To confirm the gene deletion, transformants were screened by colony PCR. One of the

transformants yielded a product of about 1400 bp with the primer pair 1–1′, a product of about 1300 bp with the primer pair 2–2′ and a product of about 1700 bp with the primer pair 3–3′. PCR products of these sizes are expected in the case of a gene deletion of ku80 (see Materials and methods and Fig. 1). The Δku80 monokaryon was phenotypically indistinguishable from the wild type. The Δku80Δku80 dikaryon formed normal fruiting bodies that produced similar numbers of spores with a similar viability when compared Nintedanib supplier with the wild type. Moreover, like in the wild type, 109 protoplasts could be obtained from 5 g wet weight mycelium (data not shown). To assess whether the HR pathway was upregulated in the Δku80 mutant, qPCR was performed. Rad52 expression (which represents a gene involved in HR) was similar in the Δku80 strain (Ct=29.24±0.33) when compared with the wild-type strain (Ct=28.90±0.16). Apparently, inactivation of the NHEJ pathway does not induce an upregulation of the HR pathway. The Δku80 strain was transformed with the knockout constructs pDelcas-sc15, pDelcas-jmjC and pDelcas-priB. The deletion constructs had been linearized with the restriction enzyme SspI (pDelcas-JmjC and pDelcas-priB) or PacI (pDelcas-SC15) before they were introduced into the Δku80 H4-8 strain. Four out of seven

transformants had a deletion

of SB203580 datasheet sc15, while one out of one and two out of two transformants had a deletion of jmj3 and pri2, respectively (Table 2). Typically, 100 transformants are obtained with protoplasts of the wild-type strain and these transformants would contain one, if any, knockout strain (see the efficiency of the inactivation of ku80). The number of transformants obtained with the Δku80 strain is 100-fold lower (Table 2). However, most of these transformants have a gene deletion. Transformation of a Δku80 strain in which a wild-type ku80 gene had been reintroduced by crossing had a transformation frequency similar to that observed for the wild type. This confirms that the low transformation frequency was due to the deletion of the ku80 gene. The deletion of ku70 in Aspergillus oryzae (Takahashi et al., 2006) DNA Damage inhibitor and Sordaria macrospora (Pöggeler & Kück, 2006) also led to a reduction in the transformation frequency. In these cases, a seven- and 40-fold reduction in the number of transformants was obtained. Also in these cases, it may well be that the HR machinery is not upregulated when the NHEJ machinery is inactivated. The phenotype of the Δsc15 strain has been described (Lugones et al., 2004). Before determining the phenotypes of the Δjmj3 and Δpri2 strains, the wild-type ku80 gene was reintroduced by crossing. Monokaryotic and homozygous dikaryotic Δpri2 strains showed no phenotypic differences when compared with the wild type.