagent manufacturer instructions. The autonomous plasmid containing the AMA1 origin of replication to generate promoter-reporter gene fusions for propagation in A. fumigatus was derived from pPRTII (25). To generate a gpdA2-luciferase fusion construct in pPTRII, the following fragments were PCR amplified: CEN6-ARSH and ScURA3 genes from pRS316 (49), the gpdA2 promoter from A. fumigatus strain Af293, either Renilla reniformis luciferase from pH12.7 (55) or firefly luciferase from pLG3 (Promega), and the gpdA2 transcription terminator region from the Af293 genome.…”

Section: Methodsmentioning

confidence: 99%

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Analysis of Promoter Function in Aspergillus fumigatus

2012

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ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Analysis of Promoter Function in Aspergillus fumigatus

2012

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“…pPTRII (Supplementary Table S2), carrying the AMA1-based elements together with the pyrithiamin-resistance gene ptrA from Aspergillus oryzae (Gems et al, 1991;Kubodera et al, 2000Kubodera et al, , 2002, was modified by introducing complementary annealed oligonucleotides (Nhe_1 and Nhe_2) into the KpnI-HindIII restriction sites. This construction generated a single NheI site in plasmid pPTRII-NheI.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

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RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific 32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the ΔPcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

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“…The hosts used in this study were A. oryzae NSAR1, a quadruple auxotrophic mutant (niaD À , sC À , ÁargB, adeA À ) 22) for fungal expression, and S. cerevisiae BY4743 pep4Á prb1Á trp1Á (MATa/MAT, his3Á1/his3Á1, leu2Á0/leu2Á0, lys2Á0/þ, met15Á0/þ, ura3Á0/ura3Á0, pep4Á/pep4Á, prb1Á/ prb1Á, trp1Á/trp1Á) was produced by disrupting the PEP4, PRB1, and TRP1 genes in the BY4743 parental strain, 23,24) according to the protocol provided by Güldener et al 25) for yeast expression. The vectors used in this study were pTAex3, 5,6) pUSA, 26) pAdeA 27) and pPTRI 28) for fungal expression, and pLTex321sMHTRP and pLTex321sV5H 29) for yeast expression. The pLTex321sMHTRP vector was constructed by respectively replacing the V5-epitope tag and URA3 marker in pLTex321sV5H with a c-Myc-epitope tag and TRP1 marker.…”

Section: Methodsmentioning

confidence: 99%