Todd, R.B., Davis, M.A. & Hynes, M.J. Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction. Nat. Protocols 2, 811-821

Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia.
Nature Protocol (Impact Factor: 9.67). 02/2007; 2(4):811-21. DOI: 10.1038/nprot.2007.112
Source: PubMed


The multicellular microbial eukaryote Aspergillus nidulans is an excellent model for the study of a wide array of biological processes. Studies in this system contribute significantly to understanding fundamental biological principles and are relevant for biotechnology and industrial applications, as well as human, animal and plant fungal pathogenesis. A. nidulans is easily manipulated using classical and molecular genetics. Here, we describe the storage and handling of A. nidulans and procedures for genetic crossing, progeny analysis and growth testing. These procedures are used for Mendelian analysis of segregation of alleles to show whether a mutant phenotype segregates as a single gene and independent assortment of genes to determine the linkage relationship between genes. Meiotic crossing is used for construction of multiple mutant strains for genetic analysis. Genetic crossing and analysis of progeny can be undertaken in 2-3 weeks and growth testing takes 2-3 days.

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    • "These features permit dispersal and survival under adverse environmental conditions (Gregory 1966). The asexual cycle in A. nidulans is normally followed by the production of sexual reproductive structures or cleistothecia, which contain meiospores called ascospores (Pontecorvo et al. 1953; Todd et al. 2007). The implementation of fluorescence microscopy 20 years ago for the study of filamentous fungi has significantly improved our knowledge of the morphogenetic processes described above. "
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    ABSTRACT: Filamentous fungi comprise a large group of agriculturally, industrially, and clinically important eukaryotic organisms. They are used as factories in the production of useful enzymes, secondary metabolites, pigments, vitamins, and antibiotics. Other species are pathogens and can, therefore, cause dire diseases in humans, animals, and plants. Both their useful and detrimental manifestations are the consequence of characteristic growth and developmental patterns as well as their ability to rapidly and efficiently adapt to changing environments. The implementation of fluorescence microscopy to study the subcellular localization of filamentous fungal proteins and organelles in the beginning of the 1990s made an important contribution to our knowledge of mechanisms that control growth, reproduction, stress response, cell cycle, secretory pathways, and cargo transport. Standard procedures are currently available for labeling fungal factors with fluorescent proteins. Furthermore, a new battery of advanced fluorescence microscopy techniques is being adapted for a deeper and more accurate study of protein localization, interaction, and dynamics in many filamentous fungal species. This chapter provides an overview on these novel fluorescence microscopy methods for filamentous fungi, focusing mainly on the model ascomycete Aspergillus nidulans.
    Advanced Microscopy in Mycology, 1st edited by Tanya E.S. Dahms, C.J. Czymmek, 11/2015: chapter Fluorescence-based Methods for the Study of Protein Localization, Interaction, and Dynamics in Filamentous Fungi: pages 27-46; Springer., ISBN: 978-3-319-22436-7; 978-3-319-22437-4
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    • "Moreover, Aspergillus nidulans provides a genetically tractable model system, in particular to study telomere length in a number of different cell types such as asexual conidia and sexual ascospores. A. nidulans has ascospores that are uniquely housed in a separate structure called the cleistothecium. One cleistothecium holds 10,000 or more ascospores [17], but unfortunately the external surfaces of cleistothecia are covered by conidia and other cell types. These cells are extremely labor intensive to remove, and thus pure ascospore DNA can be obtained only in small quantities. "
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    ABSTRACT: Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed "telomere-anchored PCR," measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.
    PLoS ONE 06/2014; 9(6):e99491. DOI:10.1371/journal.pone.0099491 · 3.23 Impact Factor
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    • "Aspergillus nitrogen-free minimal medium (ANM), appropriately supplemented for auxotrophies, contained 1 % (w/v) glucose as the carbon source and nitrogen sources at a final concentration of 10 mM. Genetic analysis was performed as described by Clutterbuck (1974) and Todd et al. (2007). "
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    ABSTRACT: NADP-dependent glutamate dehydrogenase (NADP-GDH) is a key enzyme in the assimilation of alternative nitrogen nutrient sources through ammonium in fungi. In Aspergillus nidulans NADP-GDH is encoded by gdhA. Several transcription factors are known to regulate gdhA expression, including AreA, the major transcription activator of nitrogen metabolic genes, and TamA, a co-activator of AreA. TamA also interacts with LeuB, the regulator of leucine biosynthesis. We have investigated the effects of leucine biosynthesis on gdhA regulation and found that leucine regulates the levels of NADP-GDH activity and gdhA expression. We show, using mutants with perturbed levels of α-isopropylmalate (α-IPM), that this leucine biosynthesis intermediate affects gdhA regulation. Leucine regulation of gdhA requires a functional LeuB with an intact Zn(II)2Cys6 DNA binding domain. By analyzing prevalence of putative LeuB DNA binding sites in promoters of gdhA orthologs we predict broad conservation of leucine regulation of NADP-GDH expression within ascomycetes except in the Fusaria and fission yeasts. Using promoter mutations in gdhA-lacZ reporter genes we identified two sites of action for LeuB within the A. nidulans gdhA promoter. These two sites lack sequence identity, with one site conforming to the predicted LeuB DNA binding site consensus motif whereas the second site is a novel regulatory sequence element conserved in Aspergillus gdhA promoters. These data suggest that LeuB regulates NADP-GDH expression in response to leucine levels, which may act as an important sensor of nitrogen availability.
    Microbiology 09/2013; 159(Pt_12). DOI:10.1099/mic.0.071514-0 · 2.56 Impact Factor
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