Neurospora heterokaryons involving a thymidine kinase-positive "Helper": use in storing poorly viable strains or crossing strains of limited fertility

Many of the most interesting and useful strains encountered in research readily die in storage and/or are semisterile. If such strains are Oak Ridge-compatible, they can be carried and even crossed as heterokaryons with the sterile but vigorous strain from the Griffiths lab, am1 ad-3B cyh-1 (FGSC #4564 Perkins, Neurospora Newsl. 31: 41-42, 1984). 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/iss1/10 Number 49, 2002 19 Neurospora Heterokaryons Involving a Thymidine Kinase-positive "Helper": Use in Storing Poorly Viable Strains or Crossing Strains of Limited Fertility Robert L. Metzenberg and Matthew S. Sachs Department of Biological Sciences, Stanford University, Stanford , California 94305-5020; present address, Dept. of Chemistry and Biochemistry, University of California, Los Angeles CA 90095-1569; Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Beaverton, OR 97006 Many of the most interesting and useful strains encountered in research readily die in storage and/or are semi-sterile. If such strains are Oak Ridge-compatible, they can be carried and even crossed as heterokaryons with the sterile but vigorous strain from the Griffiths lab, a ad-3B cyh-1 (FGSC #4564 Perkins, Neurospora Newsl. 31: 41-42, 1984). This strain has a mutation in the mating type gene that prevents it from engaging directly in fertilization and also eliminates the heterokaryon incompatibility that is associated with a difference in mating type. We call this strain "Helper 1" and have used it extensively for improving the fertility of crosses. However, it has one shortcoming as an aid for promoting abundant conidiation and thus preservation of poorly viable strains. When one wants to retrieve a homokaryotic culture of the fragile strain for purposes other than crossing, it is at best a nuisance and often even a challenge to isolate it from the heterokaryon. We have deposited at the FGSC three new strains, Helpers 2, 4, and 5, that have a deletion of the mat locus and, in addition, contain an insertion of the herpes simplex thymidine kinase gene, tk. The encoded enzyme causes 5-fluorouracil-2'-deoxyriboside (FUDR) to be phosphorylated, turning a minimally toxic pro-drug into a powerful inhibitor. Thus the tk can be regarded as a dominant sensitivity gene (Sachs, M. S., E. U. Selker, B. Lin, C. J. Roberts, Z. Luo, D. Vaught-Alexander and B. S. Margolin, Nucleic Acids Res. 25: 2389-2395, 1997), and this can be used to select strongly against the outgrowth of heterokaryotic conidia or germlings. The new helpers are: FGSC #8745 mat tk cyh-1; Bml pan-2; inl "Helper 2" FGSC #8746 mat his-2 tk cyh-1; Bml pan-2; inl "Helper 4" FGSC #8747 mat his-3; hyg tk Bml pan-2 "Helper 5" In our hands, optimal conditions for isolating strains not bearing tk from heterokaryons with a strain that is tk (sensitive) are as follows: FUDR, 2 micromolar, and uracil, 1 millimolar. The rationale for adding uracil is as follows. Wild type is itself appreciably inhibited by FUDR, especially in germination rather than growth, though not nearly as severely as are tk strains. This fact limits the robustness of the method. The inhibition of wild type by FUDR has long been known, as is evident by the existence of a mutant, ud-1 , which is resistant to it by virtue of its inability to take up the FUDR (Perkins, Radford, and Sachs, The Neurospora Compendium, 2001). However, it is obviously not practical to build ud-1 into every strain that one would wish to separate from a tk strain. Our operating hypothesis is that the toxicity of FUDR to wild type is not due to traces of thymidine kinase, but to the successive action of nucleoside phosphorylase on FUDR to give the free base, 5-fluorouracil (FU) followed by salvage of FU with 5-phosphoribose-1-pyrophosphate (PRPP ) to give pyrophosphate and fluorourid ine-5’-phosphate (FUR-5'-phosphate). The latter would probably be toxic in itself by incorporation into RNA. In addition, it would almost certainly be reduced by ribonucleotide reductase to the toxic FU DR-5’-phosphate, which of course is what is made from FUDR in tk strains. A simple remedy, if this rationale is correct, would be to add a relatively large supply of uracil (not uridine) to compete with the FU for salvage via PRPP. That this does very substantially increases the resistance of wild type to FUDR while leaving the tk strains fully sensitive suggests that the rationale may be correct. Whatever the explanation, streaking or plating a water-suspension of conidia from such a heterokaryon to appropriately-supplemented sorbose-glucose-fructose agar medium results in colonies which are homokaryons of the component that does not carry tk. In our hands, 1-2 micromolar FUDR is the minimum concentration which gives only homokaryons, but at least 10 micromolar FUDR works equally well and may be preferred for the most exacting work. The uracil concentration is 1 millimolar. The method works about equally well at 23°C or 34°C with either the salts base of Vogel or of W estergaard-M itchell. Published by New Prairie Press, 2017