Richard L Weiss | PubMed Health

PubMed2006

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

DNA Primers↗

PubMed2006

Developmental expression of two forms of arginase in Neurospora crassa.

N. crassa has two forms of arginase. The physiological role of multiple arginases is not understood. The two forms were shown to be differentially expressed from a single locus (aga) and both proteins are localized to the cytoplasm. The 36-kDa protein was expressed in minimal and arginine supplemented medium, whereas the 41-kDa form was detected only in the presence of arginine. In this study we examined developmental expression of the two arginase transcripts and proteins in conidia and during conidial germination. Two novel observations are revealed, storage of both arginase proteins in conidia and temporal expression of aga transcripts during early germination. To better understand the role of arginase in conidia and the nature of the temporal expression, we examined the effects of related metabolites, arginine, ornithine, proline, glutamate and glutamine on protein storage and temporal expression. These metabolites were used as supplements or sole nitrogen sources. Storage of arginase protein was detected in all conidial samples examined except when glutamate was used as the nitrogen source. The aga temporal RNA expression early in germination was abolished when arginine related metabolites were used as nitrogen sources. The exception to this result is observed with glutamate where temporal expression was seen when glutamate was the sole nitrogen source and abolished with glutamate supplementation. The temporal expression result supports a unique role for arginase in glutamate accumulation early in germination whereas the protein storage result supports the existence of a novel pathway utilizing arginase for glutamate synthesis in asexual spore development.

Arginase↗