A system for studying aneuploid production in Neurospora crass

Smith, B. R.

Yorston, M. E.

English

Kansas State University

Fungal Genetics Reports Volume 28 Article 14 A system for studying aneuploid production in Neurospora crassa B. R. Smith M. E. Yorston 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 Smith, B. R., and M.E. Yorston (1981) "A system for studying aneuploid production in Neurospora crassa," Fungal Genetics Reports: Vol. 28, Article 14. https://doi.org/10.4148/1941-4765.1659 This Research 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. A system for studying aneuploid production in Neurospora crassa Abstract A system for studying aneuploid production in Neurospora crassa This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol28/iss1/14 formation in some species, but not in others. Since perithecial production is significant for laboratorygenetics as well as for the photobiology and ecology of Neurospora, observations should be extended tothe four known heterothallic species. (Supported by Public Health Service Research Grant AI 01462.)- - - - Department of Biological Sciences, Stanford University, Stanford, California 94305.Selitrennikoff, C. P.A new allele of csp-2 whichdoes not complement csp-1.The conidial separation mutations [ csp-l (IL) and csp-2 (VII)]confer upon Neurospora strains reduced ability of conidial chains toseparate; double mutant strains are even further impaired (seeSelitrennikoff et al. 1974 Genetics 78: 679). The one csp-2 alleleand the three csp-2 alleles complement in heterokaryons and form awild type number of free conidia. A new allele of csp-2 is describedwhich does not complement csp-2,  yet is recessive to wild type.A strain of   [poky  f ] a which did not form free conidia was obtained from John Chalmers (University ofCalifornia, Berkeley) and was found to contain a conidial separation mutant. This mutant was crossed towild type (Oak Ridge) four times (as the male parent) and the csp phenotype segregated 1: 1. This mutantstrain, designated UCLA 102, was found to grow in both liquid stationary culture and "race" tubes identi-cally with wild type. Inter se crosses among the csp strains showed that UCLA 102 was allelic with csp-2(0 recombinats/69 with FS 590; 0/211 with FS 591; 9176 wih UCLA 101) but was not allelic with csp-1(11/40). The double mutant, csp- 1;csp-2 (UCLA 102) was found to produce a very low level of free conidia,similar to other combinations of csp- 1;csp-2 alleles. The phenotype of forced heterokaryons containingcsp- 1 and csp-2 alleles is essentially wild type as judged by either counting the number of free conidiawith a haemocytometer or inverting agar-slant cultures and tapping to release free conidia (the "tap-test";Selitrennikoff and Nelson 1973 Neurospora Newsl           20: 34). In sharp contrast, the forced heterokaryoncsp-1/csp-2 (UCLA 102) produced a number of free conidia equivalent to the csp-2 level, i.e., csp-2 (UCLA102) did not complement with csp-1..   However, csp-2 (UCLA 102) was found to be recessive to csp-2+ .These results indicate that the interaction of the csp genes (or gene products) is not as autonomousas previously described. The new allele of csp-2 is available from the Fungal Genetics Stock Center.- - - - Department of Anatomy, University of Colorado Health Sciences Center, 4200 East Ninth Avenue,Denver, Colorado 80262.---------em--Smith, B. R. and M. E. Yorston   Crosses between the complementing histidine-5 allele K78 andK746 yield two kinds of his+ progeny. One results from recombin-A system for studying aneuploid ation between the alleles and the other primarily from nondisjunc-tion of chromosome IV giving disomic ascospores. Less frequently,production in  Neurospora crassa. disomic progeny may be produced as a result of extra chromosomereplication or chromosome non-conjunction (pairing failure). Thedisomic spores form pseudowild colonies on minimal medium that aredistinguishable from the much rarer his+ recombinants by their slower growth rate.The inclusion in the parent strains of linked auxotrophic markers, flanking the his-5 locus, permitseasy recognition of parental and pseudowild types among conidia formed by pseudowilds. In contrast, conidiafrom his+ recombinants are homokaryotic and do not show marker segregations.Genotypes of parent strains.Parent A. pyr-3 (1298), his-5 (K78), aParent B. his-5 (K746), leu-2 (37501), AReciprocal crosses were prepared, some of which were treated with p-fluorophenylalanine. The progenywere then screened on selective media to estimate the frequency of pseudowilds. The amino acid analoguep-fluorophenylalanine is known to increase meiotic non-disjunction of chromosome I of Neurospora (Griffithsand DeLange. Mutat. Res.these crosses.1977, 46: 345) and should significantly increase frequencies of pseudowilds inPreparation and treatment of crossesPetri dishes (90 mm diameter) containing 20 ml of Westergaard's crossing medium supplemented withuracil, histidine and leucine, were inoculated with drops of conidial suspension of one parent. To in-crease the fertility of the crosses, macerated Whatman's No. 1 filter paper was added to the crossing medi-um at the rate of 270 cm2/1. Petri plates were incubated for five days to allow protoperithecia to form.During this period, the petri dish walls were wiped with alcohol twice daily to prevent the mycelium spread-ing over the sides of the plates.conidia.Fertilization was then effected by the addition of a dense suspension ofExcess water was removed after 30 min and 41/2 h later, 5 ml of water or 5 ml of p-fluorophenyl-alanine solution (0.05 mg/ml) was added to each petri plate. The water or p-fluorophenylalanine  solution171_-was drained off after 16 h incubation and the plates were then incubated in an inverted position for 21days. Ascospores were subsequently collected in sterile water from the petri dish lids.Crosses treated with p-fluorophenylalanine  show dramatically increased frequencies of pseudowild typeprogeny - 3.5 times the control value in the cross AP x Bet, and 7.1 times in the cross B? x Ad. Theseincreases are of the same order as those observed by Griffiths and DeLange.Table 1Analysis of Crosses% Recombinationthat results between pyr-3do not differB x A-Overall frequency of his+ allelic recombinants = 9.5 per l05 ascospores- - -p-f - p-fluorophenylalanine treated,The final column in Table 1 shows estimates for recombination between pyrimidine-3 and leucine-2 in thecrosses ( The estimates are based on frequencies of pyr+ leu+ colonies detected on histidine supplementedmedium (values were corrected by substracting pseudowild type frequencies).significantly (heterogeneity χ2 = 0.862 p =These frequencies do not differfluence crossing-over.0.08-0.09) indicating the p-fluorophenylalanine does not in-This simple test system seems ideal for studying,aneuploid production, and for detecting chemical agentsthat might influence formation of aneuploid products during meiosis.University of Aberdeen, Aberdeen, Scotland, United Kingdom.- - - - Department of Genetics,Stadler, D.Thirty-four temperature-sensitive mutants which were isolatedTemperature-sensitive  mutants. by the inositof-fess death enrichment procedure have been depositedin the Stock Center. Conidia of strain inl a Were treated with suf-ficient UV for 50-80% kill and then plated in minimal sorbose agarand incubated 3-5 days at 33°C or 37°C. The plates were then trans-ferred to 22°C and supplemented with inositol. Colonies arising subsequently were isolated and testedfor temperature sensitivity. Three to five percent of the isolated colonies were temperature sensitive,but half of these were discarded because of poor growth or conidiation even at the permissive temperature*No mutant was found which was blocked specifically in Gonidial germination. That is, when the mutantconidia were permitted to germinate and start growing in race tubes at 22°C and then shifted to 37°C,growth was arrested in all cases. Most of the mutants stopped quickly (2-4 h) after the temperature shift,though one (165C) continued to grow for many hours before finally stopping.Results of linkage studies and some other properties of the mutants are summarized in Table 1.There are five cases in which two or more mutants appear to be allelic. These groups are indicatedin Table 1 by the spacing.each.They include two groups of four mutants each and three groups of two mutantstion,Mutants grouped in this manner failed to complement each other in heterokaryon tests for ts+ func-Complementation tests were performed for nearly all pairwise combinations of the 34 mutants. Theresult was positive in all cases except those indicated here. In four of the groups the Gross tests andthe heterokaryon tests were all consistent with the members of a group being aflelic.up of 33, 134C, 16J and 21T, the evidence is doubtful.In the group madeHeterokaryon tests were incomplete with 16J be-

A system for studying aneuploid production in Neurospora crassa

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A system for studying aneuploid production in Neurospora crassa

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