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

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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 segrega- tions), the latter type being much more frequent than the former. (2) The evi- dence 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 segre- gations may involve either one allele or both alleles simultaneously. The mecha- nism suggested to explain these instances of so-called “gene conversion” is one in- volving 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, re- combination, 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 per- cent Clorox solution. Dissected ascospores were transferred to tubes of supplemented Fries mini- mal 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 com- bination. 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 for- mation, 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 unde- tected. 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 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 gen- eral 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.
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