Transformation of Aspergillus nidulans by using a trpC plasmid.

Abstract

We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements thpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpCstrain to trpC' at frequencies of >20 stable transformants per ,ug of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the tipC-allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.The Ascomycete fungus Aspergillus nidulans has been used extensively for the study of eukaryotic gene structure, organization, and regulation (1-4) because features of its life cycle make it particularly amenable to biochemical and genetic analysis. A. nidulans has been especially valuable for investigating the genetic and molecular processes controlling fungal cell differentiation (5-9). In contrast to the situation with its close relatives Saccharomyces cerevisiae (yeast) and Neurospora crassa, however, investigations of A. nidulans have not been facilitated by the availability of a tractable DNA-mediated transformation system.We recently isolated the trifunctional trpC gene from A. nidulans (10) for use as a selective marker in transformation experiments with this organism. Here we report construction of a chimeric plasmid, designated pHY201, consisting of a complete wild-type copy of the Aspergillus trpC gene inserted into the unique Sal I site of pBR329 (11). We find that pHY201 DNA, either in circular or linear form, transforms a trpC-A. nidulans strain to trpC+ at frequencies of >20 stable transformants per pg of DNA. The transforming DNA becomes integrated into the host genome, frequently at the site of the resident gene. In several instances, we were able to recover pHY201 in native or altered form from Aspergillus transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of Escherichia coli to ampicillin resistance. Thus, pHY201 has properties that suggest that it may serve as a valuable prototype for the development of more sophisticated Aspergillus cloning vectors. (15) of E. coli MC1066 by using a recombinant phage library formed between A. nidulans nuclear DNA and X Charon 4 (9, 10). We determined that a 4.1-kilobase Xho I restriction fragment present in a phage designated XAn trpC12 contained the entire trpC gene plus about 0.4 kilobase of 3' and 5' flanking sequences (unpublished results). This fragment was recloned into the unique Sal I site of pBR329 (11) to form pHY201 (Fig. 1). MATERIALS AND METHODS MaterialsIsolation and Labeli...

Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data

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A single gene encodes a selective toxin causal to the development of tan spot of wheat.

“…Mycelia (1 to 1.5 g) were added to 8 mL of 1.2 M MgS04, 10 mM sodium phosphate containing 80 mg of mureinase, 40 mg of driselase, 4 mg of chitinase, and 8500 units of P-glucuronidase and incubated with gentle agitation at 25°C for 2 to 3 hr. The protoplast suspension was filtered through a 20-pm Nitex (Tetko Inc., DePew, NY) membrane and rinsed with STC (1.2 M sorbitol, 10 mM Tris-HCI, pH 7.5, and 10 mM CaCI2) (Yelton et al, 1984). The suspension was centrifuged (4500 rpm for 5 min) in an SS34 rotor (Beckman Instruments, Palo Alto, CA).…”

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

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