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 tipC'. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpCq allele in a minority of the strains. (ii) An A. nidulans tipC' 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 tipC'.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 SpoCl Cl-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 Cl-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 Cl-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.The original demonstration of DNA-mediated transformation of the unicellular yeast Saccharomyces cerevisiae (13) was followed rapidly by the construction of numerous transformation vectors with different properties and the development of a variety of experimental strategies that now make it possible to manipulate the yeast genome with great precision (reviewed in references 7, 20, and 29). The availability of this technology has resulted in many important discoveries about the molecular and genetic mechanisms regulating the activities of eucaryotic cells.One of the most prominent and important properties of the yeast transformation system is that cloned DNA sequences can be introduced into chromosomes by homologous recombination (13, 22). This property allowed the development of one-step (24) and two-step (25) procedures for precise replacement of chromosomal regions with DNA sequences that have been altered in vitro by using recombinant DNA methods. These techniques make it possible to examine the biochemical and biological conse-

Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

“…In the yeasts S. cerevisiae and S. pombe, gene replacements are often carried out by simple transformation with a linear fragment carrying the replacing allele and a selectable marker. Such onestep gene replacements are possible in A. nidulans, but problems arise in that the replacements are sometimes incomplete and, in other instances, the transforming DNA inserts in other places in the genome creating insertional mutants (Miller et al, 1985). This creates a very high background of irrelevant mutants, which can be detected by Southern hybridizations but only with significant effort.…”

Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation

“…The gene reversion approach used in this study was based on observations that, during meiosis in other filamentous fungi (i.e. A. nidulans, G. pulicaris and Neurospora crassa) homologous recombination between closely linked copies of a duplicated sequence can lead to the loss of one copy of the sequence as well as the sequence between the two copies Miller et al, 1986;Selker et al, 1987). Disruption mutant GZT33 has two copies of a 1.1 kb region of the Tri5 coding region separated from each other by approximately 3 kb of sequence corresponding to a bacterial cloning vector ( Fig.…”

“…The first approach was based on the observation that in filamentous fungi closely linked duplicated sequences can recombine when the fungi pass through the sexual phase of their life cycle and as a result of this recombination one copy of the duplicated sequence can be deleted in meiotic progeny Miller et al, 1986;Selker et al, 1987). We exploited this phenomenon to generate a trichotheceneproducing, meiotic progeny with a wild-type Tvi5 from a disruption mutant that had two closely linked, mutated copies of the TriS coding region.…”

Regulation of the Aspergillus nidulans pectate lyase gene (pelA).

“…Figure 5A. To establish formally that pRD301 contained the PL gene (which we designate pelA) and to determine what proportion of PL activity produced in culture was due to expression of the gene, we inactivated it by site-directed mutagenesis (Miller et al, 1985). The polarity and approximate position of the pelA transcription unit shown in Figure 5A was determined from the restriction map of pRD091.…”

“…nidulans trpC region cloned into plC19H (Marsh et al, 1984) was used as a trpC-specific probe (Miller et al, 1985; T. Adams and W. E. Timbedake, unpublished results). Other probes are described in "Results.…”