Figure 9.2 (a) Map of plasmid used to obtain HygR transformants of Ascobolus immersus. Black thick lines correspond to hph genes, dotted lines are regions containing regulatory elements gpd promoter and trpC terminator from Aspergillus nidulans and restriction sites for Xbal (Xb), BamHl (B), EcoRI (E) and Seal (Sc), ampicilllin resistance (amp). (b) Southern hybridization analysis of transformants using a hph probe corresponding to a Sca1-Sca1 fragment. Transformants (lane 1 and 4) have integrated one copy of the transgene. Transformants (lane 2) and (lane 3) have integrated three and four copy of the transgene, respectively. Based on Rhounim et al. (1994).

the selectable marker gene, hygromycin phosphotransferase gene (hph) from Escherichia coli and the control regions (promoter and terminator). This vector was used to transform Ascobolus immersus (Rhounim et al., 1994). On average, the transformation frequency is 25 per jag of plasmid DNA. The recipient may be transformed by one or more ectopic integrations of the plasmid DNA at random sites in the genome (ectopic integration). The mode of integration is determined by Southern blot analysis of transformants (Figure 9.2b). Transformants, which have integrated only one copy of the transgene, will exhibit only one Xbal band (lane 1 and lane 4). Multiple integrations are seen in transformants in lanes 2 and 3, which have three and four integrations, respectively. Note that the Xbal site is located in the 3 flanking sequence of the hph gene. The number of Xbal fragments will correspond to the number of transgenic copies if the multiple integrations are dispersed in the genome. This will also be seen if the multiple copies integrate in tandem at a single site.

Transformation is now routinely done in several fungi (Fincham, 1989; Hynes, 1996). Chapter 8 illustrated the use of transformation in understanding the mating process in the pathogenic fungus Ustilago maydis; here, the bias is heavily toward Neurospora crassa.

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