ALLELIC RECOMBINATION in NEUROSPORA: TETRAD ANALYSIS of a THREE-POINT CROSS WITHIN the Pan-2 LOCUS1

ALLELIC RECOMBINATION IN NEUROSPORA: TETRAD ANALYSIS OF A THREE-POINT CROSS WITHIN THE pan-2 LOCUS1

MARY E. (CASE AND \NORMAN H. GILES

Department of Biology, Josiah Willard Gibbs Research Laboratories, Yale University, New Haven, Connecticut Received October 28, 1963

HE analysis of tetrads from crosses between allelic mutants at the pan-2 locus in Neurospora crassa has provided considerable information concern- ing allelic (intragenic) recombination mechanisms ( CASEand GILES1958a,b). Previous investigations involved either a cross of two single mutants or a cross of a double mutant with wild type. The general results of those studies can be summarized as follows: (1) Recombination events within the pan-2 locus are either apparently reciprocal, or clearly nonreciprocal (giving atypical segregations), the latter type being much more frequent than the former. (2) The evidence indicates that the great majority of such nonreciprocal tetrads cannot be explained on the basis of orthodox although unusual chromosome behavior, such as heteroploidy, since segregation for adjacent markers is normal in most such tetrads. (31 Atypical segregations have been largely 3:l (6:2) types, only one 4: 0 (8: 0) segregation having been obtained and this involved an allele known to be capable of reverse mutation. The original studies, however, were not designed to detect 5:3 types, but on the basis of the results of the three-point cross only one or two asci of this type would have been expected. (4) In tetrads where mutant or wild-type alleles are represented more than the expected number of times, such alleles have so far proved indistinguishable from parental ones on the basis of mutation, recombination, and complementation tests. (5) Atypical segregations may involve either one allele or both alleles simultaneously. The mechanism suggested to explain these instances of so-called “gene conversion” is one involving copy-choice, such that a miscopying event may involve one or the other or both regions of the pan-2 locus. (6) The data were interpreted as indicating that there is a correlation between the occurrence of conversion at the pan-2 locus and recombination between marker genes located on either side of the locus. In order to obtain additional information on allelic recombination mechanisms, further studies have been made of crosses in which it is possible to follow the behavior in tetrads of three pan-2 alleles segregating simultaneously. Two of these alleles are located near opposite ends of the locus and the third one is near the middle. Hence, it has been possible to obtain further evidence relative to the occurrence of intra-locus copy-choice events and to determine whether such

1 This research has been supported in part under contracts AT(30-1)-872 and AT(30-1)- 3098 with the Atomic Energy Commission.

Genetics 49: 529-540 March 1964 530 M. E. CASE AND N. H. GILES events affect one, two, or three alleles individually or simultaneously. Certain of these results have already been described briefly ( GILES1963).

MATERIALS AND METHODS

Three pan-2 alleles (designated B23, B36, and B72) have been employed in the present studies. These are all complementing pan-2 mutants (pantothenic-2, requiring pantothenic acid) which have been located previously on both the complementation and recombination maps of the locus (CASEand GILES1960). In the cross used, a double mutant (B25B36) composed of two single mutants located near opposite ends of both the recombination and complementation maps was crossed with a single mutant (B72) located near the middle of both maps (Figure 1). In asci, individual alleles can be identified alone or in combination by means of complementation, recombination, phenotypic, and mutation tests. In addition, outside marker genes located on either side of the locus have been employed such that segregation and recombination can be followed simultaneously for these markers as well as for the mutant alleles. Crosses were made on Westergaard and Mitchell's synthetic crossing medium supplemented with 2 pg calcium pantothenate/ml, 37.5 pg L-tryptophan/ml, 50 pg adenine sulfate/ml, and 10 pg nicotinamide/ml. Serial isolations were made on a 5 percent agar block treated with 50 percent Clorox solution. Dissected ascospores were transferred to tubes of supplemented Fries minimal medium and incubated at 25% for one week before shocking in a 60°C water bath for 30 minutes. The genotypes of individual isolates from all asci were determined subsequently on ap- propriately supplemented Fries minimal media. The genotype of the cross was: ylo '(yellow, Y30539y), ad-l (adenine-requiring, 3254), B23-B36. a (mating type) x B72, tryp-2 (tryptophan-requiring, 75001), A (mating type). These tetrad data were obtained from two crosses (604 asci in the first cross, 853 asci in the second cross) in which the two parents were identical in genotype with the one exception that a morpho- logical marker del (delicate) was in the B23-B36 strain used in the first cross. A description of the origin of the pan-2 strains and the method of plating analysis have been published previously

COMPLEMENTATION MAP of the PAN-2 LOCUS

1 Illtlml~lPI~I 5 3

23 72 36

GENETIC MAP 23 5 3 72 36 I I I I

% PROTOTROPHS in TOTAL VIABLE SPORES .OOl .I4 .04 .I3

LINKAGE GROUP RECOMBINATION VALUES

5x3 5-3xWT YLO 1.1 AD 0.2CENT. 1.7 PAN 7.7 TRYP

I- 8 8) I 23-36 172 3.1 0.5 5.3 14.7

FIGURE1.-Complementation and genetic maps of the pan-2 mutants used in the 2-point and 3-point crosses. The recombination values of the outside markers with the pan-2 locus are given for 2-point crosses above the line and for the 3-point cross below the line. ALLELIC RECOMBINATION 531

(CASEand GILES1960). The complementation tests were done in the manner described by DE SERRES( 1956). In the majority of the asci all eight spores germinated and all isolates were analyzed. Initially all isolates were tested in a supplemented medium lacking pantothenate in order to detect the B72 allele which grows slightly in two to three days. The presumed double mutants were then tested for their complementation responses with the single mutants B23 and B36 in both mating types. In this manner any deviation from the normal 4:4 segregation for the two parental types could easily be detected. The complementation responses of all single and expected double mutant combinations are shown in Figure 2. The order of the mutants on the complementation map as well as on the genetic map is B23-B72-B36 with all single mutants complementing well in any pair-wise combination. All three double mutants complement with the third single mutant. The B23-B72 heterocaryon with B36 and the B72-B36 heterocaryon with B23 grow at a normal rate; however, the heterocaryon between double mutant B23-B36 (the two outside alleles) and B72 (the middle allele) grows very slowly, as indicated by the 2 response on the complementation matrix. Tests for this type of response are best noted by plating crosses and looking for pseudo-wild type formation, since the growth rate of the heterocaryon on a liquid medium is scarcely distinguishable from the B72 isolate alone. The following tests were used to characterize all isolates in the exceptional asci: (1) Crosses were made of the presumptive single mutant types, B23 and B36, as well as presumptive double mutant isolates B23-B36, to both B23 and B36. Presumptive B72 isolates were crossed to wild type and checked for the presence of B72. Selfings of B72 are difficult to analyze because of the leaky growth habit of B72 alone on a minimal medium. (2) Reverse-mutation tests of presumptive double mutants were made with ultraviolet treatment. All single mutants revert spontaneously and following ultraviolet treatment (CASEand GILFS 1958b and unpublished), while double mutants are stable. (3) Conidial platings were made of presumptive single mutants, and isolates from such platings were checked by heterocaryon tests with B23 and B36. This last procedure was necessary to test for the initially unexpected possibility (which became apparent in the course of the tests of exceptional asci) that cultures derived from single ascospores could be heterocaryotic. Conidial platings permit the detection of such heterocaryons, since segregation of different nuclear types can occur in the formation of those macroconidia which are uninucleate and give rise to homocaryotic cultures. Without such conidial platings various types of possible heterocaryons would be difficult to detect. For example, the leakiness of B72 on minimal makes difficult the detection of a true heterocaryon response between B72 and B23-B36. An isolate originally characterized as B72 could also contain B23-B36 nuclei which would remain undetected. Furthermore, the double mutant B23-B36 could be carried undetected in isolates originally characterized as B36 or as B23. All cultures found to be heterocaryotic by conidial platings were also crossed to wild type. Isolates from these crosses were then tested for their heterocaryon responses with B23 and B36.

RESULTS AND DISCUSSION Segregations for the three pan-2 alleles and their adjacent marker genes were

23 72 36

23-72

FIGURE2.-Complementation matrix of B23, B72, and B36 as single and double mutant roni binations. + = a positive response, & = a weak response, and - = a negative response. 532 M. E. CASE AND N. H. GILES analyzed in a total of 1457 asci. A number of hfferent categories of interest with respect to recombination mechanisms were detected and each of these general categories will now be discussed. Asci producing only homocaryotic cultures: Of most significance for the present study were those asci in which there was evidence that a recombination event had occurred within the pan-2 locus and that this event had given rise to asci in which all eight ascospores produced only homocaryotic cultures. Thirteen such asci were obtained, of which the complete genotypes are given in Table 1. In two of these 13 asci, 5:3 or 3:5 segregations were present for certain alleles. A discussion of these types will be deferred until later. The remaining 11 asci in this group are characterized by segregation patterns which indicate either apparent reciprocal recombination at the four-strand stage between two alleles giving in a single ascus each parental type plus two new recombinant types (two asci-Nos. 98 and 71 0), or nonreciprocal recombination resulting in segregations for one or more allelic pairs. The absence of 8: 0 segregations is noteworthy. It is also clear that the majority of recombination events are clearly nonreciprocal and that such asci are characterized by a restricted number of allelic segregation patterns. In individual asci, 6:2 (or 2: 6) segregations may involve single allelic pairs, two adjacent pairs, or all three pairs simultaneously. No instances of simultaneous 6: 2 segregations at two nonadjacent allelic sites were found (i.e., for B23 and B36, but not B72). No case was noted of two simultaneous apparent reciprocal recombination events. In all asci, all tests (complementation, mutation, recombination) used to characterize segregating cultures indicated the presence, in either the original parental or in new recombinant combinations, of only the original three types of pan3 alleles entering the cross (i.e., no new types of alleles clearly distinguishable from the original ones were detected by the tests employed). These results are in agreement with previous ones obtained in crosses involving two other alleles (B3 and B5) at this locus (CASEand GILES 1958a,b) and support the conclusion based on those results that interallelic recombination involves a copy-choicemechanism rather than one of directed “gene conversion.” Although the two asci exhibiting apparent reciprocal recombination could be interpreted as arising from classical crossing over, they may also be interpreted on the basis of a copy-choice mechanism. Since the great majority of interallelic recombination events are nonreciprocal in Neu- rospora (MITCHELL1955a; CASEand GILES1958a,b; STADLER1959; STADLERand TOWE1963; SUYAMA,MUNKRES, and WOODWARD1959) as well as in other organisms (LISSOUBA,MOUSSEAU, RIZET, and ROSSIGNAL1962), the view will be taken that only this type of recombination occurs between alleles (GILES1963). The most satisfactory specific hypothesis to explain these results appears to be one proposed by BERNSTEIN(1962; 1963, in preparation). This hypothesis, which is similar in a number of respects to one proposed by PRITCHARD(1960), main- tains that during meiosis homologous genes pair, and in every nucleus each homologous gene pair replicates in a polarized fashion in the same direction. During replication a newly forming strand may switch from the parental gene (template) and begin copying along the other gene. The ensuing unstable two- ALLELIC RECOMBINATION 533

TABLE 1 Genotypes of asci producing only homocaryotic cultures in which there is a recombination event at the pan-2 locus

Ascus 43 Ascus 98 ylo ad 23 + 36 + a + + 72 +trm a ylo ad 23 + 36 + a + + 72 +trw a ylo ad +72++a + ++%+a ylo ad ++36+a + ++%+a +++ 72 +tr~pA ad 23 72 +tryp A +++ 72 36trypA ad 23 72 + tryp A ++23 72 +tryp A 'ad 23 f 36 + A ++23 72 + tryp A 'ad 23 + 36 + A Ascus 153 Ascus 497 Ascus 529 ylo ad 23 36tryp a YlO + + 72 +trYPA +++ 72 4-t~~(1: + YlO + + 72 +trYPA +++ 72 +tvp a ylo ad 23 + 36tryp a +++ 72 +~~YPA ++ + 72 + tvp a ylo ad ++36+a +++ 72 +@YPA +++ 72 +trrp a ylo ad ++36+ a ylo ad 23 + 36 + a ylo ad ++36+A +++ + 36trrrvA ylo ad 23 + 36 + a ylo ad ++36+A +++ 72 +trm A +ad ++36+ a ylo ad 23 + 36 + A ++ +72++~ +ad ++36+a ylo ad 23 + 36 + A +++72++A Ascus 581 Ascus 583 Ascus 614 ylo ad +72++a +++ 72 +t~pa + + + 72 +trm a ylo ad +72++a ++ + 72 + tryp a + + + 72 + trYP a ylo ad 23 + 36 + a ++23 + 36tryp a +++7236+a ylo ad 23 + 36 + a ++23 + 36tryp a +++7236+a ++ + 72 +trYP A ylo ad 23 + 36 + A yload 23 + 36 + A +++ 72 +trYP A ylo ad 23 + 36 + A yload 23 + 36 + A +++ 72 + tryp A ylo ad 23 72 + + A yload 23 + 36tryp A ad $+++ 72 +trYPA ylo ad 23 72 + + A ylo 23 + 36tryp A Ascus 1021 Ascus 545' Ascus 565' yload 23 + 36 + a ylo ad + 72 + tryp a del ylo ad 23 + 36 + A del yload 23 + 36 + a ylo ad + 72 + tryp a del ylo ad 23 + 36 + A del yload + + 36 + a ylo aul 23 + 36 + A del ylo ad + 72 + tryp A del ylo ad + + 36 + a ylo ad 23 + 36 + A del ylo ad + 72 + tryp A del 4- + 4- 72 +trw A ...... + + + 72 +tTYP a + + + + 72 +trm A +' + + 72 try^ (2 + + + + 72 +t~pa + + + + 72 + tryp A ...... + + + 72 + + a + + + + 72 + trw A +' + ++36+A+ +++72++a+ Ascus 710' + + + 72 + tryp a del + + + 72 + tryp a del + 4- 4- 4- 36 + adel + 4- + 4- 36 + adel ...... yload 23 72 +tryp A + yload 23 + 36 + A + yload 23 + 36 + A + Recombination in these asci may be apparently reciprocal, or may give 63, 2:6, 5:3, or 3:5 ratios for a single allelic pair, for two adjacent pairs, or for all three pairs simultaneously. Ascospores are arranged in linear order 1 through 8, spore pairs being adjacent. Ungerminated ascospores are indicated by dots. * Cross 1. on-one situation is resolved when either of the two newly forming strands switches back. This model predicts eight different basic types of tetrads for a two- allele cross (BERNSTEIN1963). With three-allele crosses, certain additional types are possible, but these represent principally variants of the eight basic types. All 534 NI. E. CASE AND IV. H. GILES

E ITRANSITIONAL UNITRANSITIONAL

FIGURE3.-Diagrams of recombinant tetrad types at the pQn-2 locus based on the model proposed by BERNSTEIN(1963). Specific asci (from Table 1) of each type are indicated by numbers in parenthesis to the right of each diagram. Type numbers are given to the left of each diagram.

11 of the present tetrads can be classified on the basis of this model, as shown in Figure 3. Variant types are primarily recombinant asci which could be placed in either of two classes depending upon whether the left two or the right two sites are considered. In the present case, ascus 78 could be designated as either Type 2 or Type 3 on the original model. Three-allele crosses also permit the detection of tetrads in which 3:1 segregations have occurred at the middle site alone (involving allele 2372). These represent variants of Type 4 in which a bitransitional event has involved the middle site rather than having occurred between the left two sites (B23, B72) as in the case of analogous unitransitional events of type 8. Although no tetrads involving 3:l segregations for B72 were detected in this study, the occurrence of such types is indicated by data from random spore platings from similar three-allele crosses in which prototrophs are recovered carrying, in the majority of isolates, both parental markers entering the cross with the B72 parent (CASEand GILES1960). The presence of markers on either side of the pan-2 locus makes it possible to determine whether recombination between such markers is correlated with recombination events at the pan-2 locus. Previous interpretations of tetrad data from two-allele (CASEand GILES1958a,b) and three-allele (GILES1963) crosses at the pan-2 locus have assumed that in each copy-choice event the same single strand was involved in both the initial and return switches. On this basis a cor- ALLELIC RECOMBINATION 535 relation effect (FREESE1957) is indicated with crossing over in intervals adjacent to the pan-2 locus. On the present model, however, a return switch may involve either of the two replicating strands. When the original strand does not switch back (unitransitional types) the copy-choice event results in recombination between markers on either side of the locus. On the basis of this model, Table l indicates the two asci (Nos. 78 and 583) in which additional crossover events have occurred in intervals immediately adjacent to the pan-2 locus. Calculations based on the observed recombination values from the present cross (Figure 1) for the distal interval indicate that the expected number of asci is 2.9; the observed number is 2. No recombinant asci were found for the proximal interval; the expected number of asci is 0.8. Thus on the basis of the present model, neither these tetrad data nor those obtained previously at the pun-2 locus (CASEand GILES 1958a), indicate the existence of a correlation between allelic recombination and reciprocal recombination in adjacent intergenic intervals. The present tetrad data are also of interest in relation to the concept of a polarized unit of replication postulated on the basis of tetrad data in Ascobolus and called the “polaron” ( LISSOUBAet al., 1962). In this cross, the relationship of the “majority” to the “minority” parental type in relevant tetrads suggests a polarity of replication with an allele order B36+B72+B23. If this polarity is correct, it indicates that replication of this chromosome segment is initiated distally and proceeds proximally with respect to the centromere. However, tetrad data from previous twwallele crosses involving mutants B3 and B5 (Figure 1) do not indicate a clear majority-minority relationship for those two alleles (CASEand GILES 1958a). Despite these differences, the results of both the two- and three-allele crosses do support the general concept of a polarity of replication, since simultaneous miscopying of two or three adjacent alleles (apparently including in the latter cases essentially the entire pan-2 locus) frequently occurs. In contrast to certain of the allelic series considered to constitute a “polaron” in Ascoblus, the pan-2 locus appears to be sufficiently long compared to the average switch dis- tance such that two events occur with some frequency within the locus. This condition results in the occurrence of both apparent reciprocal crossover tetrads (Figure 3, Type 8) and less clear-cut majority-minority relationships. Although in general the pun-2 tetrad data appear to support the concept of polarized replication, they also suggest that the “polaron” probably does not correspond to a specific unit of polarized replication, but rather to any short segment of a single gene within which usually only initiations but not resolutions occur (cf. BERN- STEIN 1963, in preparation). As was indicated previously, two of the 13 asci listed in Table 1 showed segregations other than 6:2 at one or more sites, i.e., in these asci sister spores were not always identical. In ascus 43, a 3: 5 (wild type:mutant) segregation is present for the middle site (B72) only. In ascus 529, mutant B23 exhibits a 6:2 segregation, whereas mutants B72 and LE36 exhibit 5: 3 and 3: 5 segregations, respectively. Segregations for all outside markers and for mating type are normal. No simple explanation of these asci based on the model utilized to explain the previous 11 asci appears possible. As discussed in detail by BERNSTEIN,the simplest copy- 536 M. E. CASE AND N. H. GILES choice models to explain 6: 2 segregations require at meiosis a conservative replication mechanism (such as that postulated for E. coli DNA by CAVALIERI, DEUTSCH,and ROSENBERG1961 ) . If semi-conservative replication is assumed to occur, copy-choice models result in mechanical difficulties and require that additional complicated events, including breakage and recombination, be postulated (TAYLOR1958). For instances of 5:3 segregations, however, models based on semi-conservative replication mechanisms are feasible, as pointed out by TAYLOR(1958) and KITANI, OLIVE,and EL-ANI (1962). The occurrence of frequent 5: 3 segregation events at various loci controlling ascospore color in Sordaria has lead KITANI,OLIVE, and EL-ANI (1962) to conclude that conversion events in general in that organism may be the result of miscopying by half-chromatids. Such 5: 3 segregations do appear to be relatively much more frequent in Sordaria than in Neurospora, but 6:2 segregations also occur in the former organism. At present no single hypothesis appears adequate to explain all of the known types of intragenic recombination events. Atypical segregQtions for the marker loci: As in the previous 2-point cross (CASEand GILES1958b) atypical segregations were detected for the marker loci in 6: 2, 2: 6, or 5: 3 ratios of wild type to mutant. As indicated in Table 2, six asci showed atypical segregations for ad-1, one having a 6:2 ratio, four a 2:6 ratio, and one a 5:3 ratio. Eight asci showed atypical segregations for tryp-2, six having a 6:2 ratio, one a 2: 6 ratio, and one a 5:3 ratio. Two asci had a 2:6 ratio for yZo. Further analysis of the phenotypically wild-type isolates provided no evidence that these isolates were heterocaryotic. Asci producing heterocaryotic cultures: The second major category of asci with respect to recombination mechanisms is one which was not anticipated when this study was initiated. However, asci of this type are now known to be relatively frequent even though they can be rather easily missed unless appropriate tests are performed to detect them. Such asci are ones in which one of the eight ascospores gives rise to a culture which is typically heterocaryotic for the two pun-2 parental types; i.e., two types of nuclei are present, one having the B72 mutant and the other the double mutant B23-B36 (Table 3). In addition, in all but one of the nine asci (No. 320 in Table 3) the exceptional isolates were also heterocaryotic for outside marker genes as well. It was, in fact, the atypical (5:3) segregations for outside markers which first focused attention on this category of asci. Since B72 is a leaky mutant, instances of complementation in heterocaryons between B72 and B23-B36 are difficult to distinguish from isolates carry-

TABLE 2 Asci having other than 4:4 segregations for marker loci adjacent to the pan-2 locus

3 wild type:l mutant 1 wild type:3 mutant 5 wild type:3 mutant ad-l tryp-2 ylo ad-l iryp-2 ylo ad-l tryp-2 ylo Cross 1 010 2 01 0 000 Cross 2 150 e12 110 Totals 160 412 110 ALLELIC RECOMBINATION 537 TABLE 3

Genotypes of asci in which one of the eight ascospores produces a heterocaryotic culture

Ascus 320 Ascus 370 Ascus 61 1 ylo ad 23 + 36 + A + + + 72 + +a yload 23 + 36 + A ylo ad 23 + 36 + A +++72++a rload 23 4- 36 4- A yload + 72 + + A + + + 72 +tryp a ylo ad 23 + 36 + a ylo ad 23 + 36 + A + + + 72 +trrp a yload 23 + 36 + a yload + 72 + + A yload 23 f 36 + a + + + 72 +trYP a + + + 72 + tryp a ylo ad 23 + 36 tryp A + + + 72 +trYp a + + + 72 +tvp a ylo ad 23 + 36 tryp A + + + 72 ftryp A + + + 72 +tryp a yload 23 + 36 + A ylo ad 23 + 36 + A + + + 72 + tryp a ylo ad 23 + 36 + A + + + 72 +trYPA Ascus 634 Ascus 6.F9 Ascus 666 ylo ad 23 + 36 + A + + + 72 + +a Y~O+ + 72 +tr~pa ylo ad 23 + 36 + A + + + 72 + + a Y~O+ + 72 +tvp a + ad 23 + 36 + A + + + 72 +trYPA + + + 72 +trrp A + ad 23 + 36 + A + + + 72 +t~pA + + + 72 +trYp~ YlO + + 72 + trYP a yload 23 + 36 + A yload 23 + 36 + a yload 23 + 36 + a Iyload 23 + 36 + A yload 23 + 36 + a 1Y~O + + 72 +tvp a yload 23 + 36 + A + + 23 + 36 + A + + + 72 +trm a ylo ad 23 + 36tryp a + + 23 + 36 + A + + + 72 +tryp a ylo ad 23 + 36tryp a iylo ad + 72 + tryp A Ascus 745 Ascus 752 Ascus 900 + + + 72 + tryp A yload 23 + 36 + a yload 23 + 36 + a + + + 72 +t~p~ylo ad 23 + 36 + a + + + 72 +WA ++23+36+a ip$? st2 XPnn + + + 72 +~WA + + + 72 +tryp a yload 23 + 36 + a yload 23 + 36 + A yload 23 + 36 + a + + + 72 +tryp a Iyload 23 + 36 + a +crd23+36+a ylo ad 23 + 36tryp A yload 23 + 36 + a + + + 72 ftryp A ylo ad 23 + 36tryp A yload 23 + 36 + a + + + 72 +~VP A +++72++A yload 23 + 36 + a Y~O+ + 72 +t~pA +++72++~ YlO + + 72 +tTYPA

~~ ~~ Genotypes of these spores were determined by conidial platings and by crossing to wild type. Ascospores are arranged in linear order 1 through 8, spore pairs being adjacent. Isolates with more than one genotype are bracketed. ing B72 alone, and additional asci similar to No. 320 may have been missed. Table 3 gives the genotypes of the nine exceptional asci found. These genotypes were determined by conidial platings and by crosses of the original isolates to wild type, as discussed previously. In the crosses, only random ascospore isolations were made. These methods make it possible to detect the presence of more than one genotype in the nuclei of heterocaryons. However, they do not indicate whether any nuclei exist which tend to give rise to additional heterocaryotic single-spore cultures in further crosses, as might be anticipated if some type of continuing but unstable aneuploidy is present. To detect such a possibility further tetrad analyses in the next generation would be required. Extensive plating and crossing data indicated that for one exceptional isolate (No. 752.2) three types of nuclei with respect to genotype were present. Two types were in excess sug- gesting that these may represent the original nuclear types. As indicated in Table 3, in seven of the asci crossing over had occurred between the outside markers. However, in most instances the heterocaryotic isolate and its sister spore had not been involved in this crossover event. In six asci the heterocaryotic isolates were composed of the two parental chromosomes originally entering the 538 M. E. CASE AND N. H. GILES cross, while in the other three asci either one or both of the isolates were recombinant for outside markers. Although asci of the parental type may have been the result of contamination, this seems less probable for those asci recombinant for outside markers. Furthermore randomly isolated pseudo-wild types from pun-2 crosses (CASE, unpublished) arising from single ascospore isolates are heterocaryotic and carry parental rather than recombinant linkage-group VI chromosomes. In certain respects these results resemble the data of PITTENGERand COYLE (1963) on the origin of so-called pseudo-wild types from randomly isolated ascospores utilizing linkage-group VI markers similar to those employed here. These authors obtained evidence that pseudo-wild types are regularly heterocaryotic and may contain more than two types of nuclei. In addition to nuclei having one or the other parental-type chromosome present, two further reciprocally related recombinant-type chromosomes were often detected. These authors in- terpret their observations as supporting the hypothesis that pseudo-wild types result from nondisjunction events at meiosis in diploid nuclei. These events pro- duce unstable disomic nuclei in which somatic recombination can occur prior to nuclear breakdown to give heterocaryons. In the present studies the heterocaryotic cultures derived from single ascospore isolates from complete asci have all the characteristics of pseudo-wild types such as those studied by PITTENGER and COYLE(1963). These results indicate that pseudo-wild types may regularly arise in complete asci (i.e., those lacking aborted ascospores) and hence make it very improbable that such types originate exclusively as a result of nondisjunction at meiosis in diploid nuclei. The present results are also somewhat similar to those obtained by THRELKELD (1962), but differ in important respects. The major difference is that in the present studies in all but one ascus (No. 320) the exceptional cultures are heterocaryotic for markers at more than one locus in linkage group VI. Actually, of the two aberrant asci studied by THRELKELDwhich involved most of the same linkage-group VI markers used in the present study, one (AX 422) was heterocaryotic for the pun locus only, thus being similar to ascus 320 in this study, while the second (AT 385) was heterocaryotic for the pun-2 marker and for the ylo tryp markers as well, thus resembling the remaining eight asci in the present study. The asci producing heterocaryotic cultures in the present study can be interpreted on the basis of a mechanism similar in principle to that suggested by THRELKELD(1962) to explain his data, namely, some type of heteroploidy involving the chromosome carrying linkage-group VI markers. Although in the present studies the presence of an extra chromosome or chromosome fragment seems likely, interpretations based upon isochromosomes or telocentric chromosomes do not seem probable, since the markers for which the exceptional cultures are heterocaryotic are on opposite sides of the centromere. The present results may be interpreted on the basis of an entire extra chromosome or of an extra centric fragment which has parts of both chromosomes arms present. Even this interpretation is clearly tentative, and it is evident that at present no single hypothesis seems capable of accounting for all of the characteristics of atypical asci in which certain ascospore isolates are heterocaryotic. ALLELIC RECOMBINATION 539

SUMMARY An analysis has been made of allelic (intragenic) recombination in 1457 tetrads obtained from a cross involving three different complementing alleles at the pan-2 locus in Neurospora crassa. A double mutant (B23-B36) composed of two single mutants located near opposite ends of both the recombination and complementation maps was crossed with a single mutant (B72) located near the middle of both maps. In segregating asci, individual alleles could be identified alone or in combination by means of complementation, recombination, phenotypic, and mutation tests. In addition, outside marker genes located on either side of the locus were employed such that segregation and recombination could be followed simultaneously for these markers as well as for the mutant alleles. In the majority of asci all eight spores germinated and all isolates were analyzed. With respect to recombination mechanisms, two distinct categories of asci were detected-those in which all cultures derived from single ascospores were homocaryotic and those in which some cultures from single ascospores were heterocaryotic. Among asci producing only homocaryotic cultures, some type of recombination had occurred at the pan-2 locus in 13 asci, and at adjacent marker loci in 16 asci. At the pan-2 locus, 5: 3 or 3: 5 segregations were present for certain alleles in two asci. The remaining 11 asci were characterized by segregation patterns which indicated either apparent reciprocal recombination between two alleles (two asci) or clearly nonreciprocal recombination resulting in 6: 2 or 2: 6 segregations for a single allelic pair, for two adjacent pairs, or for all three pairs simultaneously. These results appear to be best interpreted on the basis of a copy- choice recombination mechanism, utilizing the specific model recently proposed by BERNSTEIN(1962; and in preparation). On the basis of this model, tetrad data from pan-2 crosses do not indicate a correlation between interallelic recombination and crossing over in adjacent chromosome regions. In nine asci, one of the eight ascospores gave rise to a culture heterocaryotic for the two pan-2 parental types and (in all but one ascus) for adjacent marker genes as well. These unexpected results have been tentatively interpreted on the basis of some type of heteroploidy involving the chromosome carrying linkage- group VI markers. They also indicate that cultures having all the characteristics af pseudo-wild types may arise in complete asci (i.e., those lacking aborted ascospores), which makes it very improbable that all pseudo-wild types originate as a result of nondisjunction at meiosis in diploid nuclei.

LITERATURE CITED

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