Deletion and allelic exchange of the Aspergillus fumigatus veA locus via a novel recyclable marker module

被引:101
作者
Krappmann, S [1 ]
Bayram, Ö [1 ]
Braus, GH [1 ]
机构
[1] Univ Gottingen, Dept Microbiol & Mol Genet, Inst Microbiol & Genet, D-37077 Gottingen, Germany
关键词
D O I
10.1128/EC.4.7.1298-1307.2005
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter) selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.
引用
收藏
页码:1298 / 1307
页数:10
相关论文
共 62 条
[1]   Mapping of the nitrate assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus [J].
Amaar, YG ;
Moore, MM .
CURRENT GENETICS, 1998, 33 (03) :206-215
[2]   PipeOnline 2.0: automated EST processing and functional data sorting [J].
Ayoubi, P ;
Jin, XJ ;
Leite, S ;
Liu, XH ;
Martajaja, J ;
Abduraham, A ;
Wan, QL ;
Yan, W ;
Misawa, E ;
Prade, RA .
NUCLEIC ACIDS RESEARCH, 2002, 30 (21) :4761-4769
[3]  
Baganz F, 1997, YEAST, V13, P1563, DOI 10.1002/(SICI)1097-0061(199712)13:16<1563::AID-YEA240>3.0.CO
[4]  
2-6
[5]   Lessons from the genome sequence of Neurospora crassa:: Tracing the path from genomic blueprint to multicellular organism [J].
Borkovich, KA ;
Alex, LA ;
Yarden, O ;
Freitag, M ;
Turner, GE ;
Read, ND ;
Seiler, S ;
Bell-Pedersen, D ;
Paietta, J ;
Plesofsky, N ;
Plamann, M ;
Goodrich-Tanrikulu, M ;
Schulte, U ;
Mannhaupt, G ;
Nargang, FE ;
Radford, A ;
Selitrennikoff, C ;
Galagan, JE ;
Dunlap, JC ;
Loros, JJ ;
Catcheside, D ;
Inoue, H ;
Aramayo, R ;
Polymenis, M ;
Selker, EU ;
Sachs, MS ;
Marzluf, GA ;
Paulsen, I ;
Davis, R ;
Ebbole, DJ ;
Zelter, A ;
Kalkman, ER ;
O'Rourke, R ;
Bowring, F ;
Yeadon, J ;
Ishii, C ;
Suzuki, K ;
Sakai, W ;
Pratt, R .
MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 2004, 68 (01) :1-+
[6]   Menacing mold:: The molecular biology of Aspergillus fumigatus [J].
Brakhage, AA ;
Langfelder, K .
ANNUAL REVIEW OF MICROBIOLOGY, 2002, 56 :433-455
[7]   Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus [J].
Brand, A ;
MacCallum, DM ;
Brown, AJP ;
Gow, NAR ;
Odds, FC .
EUKARYOTIC CELL, 2004, 3 (04) :900-909
[8]  
Braus GH, 2002, MYCOLOGY S, V15, P215
[9]   Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity [J].
Brown, JS ;
Aufauvre-Brown, A ;
Brown, J ;
Jennings, JM ;
Arst, H ;
Holden, DW .
MOLECULAR MICROBIOLOGY, 2000, 36 (06) :1371-1380
[10]  
Brown T., 1997, CURRENT PROTOCOLS MO