Direct and Indirect Gene Replacements in Aspergillus nidulans

Abstract

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. (i) A linear DNA fragment containing a heteromorphic trpC⁺ allele was used to transform a trpC⁻ strain to trpC⁺. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC⁻ allele in a minority of the strains. (ii) An A. nidulans trpC⁺ gene was inserted into the argB⁺ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC⁻ argB⁺ strain to trpC⁺. Approximately 30% of the transformants were simultaneously argB⁻. The null argB allele had replaced the wild-type allele in a majority of these strains. (iii) The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC⁻ strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC⁻ progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.