An efficient procedure to isolate fungal genes from an indexed cosmid library

Abstract

Filamentous fungi with low transformation frequencies require specific cloning strategies to isolate nuclear genes. Here we describe the construction of an indexed (ordered) cosmid library as a prerequisite for the isolation of any gene from the ascomycete Sordaria macrospora. Cosmid clones of the library are kept individually in microtiter dishes, to ensure equal representation of each clone. A method for the rapid screening of pooled cosmid DNA is presented allowing efficient gene isolation. The S. macrospora α-tubulin gene was isolated using a heterologous Aspergillus nidulans probe to screen colony filters carrying DNA from individual library clones. One positive clone was subsequently sequenced for further DNA analysis. In a second approach, we developed a new and rapid screening procedure to isolate the S. macrospora ura5 gene from pooled cosmid DNA. The identity of the ura5 gene was confirmed by DNA sequencing, and its functional activity was demonstrated by successful complementation of an auxotrophic S. macrospora uracil mutant.