Three Kinds of Transreplication in Sordaria Fimicola

JAP. JOUR. GENET. Vol. 37, No. 2: 131-146 (1962)

Three Kinds of Transreplication in Sordaria fimicola

YOshiaki KITANI Kyoto University, Kyoto Received September 15, 1961

Introduction

Since Lindegren originally found aberrant gene segregation in Saccharomyces (1953), similar phenomenon (gene conversion, copy choice, transmutation, transreplication, etc.) has been studied in various materials, in Saccharomyces by Roman (1956), in Neurospora by Mitchell (1955, 1959), Case and Giles (1958) and Stadler (1959), in Sor- daria by Olive (1956, 1959), in Aspergillus by Strickland (1958) and in Ascobolus by Rizet et al. (1960). Regarding mechanisms of the occurrence of this phenomenon, several assumptions have been forwarded (Freese 1957; Taylor 1958; Lewis and Lewis 1959; Stadler 1959; Pritchard 1960 etc.). However, most of these authors have domon- strated only 3:1 or 6:2 segregation and have discussed the mechanisms on the basis of 4-strand model of meiotic prophase. Taylor (1958) discussed the mechanism of aberrant segregation for both 5:3 and 6:2 asci based on 8-strand mechanism. Olive (1959) demonstrated 5:3 segregation of the gray spore color locus in Sordaria fimicola and Mitchell (1959) may have had 5:3 asci in Neurospora crassa but the asci have not been considered as 5:3 asci by the reporter herself. Recently, Kitani et al, demonstrated good number of both 5:3 and 6:2 asci in Sordaria fimicola and intensively analyzed the relationship between transreplication and crossing over. For 5:3 asci, their analysis is clear and their data agree with Taylor's diagram in most part, however, many questions are still left for 6:2 asci mainly because of the difficulty in detecting double crossing over expected to occur adjacent to the transreplication locus. The discovery of 7:1 ascus, which can not be explained to be a product of a single transreplication event in meiotic process, opened a new vision to look at another process to involve a transreplication event other than meiosis. In the present work, the difference of crossing over situation between 5:3 asci and 6:2 acsi was intensively analyzed and the mechanism of the occurrence of 6:2 segregation was discussed referring an evidence of somatic recombination. A further analysis of reciprocal double transreplication was also done.

Materials and Methods

The g locus, of which characteristics have been reported by Olive (1956) and transreplication has been studied by Kitani et al., was studied here. Morphological markers spotty (sp), milky (mi), mat and corona (cor) were mainly used. All these markers are linked with g and their linkage relationship has been reported by El-Ani et al. (1961). The positions of these mutant loci are shown in Figure 1. 132 Y. KITANI

centromere sp mi mat g cor 0 ... i _ 46 8.5 0.6 0.4 3.4 Numbers represent distances in crossover units between adjacent markers. (Kitani et at.) Fig. 1. A part of the g linkage group.

A sterile marker st-22 which is linked with g and a color marker v which is not linked with g were also used. With v, both g and g+ showed greenish spore color, and gv+, g+v+, gv and g+v were distinguished each other easily. Genotypes of progeny were distinguished morphologically except to discriminate sp mi double mutant from mi single mutant. In the latter case, the genotypes were indentified in confirm- crosses. Asci showing aberrant segregation for g locus were found under the microscope and they were dissected with the method used in the previous works. All the crosses were made on Difco cornmeal-dextrose agar plates with 0.1% yeast extract added, and incubated under 23°C until maturation, then stored in 5°C refrigerator. Various types of transreplication in g locus studied in this experiment are shown in Figure 2.

Figure 2.

Experimental Results

1. Differences between 5:3 asci and 6:2 asci in the frequencies of crossing over.

In order to analyze whether 6:2 asci differ from 5:3 asci in the frequency of transreplication-related crossing over, three groups, all of which involve both 5:3 and 6:2 sub-groups, were studied. The crossing over frequencies in the mi-g and mat-g regions (both regions are equivalent in the sence of transreplication-related crossing over) and the g-cor region are shown in Table 1, supplement. Although these ascii were already introduced by Kitani et al., their characteristics in transreplication- related crossing over are analyzed intensively here in this paper. Through all the three groups, double crossing over in the mi-g-cor region was TRANSREPLICATIONIN SORDARIAFIMICOLA 133 not detected in the 6:2 sub-groups, and the majority of single crossovers observed in 6:2 asci were not determined their location either in the mi-g interval or in the g- cor interval. So that, for the comparison of single crossing over frequencies between 5:3 asci and 6:2 asci, mi-cor interval was used instead of using mi-g and g-cor intervals separately. The result is shown in Table 1. The 5:3 sub-groups show much higher frequencies than the 6:2 sub-groups for single crossing over in the mi-cor interval in all three groups. In order to determine whether this difference is merely an accidental deviation or not, Chi-square test was applied on each of the sub-groups and on the sums of 5:3 asci and 6:2 asci. The results are in Table 2. The hypothetical frequency was derived from the total of

Table 1. 134 Y. KITANI

Table 1. Supplement

Table 2. Chi-square tests for the crossover frequencies in the mi-cor interval

both 5:3 and 6:2 asci and the total of single crossovers observed in the mi-cor interval in all the asci. Although P-values for each sub-groups are larger than 0.05, the P-value for the sum of 6:2 asci is 0.02, and in Group II, the P-values are on the limit of allowance for both 5:3 and 6:2 asci. Furthermore, the deviation of the frequency from the hypothetical value is only for the + direction in 5:3 asci and only for the - direction in 6:2 asci with the only exception of 5:3 sub-group of Group III. From these results, it is difficult to consider that both 5:3 and 6:2 asci have a common value for crossing over frequency in the mi-cor interval, as far as concerning single crossing over. The double crossing over of which one crossing over is in the mi-g interval and the other in the g-cor interval is theoretically undetectable in 6:2 asci, if present, with rare exception. Actually, no double crossing over was detected in any of 6:2 TRANSREPLICATIONIN SORDARIAFIMICOLA 135 asci in this experiment. On the other hand, about 20 percent of 5:3 asci showed double crossing over in the mi-g-cor interval. Therefore, as far as 6:2 asci are also transreplication asci, it is reasonable to consider that 6:2 asci do have transreplication-related crossing over as frequently as 5 : 3 asci do, so that, many of 6 : 2 asci could be considered to have double crossing over. However, if this is true, the total crossing over frequency, in which a double crossing over is counted as two crossovers, of 6:2 asci might be expected 92 percent which is the observed frequency of 5:3 asci. So, in Table 1, the complementing percentages 2a and a will be estimated 69.2 percent and 34.6 percent respectively. The latter value is the estimated frequency of double crossing over in 6 :2 asci. A frequency of double crossing over is usually calculated in multiplying the crossing over frequencies of two intervals, in this case mi- g and g-cor intervals, and here the observed double crossing over frequency of 5:3 asci agrees with this calculation well but the estimated 34.6 percent for 6:2 asci does not agree with it at all. Considering the above observations the situation of transreplication-related crossing over in 6:2 asci must be different from the situation in 5:3 asci. And two different explanations seem to be possible to apply on the 6:2 asci; 1) there is an un- known factor which increases the frequency of double crossing over, and 2) 6:2 ascii are not affected their crossing over frequency much by the transreplication event, so, the crossovers actually observed are nearly the all they have.

2. Presence or absence of interference In order to know whether 6:2 asci have transreplication-related crossing over or not, it is the best way to use a pseudo-allele of g locus. However, as far as no pseudo- allele has been obtained, one of the most effective way to resolve the problem is to compare the effect of interference in the sp-mat interval between 5:3 asci and 6:2 asci; for the presence or absence of interference in the sp-mat interval indicates the presence or absence of transreplication-related crossing over in the mat-cor interval. In this purpose, 96 aberrant asci were studied here. The results are shown in Tables 3 and 4. Table 3A is showing that the frequency of crossing over between sp and mat (9.1 c. u.) in the 5:3 asci is much lower than the expected and that of the correspond- ing region in the 6:2 asci (Table 3B). And in Table 3A, only one ascus shows crossing over both in the sp-mat interval and the mat-g interval, the other 20 asci which have crossing over in the mat-g interval do not show any crossing over in the sp-mat interval. Three out of four crossovers observed in the sp-mat interval are found in the asci in which the transreplication-related crossing over is not recorded. For the 6:2 asci (Table 3B),12 crossovers are observed in the sp-mat interval and the frequency of this is deviated to the + direction from the expected frequency of the normal asci and from the observed frequency of the control, but the deviation is not great (Table 4). According the Chi-square test using 18.2 percent, which is the expected frequency 136 Y. KITANI

Table 3.

TR-X Group sp+ + mat + mi + g A. 5:3 segregation

i i An ascus which has crossovers in every marked intervals is omitted from this table to be analyzed separately.

B. 6:2 segregation.

for the sp-mat interval of normal asci, as the hypothetical frequency, P-values for both 5:3 and 6:2 asci are in the allowance of significance. However, as it was already indicated in Table 1, about 28 percent of 5:3 asci do not have transreplication-related crossing over and these asci are apparently free from the interference. So that, it must be adjusted in the Chi-square test. After adjusting it, the P-value for 5:3 asci is> 0.05. Above data are indicating that the probability of obtaining the observed frequency of crossing over in the sp-mat interval is high in 6:2 asci, but very low in 5:3 asci unless some factor which decreases the crossing over frequency in 5:3 asci is assumed. TRANSREPLICATION IN SOKDARIA FIMICOLA 137

Table 4.

Table 5.

p-',111'

The factor which may be assumed to be a cause of the low crossing over frequency in the sp-mat region of the 5:3 asci is interference. In order to compare how high degrees of interference can be imagined in the 5:3 asci and the 6:2 asci in this experiment, the relationship between increasing degree of imaginary interference and the change of P-values was tested on both 5:3 and 6:2 asci. The results are in Table 5. In this calculation, the relationship between the percentage of interference and the observed crossing over frequency is: 138 Y. KITANI

a: degree of interference (/) b _ b: observed frequency of crossing over (%) 1--a/100 - c : estimated crossing over frequency under the interference-free condition (%)

For the 6:2 asci, the presence of interference is hardly allowed, because the P-value drops below 0.05 even when interference is assumed to be as small as 23 percent. For 5:3 asci, on the other hand, up to more than 60 percent of interference may be assumed, the P-value attaining its maximum when interference is about 50 percent.' Combining all the above observations and considering the fact that the crossing over frequency in the sp-mat interval of the particular group of 5:3 asci, in which transreplication-related crossing over was observed, was especially low, it is reasonable to conclude that 5:3 asci might have been affected with interference but 6:2 asci might not.

3. An evidence of somatic recombination

The increasing possibility that the transreplication-related crossing over might have occurred only rarely or not at all in 6:2 asci brought us another question : that is, whether 6:2 type transreplication event has its origin in a DNA duplication process other than meiotic one. A new material, to be considered in connexion with this question, is introduced in Table 6. This table is showing the crossing over frequencies in various regions observed in groups of asci all of which were normal in the ratio between g and g+ spores. Table 6a is for three clusters of asci obtained from a particular spot of the heterokaryotic sector of plate A. Table 6b is for clusters taken from various spots other than the particular spot of plate A. Table 6c and 6d are for other plates, L and M. All the plates contained the same cross, sp mat+ g} cor x sp+ mat g cork, and were incubated under the same conditions at the same time. The crossing over frequencies in the centromere-sp interval were 57.1 percent, 68.9 percent, 70.0 percent and 56.2 percent, respectively. These values do not differ between each other and from the expected 66.7 percent, significantly. No crossing over was observed in the mat-g interval in any asci. It is because the transreplication- related crossing over does not exist in the normal asci and the distance of this interval is only 0.4 c. u. The crossing over frequency of g-coy interval is also normal through all the groups. However, for the s p-mat interval, Table 6a shows 61.9 percent and the others show 17.8 percent, 20.0 percent and 18.8 percent, respectively against the expected 18.2 percent. Apparently, the crossing over frequency observed in Table 6a is too high to be an accidental deviation. If the cluster No. 1 of plate A is omitted for a moment, the crossover frequency roughly corresponds to 100 minus the expected frequency. This may be the result of somatic recombination occurring

1) According to Kitani et al. (in press), the P-values for the sp-mat interval were <0.01 for 5:3 asci and >0.30 for 6:2 asci if all available asci, regardless of the difference in the combination of markers, were pooled. TRANSREPLICATION IN SORDARIA FIMICOLA

Table 6. sp Cross: + + cor + mat 9 + a) Plate A, particular spot

b) Plate A, various spots other than the particular spot

c) Plate L

d) Plate M 140 Y- KITANI in the sp-mat region before the perithecia formation, and, accordingly, suggest that a few generations of diploid nuclear division existed accidentally in the heterokaryotic sector before meiosis. For the cluster No. 1 of plate A, which show 37.5 percent crossing over in the sp-mat region, two different explanations are likely to be applied ; 1) simply, the frequency is showing an accidental deviation either from the expected values in recombi-

Table 7.

Cross: sp+ _+ + cor mat g + TRANSREPLICATIONIN ,SORDARIAFIMICOLA 141 nation cluster or normal cluster, 2) the cluster was originated from the two different nuclei, one being a recombined nucleus and the other a normal one (or a pair of haploid nuclei). There is a reason for assuming the second explanation. When two strains of Sordaria fimicola are crossed, it is not rare that a single cluster contains both homo- zygous (parental) asci and heterozygous (hybrid) asci together and the proportion of the two kinds of asci is various (Olive, 1954). As at least two somatic recombination clusters were obtained from the particular spot of plate A, a small piece of the heterokaryotic mycelium of the spot was trans- ferred on a fresh plate. Out of 14 clusters examined on this new plate, only one recombined cluster was obtained. Hence, the particular portion of heterokaryotic sector might have been a mixture of two kinds of parental haploid nuclei and diploid nuclei including recombined ones. Seventeen aberrant asci were obtained from the spot where somatic recombination clusters were found. The situation of crossing over for these aberrant asci is shown in Table 7. Both 5:3 and 6:2 asci are showing their unique pattern of crossing over frequencies in the cent. -sp, mat-g and g-cor intervals. But in the sp-mat interval, they are showing a peculiar pattern of crossing over frequency which was observed in the somatic recombination clusters. Some asci in Table 7 might have been derived from unrecombined nuclei, though indistinguishable from others.

4. Reciprocal double transreplication

A new type of transreplication, reciprocal double transreplication, was first introduced by Kitani et al. (Science 1961), and was discussed by Kitani et al. (in press). Asci of this type have 4:4 ratio for the g locus, but one of the eight spores is converted its g allele to g+ and another one spore is converted its g+ allele to g. Recently, three asci of this type were obtained from two different crosses and the genotypes of their progeny were already introduced (Kitani et al., in press). The asci are shown in Table 8. Asci X-116 and X-147 were found in a group of 51 aberrant asci, and Ascus XI-11 was found in a group of 21 aberrant asci. The data of these asci are confirming the previous explanation about this kind of asci. The important characteristics of reciprocal double transreplication asci are: 1) two aberrant spore pairs were consistently appeared in opposite halves of the ascus, 2) the frequency of occurrence is not 7 x 10-8 which is calculated multiplying the frequency of 3g+:5g event (1.2 x 10-4) and the frequency of 5g+:3g event (5.8 x 10-4), but moderately lower than the frequency of 3g+ : 5g event, 3) a marker cor, the right side of g locus, is consistently found in two spore pairs showing normal arrangement. The aberrant spors pairs are observed in the opposite halves of all three asci in Table 8. This suggests that reciprocal double transreplication may not be the result of two events, one, of 5g+:3g, the other, of 3g+:5g, but may be a result of a reciprocal miscopying within a single pair of sub-chromatids. The frequency of occurrence of these three asci was 5.4 x 10 which is far greater than the expected 10_8 order of two event origin. Ascus X-11 involves another colour 142 Y. KITANI

Table 8. Reciprocal Double Transreplication, sp + mat + Cross for following two asci : _ + mi + g Ascus X-116X-11& Ascus X-147 sp + mat + sp + mat g sp + mat g sp -f- mat + ± mi + g sp ± mat + ± mi + g sp ± mat + + mi + g + mi + g + mi + + mi + g sp + mat + + mi + + sp + mat + + mi + g

Cross for following ascus: sp + + car + + mat g + v

Ascus XI-11

+ mat g + v + mat g + v sp + + + + sp + g + + + mat + cor v + mat g cor v sp + + cor + $ p + + cor +

marker v which is not linked with g. This colour marker is helpful in distinguishing reciprocal double transreplication asci from the asci which have slippage of spore arrangement before dissecting. There is a crossing over between g and cor in Ascus XI-I1, and this crossing over is involving both transreplication strands. Although Asci X-116 and X-147 and the other three asci of the same cross (discussed previously) did not show any transreplication-related crossing over, when trans- TRANSREPLICATIONIN SORDARIAFIMICOLA 143 replication-related crossing over occurs involving both of transreplication strands, it must not be detected in this cross, because, without having a marker on the right side of g locus, there is no way to know which one of the aberrant spore pairs was originally g pair. So, it would be admitted that some of these 5 asci had transreplication-related crossing over.

Discussion

Recently Kitani et al. studied the relationship between transreplication and crossing over on large number of aberrant asci and reported followings; 1) both 5: 3 and 6:2 asci are equally common 2) transreplication-related crossing over was observed very frequently in restricted regions adjacent to the both sides of g locus especially in the 5 : 3 asci, but 3) a moderately higher frequency of crossing over than to be expected in normal asci was observed in the same regions of 6:2 asci, 4) a 7:1 ascus which can not be explained its occurrence in a single process of meiosis was found and 5) a new type of transreplication, reciprocal double transreplication, was first analyzed. Taylor (1958) previously published an explanation for the mechanism of transreplication, on the basis of 8-strand model of meiosis. According to Taylor's diagram, the transreplication process is as follows; a localized intimate pairing occurs prior to zygotene synapsis, then, during DNA synthesis, miscopying of newly developping DNA occurs in the region of the intimate pairing, then the rest of meiosis is completed. In this diagram, 5:3 type transreplication is not required to have chromosome breakage and reunion, it means that transreplication-related crossing over is not immediately expected. On the other hand, 6:2 type transreplication is based on the occurrence of chromosome breakage and reunion, it means that high frequency of obligate (not only related with transreplication event) crossing over is expected. Here, the main contradiction between Taylor's diagram and the data obtained by Kitani et al. exists in the opposite relationship concerning transreplication-related crossing over of both 5:3 asci and 6:2 asci. However, about the transreplication-related crossing over in 5:3 asci, Kitani et al. explained that the intimate pairing might be expected to favour transreplication-related crossing over. Therefore, the remaining, but serious problems are whether 6:2 asci have obligate crossing over or not and whether the 6:2 event also occurs, like 5:3 event, during meiotic process or not. The presence of 7:1 segregation ascus presents a very important suggestion to resolve these problems. According to the 8-strand model of meiosis, prophase chromo- somes are composed of two sub-chromatids until new DNA is synthesized, so that, when a nucleus is heterozygous for g locus, the nucleus has two each of g and g+ sub-chromatids from the beginning of meiotic prophase. And, as miscopying is allowed only for the newly synthesizing DNA, the maximum number of spores allowed for either g or g~ genotype in a single ascus is 6. However, if diploid mitosis is assumed to occur at least once before meiosis, occurrence of localized chromosome pairing might be expected. The occasional occurrence of diploid nuclei and the presence of localized chromosome pairing in diploid mitosis were already discussed for Aspergillus by 144 Y. KITANI

Pontecorvo (1958), in connexion with somatic recombination. If a miscopying occurs in a localized pairing region of this type, 6:2 segregation ascus will appear when no transreplication event occurs in the succeeding meiotic process, and 7:1 segregation ascus will appear when 5:3 event occurs in the succeeding meiotic process. Very rare occurrence of 7:1 segregation is expected from this hypothetical process and actually very rare. Thus, any kind of transreplication event can be explained on the basis of the occurrence of localized chromosome pairing prior to DNA synthesis and miscopying in DNA synthesis, assuming that transreplication event is not limited in the meiotic process. Present studies indicated that 6 : 2 asci probably differ from 5 : 3 asci in the f re- quency of single crossing over in the adjacent regions of transreplication locus, and unless 6 : 2 asci have had a special mechanism to increase the frequency of double crossing over, they might not have double crossing over at all. Presence or absence of double crossing over in the adjacent regions of transreplication locus (g locus) would be directly informed is a pseudo-allele of g could be used together. However, as no pseudo-allele has been obtained yet, an indirect method to estimate the presence or absence of transreplication-related crossing over was employed. Comparison of crossing over frequencies in the sp-mat interval showed that an interference by the transreplication-related crossing over (in the mat-coy interval) may not have affected 6:2 asci but may have affected 5:3 asci. Although the data were not conclusive enough (the P-value of Chi-square test was larger than 0.05 for the crossing over frequency of 5:3 asci in the sp-mat interval), the test for the allowance of interference (Table 5) is still suggesting that 6:2 asci may not have had transreplication-related crossing over. On the other hand, when miscopying is imagined to occur in mitotic process of a diploid nucleus, crossing over is not necessary to be expected in the localized pairing region at least in high degree. Because, in mitosis, contrary to meiosis, centromeres are divided and sister centromeres migrate toward the opposite poles with sister chromatids, so that, the probability of having mechanical power ona the paired part of chromatids is much fewer than the meiotic process. Connecting all above considerations, 6:2 asci may not have transreplication-related crossing over much and may be initiated in a mitotic division of a heterozygous diploid nucleus. The discovery of the presence of somatic recombination presents an evidence for occasional occurrence of heterozygous diploid nucleus and the presence of localized chromosome pairing before meiosis. For the occurrence of transreplication event, only one diploid mitosis preceding the meiosis is enough, even a synchronized nuclear division of dikaryophase haploid nuclei might be also to give a chance of miscopying. Therefore, the discovery of at least a few generations of diploid nuclear stage is an evidence covering all these possibilities. Although some of the author's experiments are indirect approach to the mechanism of 6:2 type transreplication, the following process of transreplication is figured out by combining all the observations; 1) a localized chromosome pairing occurs in a dividing heterozygous diploid nucleus or between a heterogeneous pair of dikaryophase nuclei. TRANSPLICATIONIN SORDARIAFIMICOLA 145 some time before DNA duplication, 2) a miscopying occurs during DNA duplication on one of two new DNA units in the paired region, 3) the diploid nucleus which carries a miscopied DNA carries out meiotic division without successive miscopying. The only difference from 5 : 3 event is that 6 : 2 event occurs during mitosis and 5:3 event occurs in meiosis. In addition to the 6 asci of reciprocal double transreplication, which were previously analyzed, 3 asci were recently studied. From the observations of these asci, the explanations previously applied on this type of transreplication was confirmed. There is little doubt left now that these asci are not resulted from half -chromatid crossing over. A marker coy, of which location is 3.4 units right side of g locus, did not show any aberrant arrangement in all four aberrant 4 : 4 segregation asci in which coy locus was marked. If the aberrant arrangement of g is the result of half -chromatid crossing over occurred in the mat-g interval (0.4 c.u.), coy must show aberrant arrangement with g in the great majority of the case, because the mat-cor interval is too short (3.8 c.u.) to have another crossing over frequently. It seems clear now that both miscopying., one from g to g+ and the other from g+ to g, occurred in a single event of localized intimate chromosome pairing, from following factors; both of two transreplication-related crossovers observed in the 4:4 aberrant asci were involving both miscopying strands, the frequency of reciprocal double transreplication was 10-5 order in both previous and present data, and these are far greater than the frequency 10-8 expected in the free combination of the 5g+ : 3g event and the 3g + : 5g event.

Summary

The difference of the crossing over situation between 5:3 type aberrant asci and 6:2 type aberrant asci was analyzed, and 6:2 asci were considered not to have transreplication-related crossing over frequently. The presence or absence of interference by the presence of transreplication-related crossing over was also analyzed, and it was considered that the sp-mat interval may have been affected with interference is 5:3 asci but may not have been affected in 6:2 asci. Somatic recombination was found in one cross from the same of which large number of aberrant asci have heen obtained. From above observations an explanation for the occurrence of 6:2 type aberrant asci was proposed. Main point of this explanation is that 6:2 type asci occur by a miscopying of new DNA synthesized in the localized pairing region of chromo- somes during mitosis instead of meiosis.

Acknowledgement

The author wishes to express his sincere gratitude to Dr. L. S. Olive, Professor of Columbia University for the effective guidance, and to Dr. A. S. El-Ani for the supplying of mutants. This experiment was carried out in Department of Botany, Columbia University, New York, New York, U.S.A. This research was supported in part by E-2326 from the National Institutes of 146 Y. KITANI

Health, U.S., G-14263 from the National Science Foundation, U.S., and Public Health Genetics Training Grant 2G-216, U. S.

Literature cited

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