Improved selection for inositol-utilization following transformation of Neurospora crassa

Abstract

Plasmids based on the cloned inositol gene of Neurospora crassa are potentially very useful as a transformation marker, or for techniques like insertional mutagenesis. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This brief note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol49/iss1/9 18 Fungal Genetics Newsletter Improved selection for inositol-utilization following transformation of Neurospora crassa Robert L. Metzenberg Department of Biological Sciences, Stanford U niversity, Stanford , California 94305-5020; present address, Dept. of Chemistry and Biochemistry, University of California, Los Angeles CA 90095-1569 Plasmids based on the cloned inositol gene of Neurospora crassa are potentially very useful as a transformation marker, or for techniques like insertional mutagenesis. Transformation is generally done on spheroplasts stabilized with sorbitol, or by electroporation of conidia, also suspended in sorb itol. For transformation to inositol independence, however, this gives an unacceptable background of nontransformants that grow on the nominally inositol-free medium. It appears that sorbitol always contains a trace of inositol that cannot be completely removed by recrystallization. It seems possible that sorbitol, which differs from inositol only by a pair of hydrogens, spontaneously generates the latter by air oxidation. Substituting a-methyl glucoside for the sorbitol on an equimolar basis can circumvent this problem. a-Methyl glucoside works well as an osmoticum, and gives clean backgrounds in transformation of inl mutants to inositol independence. Published by New Prairie Press, 2017