An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa

Furukawa, K.

Hasunuma, K.

English

Kansas State University

Fungal Genetics Reports Volume 29 Article 9 An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa. K. Hasunuma K. Furukawa Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Hasunuma, K., and K. Furukawa (1982) "An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa.," Fungal Genetics Reports: Vol. 29, Article 9. https://doi.org/10.4148/1941-4765.1638 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa. Abstract An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa. This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol29/iss1/9 will themselves be disrupted. Because large batches require longer homogenization for comparable breakage,we recommend processing successive, smaller lots of mycelium for organelle recovery.The Bead-Beater may be used for whole-cell extracts if large amounts of mycelia need to be homogenizedextensively. These preparations may be somewhat dilute: bead-free, broken cell extracts have an approximatevolume of 15-, 40-, and 250-ml, from the three chambers, respectively. However, ammonium sulfate precipitationcan be applied to the extract, allowing soluble proteins to be concentrated by centrifugation.The Bead-Beater is available from Biospec Products, P.O. Box 722, Bartlesville, OK 74003 for about S400.The company also sells the glass beads. It is also available from Tekmar, P.O. Box 37202, Cincinnati, OH4 5 2 2 ,  (800)543-4461. Department of Molecular Biology and Biochemistry, University of California, Irvine,Irvine, CA 92717.Hasunuma, K. and K. FurukawaAn efficient isolation method for meiotic mutantscausing meiotic nondisjunction or elevated recombi-nation frequency in Neurospora crassa.Among several inositol-requiring mutants,inl (37401 and inl (83201(t)) complement to eachother. This fact gave us an antiselection methodof aneuploid ascospores in linkaqe qroup V, whichcontain meiotic mutants causing chromosomal non-disjunction. Ascospores haploid in linkage groupV will be led to inositol-less death on minimalplates at 37°C. Meiotic mutants which cause ele-vated recombination frequencies can also be se-lected by this method. Conidia from strainsKH204a (lys-1 (33933)), inl (37401, his-6 (Y152M105; nuc-2 (T28M2)) and 2309A (al-3 (RP1OO), in1 (83201(t))were irradiated with UV for 0, 30 and 60 sec (10% survival) and intercrossed. The resulting ascospores wereplated in Fries minimal plates (5.1 x 104 ascospores/plate with non-irradiated sample) and incubated at 37°Cfor 3 days. From 0, 30 and 60 sec irradiated samples 571, 1100 and 528 of colonies were isolated (40 colo-nies/plate with non-irradiated sample). Most of the strains isolated had pale orange conidia which wereintermediate in color between those of al-3 and wild type. Conidial isolation of these cultures revealedsegregation of al-3. They were crossed with strain 2125A and 2998a in 12 x 105 mm test tubes. As shown inFig. 1. strains discharqing more tha 20% of abortive ascospores were screened and 60, 167 and 56 strains wereobtained from each sample.From each of these crosses of the 60 strains ob-tained as spontaneous mutants, 50 or 100 ascosporeswere isolated. White ascospore production by theseisolates was analyzed by crossing with wild type, withthe isolates as protoperithecial parents. When morethan 30% of isolates showed more than 30% of abortiveascospores, these strains were reserved for furtheranalysis. Isolation of single conidia of appropriateisolates which showed meiotic aberration was perform-ed and those which produced more than 30% of abortivewhite ascospores when crossed as protoperithecialparents were used for further analysis. This processwas essential to avoid heterogenous genetic back-ground caused by meiotic nondisjunction. Thesestrains thus obtained were reciprocally crossed andresulting ascospores were analyzed. As shown inTable 1, these mutants were grouped into two classes.Class I includes those strains which produced 30 to90% of abortive white ascospores only when they werecrossed as protoperithecial parents. With homo-zygous crosses, higher frequencies of abortion wereTABLE IWhite abortive ascospore production in isolatedmeiotic mutants.Genotype         Ascospore shot (% of white spore)(Protoperithecia Class I Class II/conidia) N2AM152 N2AM6 N2AM195 N2AM83+/+ o-5 o-5 o-5 o-5+/+ o-5 o-5 50-90 70-90Mei / + 40-70 40-60 90 80-90Mei / Mei 90 70-90 No dis.* 90-95*Perithecia were formed but no discharge of ascosporewas observed.5;c20 40 60 60 1 00P e r  c e n t  o f  w h i t e  s p o r e sFigure 1. -- Distribution of per cent Of whiteabortive ascospores produced from crosses betweennutrition nonrequiring isolates and wild type,2125A or 2998a. Experimental details were asdescribed in the text. Crossed with 2125A(--e--l  or 2998a  (-0-j. Accumulation of 0, 30,and 60 sec irradiated samples (-A-).observed. Finally 12 meiotic mutants belonging to class I were obtained. Seven mutants belonging to class II,which produced 30 to 90% of abortive white ascospores irrespective of conidial or protoperithecial parents,were also isolated.Among class I mutants, N2AMI52 and N2AM189, were considered to cause chromosomal nondisjunction and threemutants, N2AN6, N2AM213 and N2AM229, were suggested to cause chromosomal degradation. Among class II mutants,N2AM195 was estimated to cause chromosomal nondisjunction, and N2AM83 was found to cause chromosomal degrada-tion of linkage group V and elevation of recombination frequency in linkage group V. This mutation was mappednear al-3  The frequency of spontaneous mutation to meiatic aberration was approximately 2.7 x 10-5.--- National Institute for Basic Biology, 38 Nishigonaka Myodaijicho Okazaki, 444, Japan.Nemeti, F. M., M. Schablik and G. SzaboExperiences with Metzenberg's method forpreparing Neurospora DNA.Metzenberg and Baisch (1981 Neurospora Newsl. 28: 20) re-ported an easy method for preparing high molecular weight DNAfrom Neurospora crassa RL3-8 A.  We confirmed their results:however, in our hands the nucleic acid prepared by Metzenberg'smethod contained large amounts of RNA and palysaccharides. Toreduce contamination in our DNA preparation the original pro-cedure was modifie ed as follows: (a) digestion with RNAse wasextended to 3 hours; (b) final precipitation of DNA was per-formed with 0.54 volume of iso-propanol.Since the product obtained in this way still contained equal amounts of DNA and RNA, as determined by thediphenylamine (Richards 1974 Anal. Biochem. 57: 369) and phloroglucine (Dische and Barenfreund 1957 Biochim.Biophys. Acta 23: 639) methods, respectively, an additional treatment with RNAse was performed. The RNA con-tent of the final product was less than 10%.The DNA prepared either by the method of Metzenberg or according to the above modification containedabout equal amounts of DNA and polysaccharides (w/w): the latter was determined with the anthron reagent(Roe 1955 J. Biol. Chem. 212:  335).The molecular weight of DNA was determined by agarose gel electrophoresis (Aaij and Borst I972 Biochim.'Biophys. Acta 269: 192) with λ-phage DNA used as standard, and was found to be about 49 kilobase pairs (kb).About 5% of the product were fragments of lower molecular weight (between 23 and 49 kb).In conclusion, although Metzenberg's mthod provides an easy way for obtaining high molecular weight DNA,the DNA prepared in that way is contaminated to a significant degree with RNA and polysaccharides.Institute of Biology, University Medical School, H-4012 Debrecen, Hungary.Raju, N. B.Easy methods for fluorescentstaining of Neurospora nuclei.This note is intended for Neurospora workers who have access to afluorescence microscope but do not have easy access to advice on nuclearstaining. The section on Equipment provides basic information for equip-ping and using a fluorescence microscope.Fluorescent staining of fungal nuclei is quick, easy and much simplerthan conventional methods (See e.g. Lemke et al. 1978 J. Cell Sci. 29: 77;Williamson and Fennel1 1975 Methods in Cell Biol. 12: 335; Slater l977Methods in Cell. Biol. 20: 135; Martegani and Trezzi 1979 Neurospora Newsl. 26: 20). Several articles in "FlowCytometry and Sorting" (eds. Melamed et al,  John Wiley & Sons, New York, 1979) give useful information on theuse of numerous nucleus-staining fluorochromes.I have been exploring possible applications of fluorescence microscopy to routine Neurospora cytology. Ihave used five fluorochromes that have specificity for DNA: DAPI (diamidino phenylindole), Hoechst 33258,olivomycin, auramin-0 and acriflavin. DAPI, Hoechst 33258 and olivomycin are simplest and quickest to use.Acriflavin is very good for observations on ascus nuclear cytology. DAPI and Hoechst 33258 require a mercurylight source and fluorite objectives. With auramin-0, the staining solution must be prepared fresh every time,and requires filtering. (See Lemke et al. 1975 Exptl. Cell Res. 96: 367.) Acriflavin and auramin-0 are veryinexpensive and easily available. The other three are more expensive, but their cost is not prohibitive be-cause of the very small quantities needed. Acriflavin- and auramin-O staining procedures are based on Feulgen,and use of acriflavin for quantitative DNA measurements has been reported. Some fluorochromes (DAPI, oilvomycin,Hoechst 3342) are also useful as vital nuclear stains.Based on my experience using an Olympus microscope fitted with epifluorescence accessories, the fluorescentmethods are indeed very simple, yet highly reliable for staining Neurospora nuclei in conidia, mycelia and asci.Nuclear counts are reliable and painless. In addition, because of the specificity of several fluorochromes  forDNA, they can serve a diagnostic function that is especially needed in fungi where Feulgen staining is difficultor unsuccessful.A. DAPI and Hoechst 33258 (available from Calbiochem and Sigma, respectively) are specific to AT-richregions. DAPI (and perhaps Hoechst 33258) also interacts with polyphosphate and S-adenosylmethionine, resultingsometimes in lower contrast between nucleus and cytoplasm. Stock solution may be prepared at 1 mg/ml in dis-tilled water and can be stored in the refrigerator for several months. 1) Place cells or mycelium in 3:1 ehtanol:

An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa.

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An efficient isolation method for meiotic mutants causing meiotic nondisjunction or elevated recombination frequency in Neurospora crassa.

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