Making the selectable marker bar tighter and more economical

Shan Hays and Eric Selker, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

Use of the bacterial basta resistance gene (bar) as a selectable marker in Neurospora was reported by Avalos et al (1989 Curr. Genet. 16:369-372). Unfortunately, phosphinothricin (PPT, also called glufosinate), the active ingredient in basta, is currently expensive in its pure form. PPT is a principal ingredient in the relatively inexpensive herbicide Finale (Hoechst-Roussel Agri-Vet Inc.) commonly found in lawn and garden stores, but we found that Finale prevents growth of both Bar⁺ and Bar^- Neurospora strains. Marty Pall pointed out to us that since PPT is highly soluble in water, a simple extraction may separate the PPT from the non-specific inhibitory ingredients. We found this to be the case. One simple method is to extract Finale twice with an equal volume of 1-butanol, lyophilize the solution, and then dissolve the resulting gel in water (e.g. to half of the original volume). Bioassays indicated that the extracted PPT worked as well as pure PPT and that little if any PPT was lost during the extractions.

We have also found that the Neurospora am (NADP-specific glutamate dehydrogenase) marker can be used to tighten the bar selection. PPT is known to act by inhibiting glutamine synthetase, the product of gln-1 in Neurospora (Dávila, et al, 1978 J. Bacteriol. 134:693-698) and it had been reported that am; gln-1 double mutants do not grow on media with ammonium as the sole nitrogen source (Hummelt and Mora, 1980 Biochem. Biophys. Res. Comm. 92:127-33). We therefore tested whether PPT would cause an am mutant to phenocopy an am; gln-1 double mutant. It did and only 10 ug/ml of PPT (5% of the normal level; Pall, 1993 Fungal Genet. Newsl. 40:58) completely inhibited Bar^- strains on Vogel's minimal medium N but permitted growth of Bar⁺ strains. Greater than 10 ug/ml PPT inhibited even Bar⁺ strains. In our hands, this scheme resulted in less background growth than in the original system. Addition of alanine to the medium (which promotes growth of am strains; Fincham, 1950 J. Biol. Chem. 182:61-73) completely countered the effect of am.

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