Deletion of Aspergillus nidulans aroC using a novel blaster module that combines ET cloning and marker rescue

Abstract

Blaster cassettes are of significant value in functional genomics, as they represent tools with which to inactivate duplicated or homologous genes in an individual organism. We have constructed a novel blaster module which allows repeated gene deletion in the filamentous fungus Aspergillus nidulans. Because bacterial resistance marker cassettes are employed as flanking repeats in direct orientation, the blaster cassette is suited for recombinogenic engineering by ET cloning in Escherichia coli. The functionality of the blaster module was demonstrated by deleting the chorismate mutaseencoding gene aroC of A. nidulans, followed by marker rescue based on mitotic recombination. The resulting aroCD strains are auxotrophic for phenylalanine but not tyrosine, and display a limited capacity for fruit body formation and ascosporogenesis, which depends on the phenylalanine/tyrosine supply. The data support the notion that amino acid status has a strong impact on cleistothecium development in A. nidulans.

Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality

“…However, the osmotrophic lifestyle of Aspergillus , i.e., extracellular digestion of complex substrates achieved by a plethora of secreted hydrolytic enzymes (Kobayashi et al, 2007; Monod et al, 2009) and followed by uptake of breakdown products via numerous transporters, impedes a comprehensive analysis (Yoon et al, 2009). To assist in assessing any relevance of cellular functions that are encoded by multiple genes, progress was made with respect to gene targeting approaches that allows repetitive targeting of all members of a gene family to abolish any redundantly encoded cellular function (d’Enfert, 1996; Krappmann and Braus, 2003; Krappmann et al, 2005; Nielsen et al, 2006; Forment et al, 2006; Hartmann et al, 2010). By developing genetic markers, recombinase-driven excision of which can be achieved in fungo , marker rescue became feasible to allow recurring rounds of gene targeting in a straight-forward manner.…”

Γονίδια Που Κωδικοποιούν Παράγοντες Που Συμμετέχουν Στην Ενδοκυτταρική Κυκλοφορία Και Τοποθέτηση Στη Μεμβράνη Μεταφορέων Όξινων Αμινοξέων

“…Έχει διαπιστωθεί ότι ορισµένες µεταλλαγές που προσδίδουν αυξοτροφία για κάποια ουσία (για παράδειγµα µεταλλαγές στα γονίδια trpC, hisB, yB ή argB) οδηγούν στο σχηµατισµό µικρών κλειστοθηκίων, που στερούνται ασκοσπορίων (Serlupi-Crescenzi et al, 1983;Timberlake, 1990;Eckert et al, 1999;Bruggeman et al, 2003). Η ενδοκυτταρική συγκέντρωση των αµινοξέων έχει καθοριστικό ρόλο στη ανάπτυξη των κλειστοθηκίων (Krappman and Braus, 2003 (Sanger et al, 1977 Στο πρωτόκολλο αυτό χρησιµοποιούνται τυχαία εξαµερή ως εκκινητές (random priming, Ausubel et al, 1995). Tα ραδιοσηµασµένα µόρια DNA καθαρίζονται από την αντίδραση µε κολώνα από σφαιρίδια Sephadex G-50 σε 1Χ ΤΕ (10 mM Tris-HCl,1 mM EDTA) και πριν τον εκάστοτε υβριδισµό αποδιατάσσονται στους 100 ο C για 5 λεπτά.…”

CsnA Dependent Development and Regulation of Amino Acid Biosynthesis of the Filamentous Ascomycete <i>Aspergillus nidulans</i>

“…Counterselecting against the pyrG m a r k e r g e n e t h e m a r k e r w a s r e s c u e d d u e t o m i t o t i c recombination (Krappmann and Braus, 2003). Microscopy was performed by differential interface contrast (DIC).…”

“…(5-FOA) to recycle the pyrG-marker of the deletion cassette (Krappmann and Braus, 2003) for further experiments resulting in strain AGB234 (pyrG -). A. nidulans strain AGB234 shows a similar phenotype to AGB223 on minimal medium containing uridine (Figure 8).…”