*present address: Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106
We developed a simple ascospore plating assay for aneuploidy, based on identifying disomic progeny that inherit two independently selectable mtr alleles. We validated the assay using a known meiotic mutant, mei-2. We used this assay to demonstrate that elevated frequencies of aneuploidy previously reported to be associated with reduced DNA methylation were not, in fact, due to the methylation deficiencies. A new allele of the mei-1 gene was responsible for some of the high aneuploidy.
Aneuploid progeny were initially reported to have been generated at higher than normal frequencies in crosses of Neurospora crassa mutants deficient in DNA methylation (Foss et al. 1993 Science 262: 1737-1741, Foss et al. 1995 Science 267:316). This suggestion grew from anecdotal observations of aneuploidy at frequencies higher than was expected based on literature values. The mutant examined in most detail, dim-2 (defective in methylation) has no detectable methylation, at least in vegetative tissue (Foss et al. 1993 Science 262:1737-1741) and ascospores (E. Selker and E. Kuzminova, unpublished). In an attempt to validate the finding of enhanced aneuploidy, methylation was reduced in a dim+ met-7 (methionine) auxotroph, by starving for methionine, and a high frequency of aneuploidy was observed. The criteria for identifying progeny as aneuploid were one or more of the following: 1) patchy coloration due to heterozygosity for a pigment gene; 2) the ability to self-fertilize, which requires the two mating-type idiomorphs; 3) the presence of restriction patterns indicating RFLPs from both parents at a given locus; 4) intermediate levels of methylation, reflecting heterozygosity for a dim gene. In an attempt to quantify better the effect of methylation on aneuploidy we developed a simple plate assay for aneuploid sexual progeny.
Our simple assay for aneuploidy is based on identifying progeny that are disomic for LGIV, inheriting two independently selectable alleles of the mtr gene. We cross a strain that has a functional, wild-type allele of mtr with a strain carrying an allele of mtr that is defective by virtue of an insertion of the E. coli hph gene, encoding hygromycin resistance. An aneuploid carrying one of each mtr allele will plate under conditions that select simultaneously for both alleles, whereas neither euploid strain will. All strains carry trp-2, which allows for selection of mtr+ strains. In many of our tests one parent carried a mutation (am33; A. J. Griffiths)
that eliminates the vegetative incompatibility (and death) that would otherwise result from having two different mating types in a single cytoplasm. This mutation was included because there is evidence that aneuploids produced in wild-type crosses are frequently multiply disomic (Smith 1974 Genetics 76:1-17). As it turns out, this mutation did not significantly influence the frequency of aneuploidy detected by our assay (Table 2). Plating assays employed in past studies to detect aneuploids involved crossing two strains, each carrying a different set of auxotrophic markers, and selecting for prototrophs (Smith 1974 Genetics 76:1-17). Only strains carrying both chromosomes, and rare recombinants, were able to plate. Our method is an improvement over this since homologous recombination cannot produce false positives in our assay, and our assay requires introduction of only two markers (trp-2 and mtr::hph).
Neurospora strains and genotypes are listed in Table 1. Culture and crossing were carried out as per Davis and DeSerres (1970 Meth. Enzymol. 17:47-143) using the crossing medium of Russo et al. (1985 Neurospora Newsl. 32:10-11). Crosses involving met-7 strains were done on medium containing 0.01 mg/ml methionine. Ascospore plating was on Vogel's agar with 2% sorbose and 0.1% dextrose. Permissive plating was on medium supplemented with 0.05 mg/ml anthranilic acid (and other supplements when needed). Selection for aneuploids was on medium containing 0.01 mg/ml tryptophan, 0.6 mg/ml arginine, (selection for mtr+; Stadler et al. 1991 Genetics 129:39-45) and 0.1 mg/ml hygromycin B (Calbiochem).
We tested our assay using mei-2 strains, which are defective in meiotic pairing and are known to generate aneuploids at enhanced frequencies (Smith 1975 Genetics 80:125-133, Shroeder and Raju 1991 Mol. Gen. Genet. 231:41-48). The data shown in Table 2 indicate that roughly one-third of the ascospores from crosses homozygous for mei-2 produced colonies on the aneuploid selection medium, compared to about 2% for normal strains. Spores from the dim-2 and met-7 mutant crosses, which were either heterozygous or homozygous for the methylation defect, did not display enhanced frequencies of plating under the selection for aneuploids. Crosses heterozygous for dim-3, another gene required for normal methylation and previously implicated in inducing aneuploidy when heterozygous, also showed normal levels of aneuploidy in our assay. We conclude that reduced methylation does not induce aneuploid production.
One strain, N1472, when crossed with any other strain that we tested, gave the lowest frequencies of aneuploidy. A likely explanation for this is that vegetative incompatibility killed off most of the aneuploids produced, since N1472 is not completely compatible with the other strains used here, which are Oak Ridge-compatible. Control experiments to test this hypothesis, using incompatible strains that were either mei-1, dim-2 or dim+, showed up to 10-fold reductions in frequencies of aneuploidy by our assay when compared to crosses of identical genotype involving Oak Ridge-compatible strains (data not shown). Thus, it is important to use compatible strains in order to maximize detection of aneuploids; this probably holds for any assay method.
In contrast to results from our new assay for aneuploidy, generation of aneuploids in met-7 crosses reported previously (Foss et al 1993 Science 262:1737-1741) was strikingly elevated. Because we had to construct new met-7 strains carrying appropriate genetic markers for our plating assay for aneuploids, we tested these new strains for the ability to generate al-2/al+ (white/orange) mosaic aneuploids, one of the tests for aneuploidy in the previous study, and these strains failed to generate mosaics. Reasoning that we must have lost the "aneuploidy factor" in our strain construction, we therefore crossed a met-7 strain used in the previous study with a multiply marked strain (FGSC 4453) in an attempt to map the defect causing the aneuploidy. In our mapping cross we did not follow the defect using our plating assay for aneuploidy, since this requires the introduction of additional markers. Instead, we followed another sexual-cycle phenotype that is associated with the ability to generate aneuploids; misshapen spores and a high proportion of white spores (dead, presumably due to a chromosomal deficiency). We established a linkage distance of 13 map units (200 progeny analyzed) between the defect associated with aneuploid production from the met-7 strain, and pyr-1, on LG IV.
One gene known to affect meiotic chromosome segregation, mei-1, is known to map near pyr-1. To determine whether the new defect was allelic with mei-1, we crossed mei-1 strains (FGSC 2919 and 2920) with our mutant strains. Although both mei-1 and the new defect are recessive (they appear normal in heterozygous crosses), we failed to see complementation of the defect; crosses between our mutant and mei-1 strains generated as many white spores (~80%) as crosses homozygous for our mutant or for mei-1. We conclude that the new defect is an allele of mei-1, which we name mei-1(AH). The met-7 strain FGSC 3915 was the source of mei-1(AH).
We thank Rik Myers and Jette Foss for helpful advice. This work was supported by A.C.S. grant PF-3557 to ATH and N.I.H. GM-35690 to EUS.
Table 1. Neurospora Strains
Name Genotype N568 (FGSC 8288) mtr::hph trp-2 A N569 (FGSC 8289) mtr::hph trp-2 a N1465 mtr::hph trp-2 al-2 A N1466 (FGSC 8290) mtr::hph trp-2 al-2 A N1467 trp-2 arg-3 am33 N1468 (FGSC 8291) trp-2 arg-3 am33 N1469 trp-2 arg-3 am33 N1470 dim-2 mtr::hph trp-2 al-2 A N1471 dim-2 mtr::hph trp-2 al-2 A N1472 dim-2 trp-2 arg-3 inl am33 N1473 dim-3 trp-2 a N1474 dim-3 trp-2 A N1475 trp-2 a N1476 trp-2 A N1479 mei-2 mtr::hph trp-2 al-2 A N1480 mei-2 trp-2 arg-3 am33 N1481 mei-2 trp-2 arg-3 am33 N1482 trp-2 mei-1(AH) a N1483 mtr::hph trp-2 al-2 A N1484 met-7 thi-3 mtr::hph trp-2 al-2 A N1485 met-7 thi-3 trp-2 mei-1(AH) a FGSC2919 mei-1 A FGSC2920 mei-1 a FGSC3915 met-7 thi-3 mei-1(AH) a FGSC4453 arg-5 thi-4 pyr-1 lys-1 inl nic ars AAllele numbers: al-2(R401 or Y122M38), arg-3(30300), dim-2(KC), dim-3(KC), mei-2(ALS181), met-7(NM251), thi-3(18558).
Table 2. Generation of aneuploids in crosses of Neurospora crassa
Cross Type Parent Strains1 Aneuploid Frequency (%)2 dim+ x dim+ N1465, N1467 1.77 + 0.31 (3) N1466, N1467 2.0 N1466, N1468 2.7 N1466, N1469 2.8 N1475, N568 4.7 N1476, N569 1.3 mei-2 x mei-2 N1479, N1480 40, 27 N1479, N1481 35, 26 dim-2 x dim-2 N1470, N1472 0.17 + 0.06 (3) N1471, N1472 0.2 dim+ x dim-2 N1465, N1472 0.6 N1466, N1472 0.7, 0.1 dim-2 x dim+ N1470, N1467 5.24 + 2.04 (5) N1471, N1467 1.7, 2.4 dim-3 x dim+ N1473, N568 1.7, 1.3 N1474, N569 2.5, 1.1 met-7 x met-7 N1484, N1485 3.8, 3.1* met-7 x met+ N1484, N1482 1.6, 1.2* met+ x met-7 N1483, N1485 2.8, 2.6*1 See Table 1 for strain genotypes.
2 Values listed represent the efficiency of plating on the aneuploidy selection medium relative to the permissive medium. Each number is for an independent cross, except as indicated by *, which denotes two platings from the same cross. For crosses performed more than twice, values are means + standard deviations, where numbers in parentheses indicate number of repetitions of the cross.
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