Strains:

Standard Oak Ridge wild types were used, and markers were in Oak Ridge genetic background. Allele numbers are as follows: ace-1, Y2492; adh (adherent), NM227; arg-12, UM107; cya-8, P9178; cyt-7, 20; fl (fluffy), L; nic-3, Y31881; pe (peach), Y8743m; rid-1, N2250; rip-1 (ribosome production), 4M; trp-3, td24; un-15, T54M50; un-20, P2402. The helper-1 strain a^m1ad-3B cyh-1 (FGSC 4564) was combined with cya-8 strains to form phenotypically wild-type female-fertile heterokaryons. For information on markers and on the inactive mating-type helper-1 strain, see Perkins (1984), Perkins et al. (2001), or http://www.bioinf.leeds.ac.uk/∼gen6ar/newgenelist/genes/gene_list.htm. Three mutant eas alleles were used: UCLA191 (eas^UCLA) (Selitrennikoff 1976), JD105 (eas^JD) (Bell-Pederson et al. 1992), and KH5-9 (eas^KH) (Hasanuma 1984). Translocations ALS179, OY329, and S1229 are described by Perkins (1997). Mutations at cya-8 were not known previously. Existing stocks of eas^UCLA contain a wild-type linkage group (LG) VII with the cya-8⁺ gene intact.

Genetic analysis:

Crosses were made at 25° on slants of synthetic cross medium in 150-mm tubes. Ascospores were spread on 4% agar, isolated using a platinum–iridium blade to pick up single ascospores on a small piece of the underlying agar, transferred to 10 × 75-mm slants with appropriately supplemented Vogel's medium N, and heat-shocked 30 min in a 60° water bath. For media and general methods, see Davis and de Serres (1970) or http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm. When growth was not visible to the naked eye, tubes were examined for transparent growth using a dissecting microscope at ×40–×70 magnification with transmitted light from a substage mirror. For mapping cya-8, an exceptionally vigorous transparent strain (P9178, FGSC no. 4523) was used initially (as male parent—despite its vigor, the strain is female sterile). Because cya-8 strains grow slowly and are female sterile, subsequent crosses employed phenotypically vigorous wild-type heterokaryons (FGSC 4524 A, 4525 a) combining a sparsely growing transparent strain with helper-1 (a^m1 ad-3B cyh-1) as the second component. The heterokaryons with helper-1 are fully fertile either as female or as male, but the helper component does not participate in the cross because the a^m1 mating-type allele is inactive (see Perkins 1984).

Each of the duplication strains used as a parent in Table 5 was obtained from a cross of the corresponding balanced translocation with wild type. In Neurospora, presence of a segmental duplication in one or both parents typically results in a cross being barren, i.e., producing few or no ascospores (Raju and Perkins 1978). When a duplication was present, crosses were made by growing one parent on crossing medium in a petri dish and fertilizing the lawn with a conidial suspension. After perithecia had reached full size, the cross plates were inverted over an agar surface. Usually, a few ascospores were eventually ejected, and these were isolated not <30 days after fertilization. Because two or more spores often come from the same ascus, the numbers of progeny in Table 5 probably exceed the numbers of asci from which they originated.

Insertion of fluffy into the eas^UCLA inversion:

The procedure was similar to that used by Bailey and Ebbole (1998) for obtaining a fluffy mutation in wild-type sequence by RIP mutagenesis, except that eas^UCLA strains were used instead of the Oak Ridge wild types. A plasmid (pFLUF3) that contains the fl⁺ gene on a 2.9-kb SmaI restriction fragment was used to transform eas^UCLA mat A. A transformant purified through three rounds of conidial passage was crossed to eas^UCLA mat a, and fast-growing fluffy strains were recovered among the progeny.

Molecular methods:

To isolate genomic DNA, wild type (N150, 74-OR23-IVA), mutants eas^UCLA and eas^JD, and normal and slow-growing progeny of eas crosses were grown for 48 hr in Vogel's medium N. DNA was isolated as previously described (Miao et al. 2000). To detect DNA methylation by Southern analysis, ∼1 μg of genomic DNA was digested with the cytosine methylation-sensitive Sau3AI endonuclease or its cytosine methylation-insensitive isoschizomer, DpnII (Miao et al. 2000). Gel-purified probe fragments for Southern analysis were generated by PCR from genomic DNA (probes A–D) or plasmid pS7C1 (probe E) as indicated in Figure 2. The sequences of oligonucleotides used for PCR are: no. 1 (easSalIF), 5′-GACGGAAAAGTTGTGAAGCGTCCGG-3′; no. 2 (easEcoRIR), 5′-TGACTCCAAATGGAGACGGACCAG-3′; no. 3 (79F), 5′-TTTAAACGCGTCCCCACAA-3′; no. 4 (619F), 5′-CGGAATTCACCTGACATCGCAAATCA-3′; no. 5 (253F), 5′-ATCCATTACCAGTCTGTCAGT-3′; no. 6 (646F), 5′-GCCCGAACCCGTTATGTTCAAC-3′; no. 7 (95F), 5′-ACGGTAGCGGACTGCCAG-3′; no. 8 (620R), 5′-CGGGATCCTTCTTGTGGGGACGCGTT-3′; no. 9 (252R), 5′CGCGATGATTTGCGATGTCAG-3′; no. 10 (254R), 5′-GCAAGGAATACCTCCTGAGTT-3′; no. 11 (78R), 5′-CACGACCAACGTTGTTAAC-3′.

Origin and characteristics of “transparent” isolates:

The cya-8 mutation was first identified in this study. Stable transparent cultures were sent for characterization to Helmut Bertrand, who found them to be deficient in cytochrome aa₃ (personal communication), resembling previously known cya mutants (chromosomal; Bertrand et al. 1977) and the [mi-3] mutant (mitochondrial; Kennell et al. 2004). Early observations led to the following hypotheses (Selker 1990): The transparent phenotype obtained in crosses with eas^UCLA is due to RIP, which is induced by a duplicate copy of cya-8⁺ in the eas^UCLA parent. The cya-8⁺ gene was transposed from linkage group (LG) VII and inserted ectopically at or near the eas locus in LG II at the time of the original UCLA191 mutation. Crossing the mutant by wild type replaced the cya-8-deficient donor chromosome with a normal LG VII, resulting in eas^UCLA, a progeny strain that contained two copies of cya-8⁺.

Transparent cya-8 mutant progeny are produced whenever eas^UCLA is present in one or both of the parents of a cross. In heterozygous crosses, frequencies of transparent progeny are similar regardless of whether eas^UCLA was present in the female (protoperithecial) parent or in the male. The mutant progeny show slow, thin growth, producing only a sparse network of hyphae on agar slants 2 days after ascospores are heat-shocked. By the time normal siblings have grown up and conidiated, growth of transparent germinants is still so thin that it usually cannot be seen with the naked eye. Transparents can be readily be distinguished from nongerminants, however, when cultures are examined using a dissecting microscope with transmitted light. Growth of transparent strains is somewhat faster and more vigorous on minimal medium (Vogel 1964; Davis and de Serres 1970) than on glycerol complete medium (Tatum et al. 1950). Growth is not improved by supplementing the medium with a variety of growth factors or carbon sources. Different transparent isolates vary in the degree of impairment. After several days, many have grown enough to cover the slant with a thin film that is visible to the naked eye. A few especially vigorous transparent progeny eventually produce sparse conidia, but these strains never attain wild-type growth. At the other extreme, growth of some transparent germinants is so sparse and limited that they fail to cover the agar surface before slants have dried down.

A few germinants that were originally classed as transparent revert to wild type, attaining full growth and conidiation. Conidial pigmentation in such cultures was sometimes yellow rather than orange in the original slant, but in subsequent transfers the conidia were orange. The transferred revertant strains were indistinguishable from their normal siblings, in both phenotype and genotype. During most of this study, strains of this type, i.e., with transitory slow growth, were classed as nontransparent and were pooled with other nontransparents rather than recorded as a distinct class. However, a few of the late-escaping unstable strains may have been misclassified as transparent in the early experiments. Only after the mutant rid gene, a suppressor of RIP, came into use was a careful record kept of the transient transparents, which are called “laggards” (Table 6).

Mutation frequency in crosses of different parentage:

The frequencies of transparent progeny do not differ greatly in crosses that are heterozygous or homozygous for eas^UCLA, or when eas^UCLA is used as the male or the female parent. Thirteen crosses in which only one parent was eas^UCLA produced 123 transparent progeny among a total of 608 (20%). Six crosses where the parents were both eas^UCLA produced 92 transparents among 420 progeny (22%) (Table 1). These are minimum values because some transparent progeny stop growing or grow so slowly that they may be scored as nongerminants, especially if examination is with the unaided eye rather than with the microscope. We have never found a strain containing allele UCLA191 that failed to produce transparent progeny in significant numbers. This was true for crosses parented by >25 different eas descendants of the standard eas^UCLA strain during 10 generations of outcrossing and backcrossing to the Oak Ridge wild type.

Transparent cya-8 mutations are not produced by eas alleles other than UCLA191:

Hasanuma (1984) described an easily wettable mutant of independent origin that is inseparable from a reciprocal translocation, T(IL;IIR)KH5-9. He mapped eas and the IIR breakpoint near ace-1 in crosses heterozygous for the translocation and used crosses homozygous for the translocation to show that eas is located between un-20 and un-15. For convenience, we refer to T(IL;IIR)KH5-9 eas as eas^KH.

eas^KH is allelic with eas^UCLA. Forced heterokaryons between the two eas strains were easily wettable, as were all progeny from eas^KH × eas^UCLA. Conidia did not shake loose in tap tests when ascospores from the intercrosses were heat-shocked en masse and grown to maturity on plates, or when 63 random isolates were grown to maturity on slants.

Another eas allele, eas^JD, was generated independently by RIP (Bell-Pedersen et al. 1992). Unlike eas^UCLA, the mutant alleles eas^KH and eas^JD do not act as mutators (Table 1). Transparent progeny are produced only when eas^UCLA is present in one or both parents.

Genetic basis of the transparent phenotype:

The transparent phenotype is due to a Mendelian mutation that maps in LG VII, 15 map units (MU) distal from what was previously the leftmost gene marker (Table 2). The new locus, named cya-8 (cytochrome a-8), is also left of the breakpoint of the quasi-terminal translocation T(VIIL → IVR)ALS179, in which a distal segment of VIIL is translocated to the tip of IVR (Perkins 1997). Progeny from translocation ALS179 × Normal sequence include a viable class that is duplicated for the VIIL segment. Duplication progeny from translocation ALS179 × cya-8 are heterozygous for cya-8 and are phenotypically nontransparent. cya-8 is phenotypically unlike cyt-7, the only other identified cytochrome mutant in VIIL, and 4% wild-type progeny were obtained from intercrossing cyt-7 × cya-8. Sequence of these elements on the genetic map is Tel-VIIL cya-8 T(ALS179) cyt-7 adh nic-3 … Cen-VII.

eas^UCLA is unlinked to VIIL, on the basis of 15 parental ditype (PD):14 nonparental ditype (NPD):34 tetratype (T) asci from eas^UCLA × nic-3, and 25 recombinants between eas^UCLA and cya-8 among 48 cya-8⁺ progeny from nic-3 × eas^UCLA; cya-8. All eight previously described cya mutants are located in linkage groups other than VII.

cya-8 is recessive both in partial diploids and in the heterokaryons (cya-8 A + a^m1 ad-3B cyh-1) and (cya-8 a + a^m1 ad-3B cyh-1), which are phenotypically wild type. Crosses heterozygous for cya-8 are fully fertile, but perithecia are barren, unbeaked, and completely devoid of ascospores in crosses homozygous for cya-8. Expression of the cya-8 phenotype does not depend on the presence of eas^UCLA. The two genes show independent segregation in crosses. Progeny tests show that both eas^UCLA and eas⁺ alleles are present among the cya-8 progeny from intercrosses heterozygous for eas^UCLA.

Are independently arising transparents all cya-8?

Conceivably, the new transparent progeny that originate from nontransparent eas parents in different crosses could have resulted from mutation at loci other than cya-8. To examine this possibility, transparent progeny were obtained from a series of nine crosses, each of which was parented by a different cya-8⁺; eas^UCLA strain. The eas^UCLA strains used as parents had been derived independently, mostly from a series of 10 recurrent backcrosses of eas^UCLA to the standard Oak Ridge wild types. Because transparent strains grow too poorly to form protoperithecia and serve as female parents, each putative cya-8 strain was combined into a heterokaryon with the a^m1 ad-3B cyh-1 helper and crossed with a similar cya-8 heterokaryon of opposite mating type to test for fertility. A cross was also made with adh nic-3 to test for linkage in VIIL. All nine independently arisen transparent strains proved to be cya-8 recurrences by both criteria—barrenness of perithecia in the cross with cya-8 and linkage left of adh in the three-point cross to VIIL markers (Table 3). We conclude that stable eas^UCLA -induced transparent mutations are typically cya-8 and that the mutator activity of eas^UCLA is locus specific. It has been assumed without further testing that the stable transparent progeny from subsequent eas^UCLA-parented crosses were cya-8.

The results described above were obtained with strains derived predominantly from the Oak Ridge wild types. Mutability of the cya-8⁺ gene under influence of eas^UCLA is not limited to strains in the Oak Ridge background, however. Wild-type cya-8⁺ alleles from five exotic N. crassa strains were combined with eas^UCLA. Strains from Texas (Mauriceville, P538), India (Aarey, P680), Panama (FGSC 8057), Louisiana (Welsh, P507), and Florida (Groveland, P438) were crossed by a transparent strain containing both cya-8 and eas^UCLA. Fast-growing eas^UCLA progeny, which contained the cya-8⁺ allele from the exotic parent, were then backcrossed to the exotic parent. Transparent progeny obtained from each of these crosses were tested for allelism with cya-8 by crossing each of them with a standard cya-8 strain of appropriate mating type, maintained as a phenotypically normal heterokaryon with the a^m1 ad-3B cyh-1 helper. Perithecia were barren in each of nine such testcrosses, as expected if the crosses were homozygous for cya-8 (data not shown). We conclude that wild-type cya-8⁺ genes of diverse origin are vulnerable to mutation by eas^UCLA.

Mutations to cya-8 originate in perithecia following fertilization:

Experiments using heterokaryons of cya-8 with the a^m1 ad-3B cyh-1 helper showed that homokaryotic cya-8 conidia give rise to small colonies that are recognizable as transparent under appropriate lighting and magnification. Conidia from an eas^UCLA culture that was known to generate transparent sexual progeny in frequencies of 20% or more were plated on sorbose medium. No transparents were found among 145 colonies.

If new cya-8 mutations were occurring in eas^UCLA strains during the vegetative phase, wide fluctuations might be expected in the frequency of transparent progeny from cross to cross, reflecting jackpots due to early mutations in a parent culture. Frequencies might also be related to the age and history of the eas^UCLA cultures used as parents. The fact that observed frequencies of transparent progeny are rather uniform regardless of the age of the eas^UCLA parents, combined with the failure to demonstrate the presence of transparent nuclei in vegetative cultures of eas^UCLA, suggested that mutation occurs only at fixed times during the sexual part of the life cycle. Experiments were therefore designed that would set limits to the period during which cya-8⁺ is subject to mutation.

All asci of an individual perithecium usually trace their origin to the single pair of haploid nuclei of opposite mating type that came together in the archegonium at the time of fertilization (Nakamura and Egashira 1961; Johnson 1976). If mutation occurred in one of the nuclei at the time of fertilization or before, 50% of ascospores in each affected perithecium would be mutant and all asci in the perithecium would contain four cya-8 and four cya-8⁺ ascospores. If, on the other hand, mutation occurred at some later time during perithecial development, then <50% of ascospores would be mutant. Only some of the asci in an affected perithecium would contain both normal and mutant ascospores, while in the remaining asci, eight ascospores would be normal cya-8⁺. The number of asci in which mutant ascospores are present, and the number of mutant ascospores per ascus, would depend on the stage of development when mutation occurred.

The wild-type strain ORS-6a was crossed with eas^UCLA. Ten days after fertilization, individual perithecia that had not yet expelled ascospores from the ostiole were selected. The contents of individual perithecia were squeezed out one by one into separate water drops in a sterile petri dish and asci were broken up so as to release ascospores. After aging for 10 days at 30°, the ascospores from each water drop were transferred by Pasteur pipette or loop to sorbose–minimal 3% agar medium in a petri dish. The ascospores were spread and heat-shocked 30 min at 60°, incubated at 34°, and examined 24 and 48 hr later to determine the numbers of transparent and nontransparent progeny. Transparent progeny were produced by each of the eight perithecia sampled in frequencies that ranged from 9 to 16% (12% overall for 785 colonies). It appears that transparent progeny arise from events in the perithecia subsequent to fertilization. The stage at which mutation occurred was similar in different perithecia, and jackpots were not detected.

cya-8 mutations occur prior to karyogamy:

The experiment just described sets an early limit to the time of mutation. The distribution of cya-8 mutations in individual asci enables a later limit to be defined. Mutations that occur prior to the premeiotic S-phase and karyogamy would be expected to result in asci showing 4:4 segregation for cya-8. If mutation occurred later than premeiotic DNA synthesis, then fewer than four of the eight ascospores in individual asci should produce transparent progeny.

Unordered asci from the cross eas^UCLA × adh nic-3 were obtained as groups of eight ejected ascospores, using the procedure described by Strickland (1960) and Perkins (1966). The ascospores were aged, transferred individually to slants in 10 × 75-mm tubes, heat-shocked, incubated 3 days at 34°, and examined for the presence of cya-8. Among 83 asci with seven or eight ascospores germinated, cya-8 was present in 11. Each of these showed 4:4, 4:3, or 3:4 segregation for cya-8:cya-8⁺. Mutant cya-8 progeny were also obtained from asci with only five or six ascospores germinated. The content of each of these incomplete asci was also consistent with 4:4 segregation. The overall frequency of asci that had acquired a mutant cya-8 allele was 15% (14/95).

We conclude that cya-8 mutations occur in ascogenous hyphae prior to the premeiotic S-phase, which immediately precedes karyogamy. The ascogeneous hyphae are two-component heterokaryons populated with haploid A and a nuclei that are derived from a single original pair following fertilization. Similar criteria were used by Selker et al. (1987) to establish that RIP occurs between fertilization and karyogamy in nuclei containing a duplication and by Butler and Metzenberg (1989) to determine the timing of premeiotic changes in the number of rDNA repeats in the nucleolus organizer.

Mutation occurs preferentially or exclusively in the nucleus that contains eas^UCLA:

The asci containing newly arisen mutant cya-8 alleles in the experiment just described originated from a cross in which markers linked to cya-8 were segregating. If induction of mutations was equally probable in the two nuclear components of the heterokaryon, eas^UCLA a and adh nic-3 A, then linkage of cya-8 to the nearby markers would be obscured. Results clearly indicated the contrary (Table 4A). The new cya-8 mutations were closely linked in cis to the nic-3 allele that was present in LG VII in the eas^UCLA parent. The progenitor nuclei of these asci must have been eas^UCLA; cya-8 adh⁺ nic-3⁺ and eas⁺; cya-8⁺ adh nic-3. The cya-8 and nic-3 markers segregated so as to give 10 parental ditypes, 0 nonparental ditypes, and 3 tetratypes (12% recombination), as expected for this coupling phase. Induction of cya-8 mutations is therefore restricted to nuclei that contain the mutant eas^UCLA allele.

Further evidence that mutation occurs preferentially in the eas^UCLA nuclei was obtained using random ascospore isolates from crosses heterozygous for VIIL markers (Table 4B). The newly arisen cya-8 alleles consistently showed linkage in cis phase to the marker allele that was present in the eas^UCLA nucleus.

Mutation of cya-8 in eas^UCLA strains is suppressed by large segmental duplications:

Bhat and Kasbekar (2001) have shown that when both a large duplication and a small duplication are present in a cross, RIP is suppressed in the small duplication, suggesting that the two duplications compete for access to the RIP machinery. Suppression is stronger when the large duplication is homozygous than when it is heterozygous (Fehmer et al. 2001). The hypothesis that cya-8 mutations are due to RIP was tested by crossing eas^UCLA with two strains that contain long segmental duplications. Although crosses are barren when these duplications are present, they are not completely unproductive and a few ascospores are produced. The frequency of transparent progeny is drastically reduced in crosses with eas^UCLA that are heterozygous or homozygous for either of the large duplications (Table 5). This is as would be expected if RIP is responsible for the production of cya-8 mutations by eas^UCLA.

Mutation of cya-8 in eas^UCLA strains is suppressed in the presence of a mutation that abolishes RIP:

Discovery of the recessive RIP-defective gene rid (Freitag et al. 2002) provided a good opportunity to test the hypothesis that the cya-8 mutations in transparent progeny are caused by RIP. If RIP is responsible, then crosses homozygous for rid and homo- or heterozygous for eas^UCLA should show reduced numbers of transparent progeny, or none at all. In accordance with this expectation, the frequency of transparent progeny was reduced in crosses homozygous for rid (Table 6). However, some progeny that showed the slow, transparent growth typical of cya-8 mutations were still produced. Most of these were laggards, reverting to wild type during vegetative growth, whereas most transparent progeny from control crosses were stable. Recognition that transparent progeny are of two types, stable and unstable, suggested that the unstable type was caused by something other than RIP, which introduces C:G to T:A changes that are highly stable. It seems unlikely that the unstable transparents were due to quelling, a silencing process that silences the expression of duplicated genes reversibly during the vegetative phase. As shown above, mutations to cya-8 were not detected during vegetative growth of eas^UCLA.

Mutation of cya-8 in eas^UCLA strains is not suppressed by a mutation that suppresses meiotic silencing by unpaired DNA:

Meiotic silencing by unpaired DNA (MSUD) results in epigenetic inactivation of genes that are unpaired in meiotic prophase (Aramayo and Metzenberg 1996, Shiu et al. 2001; Shiu and Metzenberg 2002). Silencing is temporary: MSUD is not known to produce stable mutations. We used a suppressor of MSUD called Sad-2 (Suppressor of ascus dominance) (Shiu et al. 2006) to show that MSUD is not involved in the production of stable transparent progeny (Table 6).

Although observations of silencing by MSUD are usually limited to genes that are expressed during the sexual phase, the possibility remains that, for some genes, function may not be restored until after ascospore germination. Further experiments will be needed to determine whether MSUD is responsible for the occasional transiently inhibited “laggard” progeny that have been classified as transparent but that are subsequently seen to revert to normal growth.

Effectiveness of eas^UCLA as a mutator after repeated exposure to RIP:

Cambareri et al. (1991) showed that the frequency of RIP decreased after duplicated segments had passed through successive crosses that subjected them repeatedly to alteration by RIP. After many generations, RIP-induced divergence was lower for unlinked duplications than for linked duplications To see whether the mutator effect of eas^UCLA declined in similar fashion, numbers of cya-8 progeny were determined after eas^UCLA had been exposed repeatedly to RIP. The eas^UCLA allele was tagged with fl^DE1, with which it does not recombine. eas^UCLA fl^DE1 was crossed with fl⁺ trp-3 to obtain a prototrophic transparent F₁ progeny of constitution eas^UCLA fl^DE1; cya-8. This, in turn, was crossed with fl⁺ trp-3 to obtain nontransparent eas^UCLA fl^DE1 backcross progeny. These were designated eas^{RIP 1} fl to signify that the eas allele had been altered by one round of exposure to RIP. The cycle was repeated eight times to obtain eas^{RIP 8} fl. When this eight-times exposed strain was crossed by wild type, 14 of 123 progeny (11%) were stably transparent, a frequency unchanged from the 14/129 (11%) transparents obtained among progeny of the parental, unexposed eas^{RIP 0} fl × wild type. Thus, mutator activity of eas^UCLA appears not to have been impaired by repeated exposure to RIP.

Cytogenetic complexity of the eas^UCLA mutation:

The point-mutant allele eas^JD was used to determine standard wild-type gene order in LG IIR. In a cross of un-20 eas^JD × ace-1, 3 progeny among 290 were recombined for ace-1 and eas. Two of these crossovers were un-20 ace-1⁺ eas⁺ and one was un-20⁺ ace-1 eas. Gene order is therefore un-20 ace-1 eas, with the eas locus right of ace-1 (Figure 1).

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Figure 1.—

Crossing-over frequencies between IIR markers in crosses of eas⁺ × eas⁺ (above the map) and eas⁺ × eas^UCLA (below the map), based on data in Tables 7 and 8. When eas^UCLA is homozygous, the order of fl and trp-3 is reversed and recombination values are ace-1 (16%) and trp-3 (11%) fl, with no double crossovers among 73 progeny, and un-20 (16%) and trp-3 (12%) fl, with no double crossovers among 76 progeny (Table 8). Markers: arg-12, ace-1, trp-3 (auxotrophs); fl: fluffy (morphological); un: unknown function (temperature sensitive conditional); rip-1: ribosome production (temperature sensitive conditional).

When eas^UCLA is heterozygous, recombination between IIR markers right of ace-1 is blocked in a region that is normally 25 MU long (Figure 1; Table 7). Crossing over left of ace-1 remains normal. These results suggest that a distal segment of IIR with one breakpoint at or near eas became inverted at the same time that the UCLA191 mutation occurred at the eas locus. Simultaneously, a copy of cya-8 might then have been inserted at one of the inversion breakpoints.

Direct evidence of inversion would be provided by crosses that are homozygous for the rearrangement and heterozygous for at least three markers—two inside the putative inverted segment and one outside. Strains were available in which eas^UCLA was recombined with the outside markers ace-1 and un-20, which are left of both eas and the recombination block. No recombinants were obtained when eas^UCLA was crossed with the rightmost linkage group II markers un-15 or rip-1, which may or may not be included in the inversion. We succeeded in inserting the included marker trp-3 by recombination. The fl (fluffy) gene is positioned to provide a second included marker. Because fluffy strains are aconidiate and fl eas cannot be distinguished phenotypically from fl eas⁺, a mutant fl allele could not be introduced by recombination. Instead, fluffy was introduced into eas^UCLA by RIP. The wild-type fl⁺ gene had been cloned and sequenced by Bailey and Ebbole (1998). Transformation was carried out using a plasmid that contained both fl⁺ and a selectable marker conferring hygromycin resistance, as described in materials and methods. A purified hygromycin-resistant transformant of eas^UCLA was crossed with an eas^UCLA strain of opposite mating type. Because transformation was by ectopic integration, the transformed parent carried two copies of fl⁺. Consequently, its progeny included RIP-induced fluffy mutations. A hygromycin-sensitive eas^UCLA fluffy strain from among the progeny was crossed to un-20 eas^UCLA trp-3 and to ace-1 eas^UCLA trp-3. The results (Table 8) show clearly that the order of fl and trp-3 is reversed in eas^UCLA. We conclude that the eas^UCLA mutation resulted from a complex rearrangement, which might be symbolized as T(VIIL→IIR) In(IIR)UCLA191 eas.

Cytological observations of meiosis:

Meiotic chromosomes were examined in asci heterozygous for eas^UCLA. If the relatively inverted segments were paired homologously, a loop might be seen at pachytene, and crossing over within the inversion would result in the formation of anaphase bridges and fragments. No obvious abnormalities were seen. Pairing appeared to be normal, and bridges and fragments were not apparent in preparations made using aceto-orcein (E. G. Barry, personal communication) or DAPI (N. B. Raju, personal communication). Bojko (1990) has used synaptonemal complex reconstructions to demonstrate that synaptic adjustment occurs in Neurospora when long inversions are heterozygous, resulting in extensive nonhomologous pairing. Thus, it might be expected that small inversions would be paired nonhomologously at late pachytene and that they would therefore go undetected when bivalents were examined using light microscopy.

Molecular evidence for RIP of the cya-8 copy that adjoins eas^UCLA:

Molecular comparisons based on restriction fragment length polymorphisms (RFLPs) detected by Southern hybridizations between eas^UCLA and its progenitor strains (Bell-Pedersen et al. 1992; Lauter et al. 1992) suggested that the eas allele is associated with an insertion of at least 2 kb, probably interrupting promoter elements required for normal transcription levels (Bell-Pedersen et al. 1996). As described above, we suspected that transposed, duplicated cya-8 sequence is part of this insertion. The molecular identity of the cya-8 gene remains unknown, even though the Neurospora genome is almost completely sequenced (Galagan et al. 2003) and many of the identified Saccharomyces or human genes involved in cytochrome synthesis have homologs in Neurospora. Cloning the cya-8 gene by complementation was a poor option because cya-8 strains grow poorly and form few asexual spores, making them difficult to transform. Instead, we attempted to isolate part of the cya-8 gene integrated at the eas locus. Standard long-range PCR with outside primers has failed. Alternative approaches (e.g., inverse PCR from genomic DNA isolated from eas^UCLA strains or construction of partial plasmid libraries with gel-purified 5.3- and 8-kb Sau3AI fragments) were also attempted repeatedly but did not yield the desired breakpoint fragments.

To provide molecular evidence for the hypothesis that the transparent progeny from eas^UCLA crosses are caused by RIP of cya-8, we assayed for DNA methylation in the eas region. While point mutations induced by RIP are usually contained within the duplicated segments, DNA methylation frequently spreads outside the duplication and has been found thousands of base pairs from the duplicated region (e.g., see Foss et al. 1991; Irelan and Selker 1997; Miao et al. 2000). We therefore reasoned that if cya-8 mutations are caused by RIP, DNA methylation may be present in the eas^UCLA promoter region. We inspected the eas promoter region for telltale RFLP and/or DNA methylation by Southern analysis using the 5-methylcytosine-insensitive DpnII and its 5-methylcytosine-sensitive isoschizomer, Sau3AI (Figure 2; data not shown). In eas^UCLA strains, probes B and C revealed RFLPs when DNA was digested with DpnII. A 395-bp band present in the wild type was replaced by an ∼480-bp band. Both eas⁺ and the nonmutator eas^JD alleles showed the expected 395-bp band as well as a 406- or 441-bp band for probes B and C, respectively. Both the 406- and 441-bp fragments appeared unchanged in the eas^UCLA strain. This suggests that both the insertion containing cya-8⁺ and the inversion breakpoint are present in the 395-bp interval (shown as a shaded box in Figure 2A). Previous molecular analyses (Bell-Pedersen et al. 1992; Lauter et al. 1992) suggested that eas^UCLA contains an insertion that interrupts important promoter elements (Bell-Pedersen et al. 1996; Rerngsamran et al. 2005). Induction of eas during conidiation requires the fluffy transcription factor FL. The strongest binding site for FL in the eas promoter lies within the 395-bp interval, 129 bp from the 3′-end of the fragment. This binding site is important for eas expression in vivo (Rerngsamran et al. 2005). We predict that one end of the insertion/inversion lies within this 129-bp interval, separating the FL-binding site from eas.

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Figure 2.—

Molecular analysis of the eas^UCLA allele. (A) Partial map of the eas⁺ gene and its transcript (arrow). Selected restriction endonuclease sites that were used in previous studies are indicated (Bell-Pedersen et al. 1992; Lauter et al. 1992). DpnII/Sau3AI sites are shown as vertical marks below the horizontal line, the size of selected fragments expected in the Southern blots in B is noted, and the position of probes A–E used for B is shown. Primers used in this study (see materials and methods) are shown as inverted flags with arrow points to indicate orientation. The shaded segment shows the region of discontinuity between normal (open) and mutated (solid) DNA in eas^UCLA. (B) Southern analysis of the proximal eas^UCLA region. Genomic DNA of wild-type strain 74-OR23-IVA (WT), eas^JD (JD), and eas^UCLA (eas) was digested with DpnII and Sau3AI; Sau3AI is sensitive to cytosine methylation. Only probes B and C detect higher-molecular-weight bands in DNA from the eas^UCLA strain, presumably caused by DNA methylation (note the concomitant disappearance of the ∼480-bp band in the eas Sau3AI lanes). Wild-type strains show no DNA methylation throughout the region tested. The eas^JD allele is methylated in the eas coding region because this allele was generated by RIP (Bell-Pedersen et al. 1992). These data suggest that the insertion/inversion occurred in the 395-bp segment shown in A and that this segment likely contains both cya-8⁺ insertion breaks and one eas^UCLA inversion breakpoint. Therefore DNA to the right of the solid box in A is inverted distally toward the telomere in eas^UCLA.

Both probe B and probe C detected Sau3AI fragments of ∼5.3 and ∼8 kb (Figure 2B). These high-molecular-weight bands presumably stem from methylation of Sau3AI sites contained in the insertion that bears cya-8⁺. The insertion is apparently heavily methylated and is perhaps longer than previously suggested (Lauter et al. 1992). We did not detect any methylation in the eas^UCLA region outside of that corresponding to the wild-type 395-bp band (probes A, D, E; Figure 2). Analyses of strains from earlier eas^UCLA × wild type crosses revealed bands resulting from RFLPs and incomplete methylation (data not shown), suggesting that the severity of DNA methylation and RIP increased in successive generations.

The cause of recurrent mutation:

The observations reported here support Selker's (1990) hypothesis that existing strains of eas^UCLA contain two copies of the cya-8⁺ gene, one at its original location in LG VIIL and the other linked to eas in IIR. This model predicts that mutant cya-8 progeny will be produced by RIP whenever the duplication-bearing eas^UCLA strain is present in one or both parents of a cross. The original UCLA191 mutation is visualized as a four-break complex rearrangement in which cya-8⁺ was removed from VII and inserted in II at one of the breakpoints of a paracentric inversion that disrupted eas (see Figure 3). About one in five translocations in Neurospora is insertional rather than reciprocal, and complex, multiple-break rearrangements are not uncommon (Perkins 1997).

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Figure 3.—

Model for the coupled insertional translocation/inversion event that produced the eas^UCLA mutation and for the production of cya-8 mutations by RIP. (A) Partial map of normal-sequence LGs II and VII with putative breakpoints, showing presumed double-strand breaks (lightning bolts), the approximate extent of the inverted segment in LG IIR (solid box), and the deleted segment in LG VIIL (shaded box). Because no crossovers have been obtained between the rearrangement and the rip-1 or un-15 loci, it is not known whether these genes are inside or outside the inverted segment. (B) Generation of the eas^UCLA mutation by a combined insertional translocation and inversion. Chromosome constitution following the coupled translocation/inversion. A fragment of LG VIIL harboring cya-8 was translocated to LG IIR and inserted centromere proximal of the eas promoter, resulting in deletion of cya-8 from LG VIIL (cya-8^Δ) and its ectopic insertion into LG IIR (cya-8^ec). Concomitantly, a segmental inversion of LG IIR inactivated eas, moving the coding region closer to the telomere. The exact extent of the translocated fragment and of the inversion is not known, nor do we know if small deletions occurred at sites of the presumed double-strand breaks (lightning bolts). Evidence that the eas locus is split is provided by our inability to amplify fragments spanning the eas promoter and the 5′ region of eas, using eas^UCLA or strains derived from it. (C) Constitution of a “transparent” backcross progeny that is mutant for cya-8. In a backcross of the eas cya-8 mutant to a wild-type strain, one-half of the mutant eas progeny are expected to be duplicated for cya-8⁺, having received one copy in an intact LG VII from the wild-type parent and carrying a second copy (cya-8^ec) in the rearranged LG II from the mutant eas^UCLA parent. Duplication of the cya-8 region results in premeiotic C:G to A:T mutations by RIP in both segments. During a backcross of one of these eas cya-8 duplication stocks, both cya-8 copies were mutated by RIP (cya-8^RIP and cya-8^ecRIP, crosshatched boxes), producing the chromosomes diagrammed here. DNA methylation is usually, but not always, found in segments mutated by RIP (for review see Selker 1990). Therefore, we expect that in many progeny both of the cya-8^RIP copies are methylated. Spread of methylation from the ectopic cya-8^ecRIP region into the adjoining promoter region of eas (short arrow) was detected by Southern analysis (see Figure 2), strongly suggesting that RIP is responsible for the recurrent mutations at cya-8.

The original UCLA191 mutant strain was not saved. Instead, eas progeny of repeated backcrosses to the standard Oak Ridge wild types were retained as stocks (Selitrennikoff 1976). Backcrossing would have replaced the original deleted donor chromosome with a wild-type copy of linkage group VII. As a result, two copies of cya-8⁺ are expected to be present in each nucleus in the eas^UCLA stocks that were retained and that were used in this study.

Predictions for RIP:

Mutations originating by RIP are expected to be stable. If the eas^UCLA-induced stable cya-8 mutations are in fact caused by RIP, testable predictions can be made:

1. cya-8 mutations should be abolished or decreased in frequency in crosses homozygous for the recessive RIP-deficient mutation rid (Freitag et al. 2002).

2. The frequency of stable cya-8 mutant progeny may be decreased when eas^UCLA is crossed to a partial-diploid strain that contains a long nonhomologous duplication. This prediction is based on the results of Bhat and Kasbekar (2001), who suggested that a long duplication out-competes a small duplication for the RIP machinery when both duplications are present in a cross. The competition occurs in dikaryotic ascogenous hyphae prior to meiosis, and it is effective regardless of whether the long and short duplications are in the same nucleus or in separate nuclei.

3. The ability of a duplicated DNA segment to trigger RIP might be expected to decrease as it is passed through successive crosses that subject it to RIP. The repeated exposure would subject it repeatedly to alteration, making it less effectively recognized by the RIP machinery (Cambareri et al. 1991).

The first two predictions have been confirmed (Tables 5 and 6). However, we saw no reduction in the frequency of stable transparent progeny after eight successive crosses in which eas^UCLA was subjected to RIP. This is perhaps not surprising, because the duplicated sequences in question are unlinked, and RIP was shown by Cambareri et al. (1991) to continue through seven generations when an unlinked duplication of an ∼6-kb sequence was passed repeatedly through crosses. We did note an increase in the size of methylation fragments from the eas^UCLA promoter region when comparing progeny from early vs. later eas^UCLA crosses (data not shown). This suggests that RIP continued to occur, resulting in more extensive DNA methylation in successive crosses.

Other possible explanations for the mutator effect:

Explanations other than RIP have been considered. Experimental results are inconsistent with origin of the stable, heritable cya-8 mutations by any of the following: quelling, meiotic silencing of unpaired DNA, mitochondrial mutation, transposition of mobile elements, or deletion at meiosis in heteroallelic repeats.

1. Quelling might be evoked to account for those transparent isolates that are unstable. Quelling, which inactivates duplicated genes during the vegetative phase (Romano and Macino 1992; Cogoni et al. 1996; Cogoni and Macino 2000), is usually detected in isolates that acquire ectopic insertions, typically with multiple copies of the sequences, following transformation. Epigenetic alterations due to quelling are unstable, in contrast to the stability of eas^UCLA-induced cya-8 mutations. We failed to find mutations to cya-8 in vegetative cultures from eas^UCLA conidia. To be responsible for the unstable transparent germinants produced by eas^UCLA, quelling would need to occur preferentially at the time of ascospore germination, which seems highly unlikely.

2. MSUD is transitory and is not known to produce stable mutations (Shiu et al. 2001). Our experiments with the Sad-2 suppressor revealed that MSUD is not involved in the production of stable transparent progeny (Table 6).

3. Loss of mitochondrial function is not a tenable explanation. Although the phenotype of the transparent strains resembles that of mitochondrial respiratory mutants produced by a mutator described in Saccharomyces by Evans and Wilkie (1978), the eas^UCLA-induced transparent progeny in Neurospora are due to the single Mendelian mutation cya-8, which is transmitted to progeny regardless of whether the eas^UCLA parent is male or female. Mitochondrial inheritance is strictly maternal in Neurospora, through the protoperithecial parent. The mitochondrial genome of the fertilizing parent is not transmitted through crosses (Mitchell et al. 1953; Mannella et al. 1979).

4. A mobile element is probably not involved. Only one active transposable element is known in any strain of Neurospora, the retrotransposon Tad (Kinsey and Helber 1989). Tad proliferates in vegetative cultures, but when strains are crossed, the copies are inactivated by RIP (Kinsey et al. 1994). Active Tad elements are absent in the wild-type strains used in this study.

The recurrent appearance of cya-8 mutations in crosses with eas^UCLA is superficially similar to what is seen with the Dotted mutator in maize, which produces red dots on an unpigmented background in the kernel (Rhoades 1948). Dotted was shown by McClintock (1950) to be identical to the controlling element Activator (Ac), and the induced gain of anthocyanin pigmentation in the mutant areas is due to relief of inhibition by excision of the nonautonomous transposable element Dissociator (Ds) from the a₁ locus. Ds moves only when transposase is provided by Ac, and this is precisely timed during development. Like eas^UCLA and cya-8, Ds and Ac show locus specificity for the target gene. Suggestive examples of transposon-induced recurrent mutation have been found in the Discomycete Ascobolus immersus, with instability of ascospore color attributed to a mobile element that is excised premeiotically (Decaris et al. 1981; Nicolas et al. 1987). No active two-element DNA transposable element system has been identified in Neurospora, however.

• 5. Deletion resulting from intrachromosomal recombination cannot be responsible for the cya-8 mutations. Behavior of eas^UCLA differs in several respects from that of buf1, a mutable gene in another Pyrenomycete, Magnaporthe grisea, with which Chumley and Valent (1990) and Farman (2002) have described a process called meiosis-associated deletion in heteroallelic repeats (MDHR). In certain crosses, 5–25% of progeny have acquired new buf1 mutations. These are all deletions that result from intrachromosomal recombination. Ascus analysis shows that, when deletion occurs, both chromatids of a chromosome are usually affected, indicating that the mutations occur prior to premeiotic DNA replication. Unlike other phenotypically normal buf1 alleles that are stable, the unstable buf1 allele contains numerous repetitive elements. The MDHR mutations occur predominantly in heteroallelic crosses, suggesting that failure of pairing may promote the recombination event that leads to deletion.

The detection of inversions:

The eas^UCLA strain analyzed here provides the first well-studied example of a paracentric inversion in Neurospora. The only other documented case is an ∼20-kb inversion detected by DNA sequencing (Micali et al. 2001; Micali and Smith 2006).

Most of the known chromosome rearrangements in Neurospora have been detected because inviable, unpigmented ascospores are produced when the rearrangement is heterozygous. The defective ascospores result from deficiencies that are generated by meiotic assortment and recombination. Unpigmented, aborted ascospores are produced abundantly by heterozygous reciprocal and insertional translocations. Long pericentric inversions also generate enough deficiency ascospores to be recognized in this way (see, for example, Newmeyer and Taylor 1967; Turner et al. 1969; Barry and Leslie 1982; Turner and Perkins 1982). The method fails to detect paracentric inversions, however. Thus, the eas^UCLA inversion, which is unrecognizable by the criterion of aborted-ascospore production, was discovered only because progeny that have undergone crossing over in a segment adjoining the eas locus are absent when eas^UCLA is heterozygous. The inversion was confirmed by showing that gene order is reversed and crossing over is no longer blocked when the mutant is homozygous (Table 8).

Synaptonemal-complex reconstructions (Bojko 1990) and orcein-stained squashes (Barry and Leslie 1982) have been used to demonstrate that heterozygous long pericentric inversions pair homologously, forming loops at pachytene. Synaptic adjustment follows, leading to disappearance of the loop (Bojko 1990). When the inverted segment is short, homologous pairing may not occur or may not be cytologically visible. Even if pairing occurs, crossing over may be too infrequent to produce enough defective ascospores to distinguished from the 5 or 10% noise level that is characteristic of crosses between highly inbred, presumably isosequential strains. Perkins and Barry (1977) have speculated that, even if pairing were effective and single crossovers occurred with an appreciable frequency in a heterozygous paracentric inversion, the dicentric bridges that were produced might result in the death and resorption of all asci. Their loss would preclude the production of enough unpigmented ascospores to provide a signal for detection.

Short inversions and other small rearrangements were shown to be at least as common as gross rearrangements in differentiating the genome of Saccharomyces cerevisiae from that of Candida albicans (Seoighe et al. 2000). It is reasonable to suggest that paracentric inversions may be present as undetected genetic polymorphisms in laboratory stocks and wild populations of Neurospora.

Complex rearrangements:

If our diagnosis is correct, eas^UCLA originated as a combination paracentric inversion and insertional translocation (Figure 3). The rearrangement had four breakpoints, one of which is likely shared between the inversion and the translocation.

Rearrangements with four or more breakpoints are not uncommon in Neurospora. Their presence is readily recognized when multiple linkage groups are involved, because <50% of ascospores are viable in crosses where a multibreak complex rearrangement is heterozygous. Genetic analysis of multibreak rearrangements is laborious. For this reason, most putative complex rearrangements have been set aside without being investigated further. A few illustrative examples have been thoroughly analyzed, however. Among these are In(IL;IR)T(IL;IIIR)SLm-1, in which an inversion and a reciprocal translocation have one breakpoint in common (Barry 1992); T(IVR → VIIL;IL;IIR;IVR)S1229, in which insertional and reciprocal translocations share a common breakpoint (Barry 1960); and Tp(IR→IL)T54M94, an inverted insertion having multiple breaks in the same chromosome (Perkins et al. 1995). Other complex Neurospora rearrangements are described by Perkins (1997).

A four-break rearrangement in the laboratory mouse resembles what we infer to have occurred with eas^UCLA: Rearrangement Is(17;In2)1Gso has a segment of mouse chromosome 17 inserted at one of the breakpoints of a chromosome 2 inversion (Beechey and Evans 1996; Lyon et al. 1996). If Neurospora conventions were used, this mouse rearrangement would be symbolized as In(2)T(17→2)Gso. Complex rearrangements in other eukaryotes may be far more common than has been revealed by classical cytogenetic methods (Savage 2002).

Nomenclature:

By convention, the hypothesized original eas rearrangement would be symbolized as In(IIR)T(VIIL → IIR)UCLA191 eas. In the derived strains that were retained and that were used here, the original linkage group VII donor with its deficiency has been replaced by a normal-sequence chromosome. Their genotype would be symbolized In(IIR)Dp(VIIL → IIR)UCLA191 eas. Extent of the inserted segment is unknown and could be as short as a single gene. In practice, the insertion is cryptic and can usually be ignored. In contrast, effects of the inversion on recombination in LG IIR are clearly manifested, especially when it is heterozygous.. Existing strains can be used as though the eas^UCLA191 mutation were a simple paracentric inversion, and this may be useful not only for investigating the behavior of paracentric inversions but also as a balancer or crossover suppressor. We propose to adopt the shortened symbol In(IIR)UCLA191 eas for the rearrangement called eas^UCLA in this article.

The eas^UCLA-cya-8 system as a model:

The experiments reported here support the following hypothesis: When a gene (the “donor”) is inserted ectopically at the locus of another gene (the “recipient”) and the recipient gene is simultaneously mutated, the mutant complex then has the potentiality of acting as a locus-specific mutator of a wild-type donor gene. Mutation results from RIP and it will occur in crosses where a second copy of the donor gene is present in its normal position in the same nucleus with the ectopic mutator complex. The mutator appears to target the normal allele of the donor gene. The eas^UCLA strain has provided the first example of this behavior, with a copy of the cya-8⁺ gene from linkage group VII inserted at the eas locus in linkage group II and with cya-8⁺ present at the original locus in linkage group VII (Figure 3). Allele eas^UCLA, which in reality is a fused complex, eas cya-8^(EC), thus appears to act as a mutator that is specific for cya-8⁺.

Mediation of locus-specific mutation in this way is probably not limited to eas^UCLA, where it happened to be discovered. When one parent in a cross has the “target” gene transposed and inserted at the locus of the putative “mutator,” genes other than eas and cya-8 should be capable of behaving as mutator/target pairs in Neurospora. Transposition of the target sequence may be induced or may occur spontaneously. Segmental rearrangements that involve transposition and ectopic insertion of essential genes are not uncommon in Neurospora, where they are readily recognized (Perkins 1997). Ectopic insertion of transforming DNA sequences is typical in Neurospora. Now that DNA sequences and the necessary molecular-genetic tools are available, it should be possible, with eas^UCLA-cya-8 as an example, to construct other mutator systems using any two well-characterized loci. A locus-specific mutator system of this type might well involve only a simple single-gene insertion without any additional complexity such as the IIR inversion in eas^UCLA, which can be considered a fortuitous distraction.