Some aspects of recombination in eukaryotic organisms.

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Annu. Rev. Genet. 1975.9:129-144. Downloaded from www.annualreviews.org by George Mason University on 04/27/13. For personal use only.

SOME ASPECTS OF RECOMBINATION

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IN EUKARYOTIC ORGANISMS P. J. Hastings Department of Genetics, University of Alberta, Edmonton, Alberta, Canada

The overall understanding of the process of recombination in eukaryotes has not changed very much since the reviews of Holliday (I, 2), Emerson (3), White house (4), and Stadler (5). The basic working model is that a recombination event consists of the formation of lengths of hybrid DNA, or heteroduplex, ' extending from outside the gene 0n one or two chromatids and ending at a variable position within the gene. Within these lengths, heterozygosity may be resolved by the removal of a length of either nucleotide chain, and its replace ment by replication that uses the other chain as a template. Failure to remove the heterozygosity results in postmeiotic segregation. The configuration is re solved so that it may lead to crossing-over or to conversion without crossing-over. Attempts to define the events of formation and resolution of the heteroduplex in molecular terms are too numerous to mention and seem to serve merely to show that the process is possible. Gajewski et al (6) questioned the assumption that postmeiotic segregation has to represent the survival through meiosis of a heteroduplex. Stahl (7) showed that it was possible to make a model that produced non-Mendelian ratios of alleles without involving excision repair of heterozygosity. Paszewski (8) offered a model that gave conversion by nucleo tide chain displacement, without involving repair mechanisms. His model is very similar to one proposed at the same time by Boon & Zinder (9) for bacteriophage recombination. It is quite common to read that Stahl and Paszewski must be wrong, because it is not possible to explain allele-specific effects without postulating that the heterozygosity induces a repair process. Since some marker effects appear to act at a level other than repair of heterozygosity (see below) it would seem that excision repair models have the same problem and that this objection to nonexcision repair models is not valid. However, the work of Leblon & Rossignol (10) reported below makes excision repair the most attractive hypothesis. Progress has been made in several aspects of eukaryote recombination that 129


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HASTINGS

are not discussed here. Recombination in yeast was reviewed by Fogel & Mor timer (II). Control of recombination was reviewed by Catcheside ( 12) and Whitehouse (4). The dissection of meiosis and recombination by mutation is receiving attention in many organisms, including Drosophila ( 13), Podospora ( 14, IS), and yeast ( 16- 18). The physiology and biochemistry of meiosis were reviewed by Stern & Hotta ( 19). Radding (20) and Hotchkiss (2 1) reviewed molecular mechanisms. Perhaps the most pleasing advance in recent years is the demonstration of several aspects of fungal intragenic recombination in Drosophila (22-24). Although it is patently impossible to demonstrate conversion in half tetrads, it is clear that conversion provides the only easy and probable explanation of the observations of Chovnick and his group (23). Thus, there is reasonable hope that the results from lower plants can be generalized to all eukaryotes. MARKER EFFECTS

The subject of marker effects was reviewed by Stadler' (5) and Catcheside ( 12). The observation that some aspects of intragenic recombination are allele-specific is now very common, and some authors ( 10) are speculating on the level at which their marker effects act. The marker effects reported seem to be falling into characteristic classes (25, 26). For the purposes of this discussion, I have adopted a different classification of the phenomena.

Type fa Frequency of Conversion Versus Postmeiotic Segregation The relative frequency of conversion and postmeiotic segregation for any marker appears to be specific for that marker in all systems in which it can be detected, including yeasts (27, 28). Many alleles are known for which postmeiotic segrega tion has not been detected. There may be also, however, a suggestion of a region-specific element in determining which alleles show postmeiotic segrega tion, because in series 46 (6) and series 19 (29) in Ascobolus immersus all alleles that show postmeiotic segregation are in the high conversion regions of the locus. The relative amount of postmeiotic segregation appears to be subject to modification by various influences, for example, by variation in genetic back ground, because Kitani & Olive (30) found that an allele of the g locus in Sordaria fimicola had a changed frequency when it was reisolated from a conversion tetrad. This does not reflect lack of fidelity in the conversion process, since the work of Fogel & Mortimer (31) using supersuppressible alleles in yeast seems to leave no doubt that conversion is faithful. In Sordaria, the relative amount of postmeiotic segregation can also be changed by the addition of DNA precursors (32) and depends on the region of a heterokaryon from which the perithecium is taken (33). Leblon (34, 35) found that in A. immersus some mutagens gave mutant alleles with high frequency of postmeiotic segregation while others gave mutants with low frequency, implying that this parameter is dependent on the nature of the heterozygosity.

RECOMBINATION IN EUKARYOTES

l31

Leblon & Rossignol ( 10) found that when a mutant allele with low frequency of postmeiotic segregation was put into a cross with an allele showing high frequency, the postmeiotic segregation of the second allele decreased, because it was co-converted under the influence of the first. Thus, it seems that conversion is epistatic to postmeiotic segregation. This is the only evidence that postmeiotic segregation is a reflection of a condition that is a precursor to conversion, and provides substantial support for the idea that conversion is a process of correction of heterozygosity.


Type Ib Relative Frequency of Conversion to Mutant and to Wild Type Inequality of conversion in the two directions is the normal observation in N. crassa, S. fimicola, S. brevicollis, and A. immersus. It has not been detected in Saccharomyces cerevisiae (36) and has been reported in Schizosaccharomyces pombe for only one allele (28). This is the clearest example of a species-specific variation in recombination pattern. Its occurrence encourages the belief in conversion as a correction mechanism, because differential response of organisms to mutagens also appears to be related to species-specific variation in the response of correction mechanisms. In A. immersus, Leblon (34, 35) found that the direction and extent of the inequality was related to the mutagenic origin of the allele. Rossignol (37) reported that conversion patterns of alleles of series 75 in A. immersus were correlated with the overall conversion frequency of each allele. This is discussed below. The separation of these two marker effects may be artificial, because it is quite likely that the two decisions are made at the same time.

Type II Frequency of Use of the "Opportunity to Convert" The expression "opportunity to convert" was introduced by Rossignol (37) to explain his observation that polarity in series 75 in A. immersus could be seen within any one class of mutant when mutants were classified according to relative frequency of conversion to mutant and to wild type. The reason that such polarity could not be seen before (38) was that mutants of different classes were mixed in the map and variation in use of the opportunity to convert was greater than any systematic change in conversion frequency. Rossignol explained his opportunity to convert in terms of relative frequency of correction of heteroduplex, so that, as in type IA marker effect above, failure to use the opportunity to convert should be seen as postmeiotic segregation. However, he gives no information on the occurrence of postmeiotic segregation for his markers, so that one does not know that polarity is revealed by the frequency of conversion plus postmeiotic segregation. Other examples discussed below reveal that failure to use the opportunity to convert does not result in postmeiotic segregation, and that, therefore, this type of marker effect is quite distinct from type IA. Series 19 in Ascobolus (29) is an unusual locus in that it shows absolute polarity. That is, within one half of the locus, recombination results only from conversion of the marker nearest the nearer end of the locus. On a heteroduplex-excision


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HASTINGS

repair model of recombination this can be interpreted as all recombination resulting from heteroduplex endings. If both markers are in the heteroduplex they must always be co-converted; otherwise some recombination would result from independent conversion of the marker farthest from the end of the polaron. In this situation the conversion frequency (in a one-point cross) of the outer marker gives the frequency of heteroduplex reaching at least to that point. The conversion frequency of the inner marker gives the frequency of heteroduplex passing the site of both markers. The frequency of ending between the markers is therefore the difference between the two conversion frequencies. In Mous seau's data (29) the frequency of tetrads that include a wild-type spore pair from two-point repulsion crosses does not even approximate this value, but is approximately an order of magnitude lower. The relevant data for the A cluster of series 19 are shown in Figure I. Either our understanding of the basis of intragenic recombination is completely wrong, or adding an additional site of heterozygosity has reduced the frequency of heteroduplex endings. Such a reduction could be caused either by preventing heteroduplex reaching either site or by extending it so that it covers both sites, resulting in co-conversion, rather than in recombination. This second possibility implies that there is a marker effect that contracts short intervals by extending the length in which conversion can occur. Because most of these alleles show no postmeiotic segregation, this extension of conversion is not at the expense of heteroduplex which would otherwise remain uncorrected. That an additional site of heterozygosity increases conversion frequency of other alleles was shown by Stadler & Kariya (39) using a temperature-sensitive allele of the mtr locus in Neurospora crassa. The presence of this silent allele in a cross increased conversion frequencies for markers in all parts of the locus, the effect being strongest in the case of alleles with lower conversion frequencies, so that the polarity within the locus was no longer apparent. Several authors have described alleles that give very much higher conversion frequency and/or recombination than "normal" alleles in the same part of the map. The behavior of one such "marker effect allele" in S. pombe is described Map

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in detail by Gutz (28). The presence of this marker increases conversion of nearby markers and increases recombination more with distant markers than with those close by. This would appear to mean that a long length is co-converted with the marker effect allele. This interpretation was used by Stadler & Kariya (39) to explain the effect of the temperature-sensitive allele of the mtr locus in Neurospora in reducing recombination frequency between other alleles. Hawthorne & Leupold (40) list several other cases of marker effect alleles in S. pombe in suppressor loci. Some, but not all of the marker effect alleles are putative anticodon mutations. Moore (25, 26) described several types of marker effect in the fir locus of Coprinus lagopus. Some of his marker effect alleles appear to involve increase in the use of the opportunity to convert, while others reduce it. In some of these cases, notably series 19 in Ascobolus (29) and ade-6 in S. pombe (28), it is clear that the difference between low and high conversion frequencies at a given point in a polaron is not made up by the occurrence of postmeiotic segregation. Consequently, there is a marker effect that acts on some precursor to conversion other than a length of heteroduplex which may, or may not, be corrected. Gutz (28) considers the possibility that alleles that do not show this marker effect are being converted back to parental genotype so that conversion is not seen. He rejects this idea in favor of the hypothesis that the marker effect allele represents a point at which events are initiated with a high frequency. Goldman (41), working with the same gene in S. pombe, presents extensive three-point intragenic cross data and concludes that initiation at the site of the marker effect allele is not a sufficient explanation. The widespread occurrence of the type II marker effect reviewed here, and particularly the forms that it takes in series 19 and 75 of Ascobolus make Gutz's first suggestion seem more attractive. I favor the idea that there is a precursor to conversion that may or may not become a free conversion heteroduplex. Type I marker effects concern the resolution of a free conversion heteroduplex, while type II marker effects act on the opportunity to convert by modifying the distribution of free conversion heteroduplex. This is summarized in Figure 2. There seems to be no evidence at present on the nature of this precursor. It is perhaps easiest to think of it as a heteroduplex that is converted back to the parental form of the chromatid on which it occurs. Some of the implica tions of this notion for recombination are discussed later in this review. There may be other marker effects, apart from the types described above, which need to be taken into account before the basis of recombination is understood. Moore (25, 26), in his work on C. lagopus described an equal and opposite increase and decrease in recombination that cancelled each other out when alleles of the two types were crossed together. I have ascribed these effects to variation in use of the opportunity to convert. But Moore also described a marker effect in which an allele gave abnormally high recombination with close markers, but normal recombination frequency with markers further away.

HASTINGS

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This may, perhaps, be compared to the situation described by Rossignol (37) in which an allele of series 75 in Ascobolus behaved like a deletion in that it appeared to occupy a length, but within the length it still showed some slight recombination, implying that it was not, in fact, a deletion. Baranowska (42) has pointed out that failure of an allele to recombine with others nearby may be a marker effect rather than evidence of a deletion.


FINE STRUCTURE MAP EXPANSION

Map expansion is a term used to describe a phenomenon seen in prototroph frequency maps from allelic crosses in which the length of a long interval exceeds the sum of the lengths of the short intervals of which it is comprised. It was first described by Holliday (43) and presented as evidence that heterozygosity interfered with heteroduplex formation.

The Fincham & Holliday Model In 1968, Holliday suggested a new explanation ( 1) which was developed by Fincham & Holliday (44). Fincham & Holliday proposed that there is a het eroduplex of fixed length h and random position. In short intervals, recombina tion was caused only by the ends of this heteroduplex because inclusion of both markers in a length of heteroduplex would always lead to co-conversion and hence could not cause recombination. The frequency of such endings would be proportional to distance d between markers, and would therefore give an Marker effect

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additive map. If, however, a length

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x, some additional recombination would result from independent conversion

of the two markers when they are both included in the same length of het eroduplex. This expansion term depends on the frequency of correction of heteroduplex for the specific alleles, and on the frequency of inclusion of both markers in heteroduplex, which will vary as (h - d), declining as d increases. Recombination within a distance exceeding the co-conversion length, x, will thus be greater than the sum of lengths less than x within it, by the amount of this additional recombination. This model does not explain polarity, but can readily be modified to do so, by having one end of the heteroduplex fixed outside the gene and allowing the length h to vary. This would not alter the conclusion from the algebra. The major objections to this model are as follows: I. When recombination in a long length r is plotted against the sum of short lengths, assumed to represent d, the points do not fall within the two phases (dx) defined by the Fincham & Holliday algebra, but in the first phase and on the line that would join the first and second phases. It may be possible to show that this objection is not serious if x is allowed to vary. This would change the discontinuity to a line with positive slope. 2. In a map sequence a - b - c - d, if (a to c) exceeds (a to b) + (b to c), it is not also possible for (a to d) to exceed (a to c) + (c to d) on the Fincham & Holliday model, because the expansion term in (a to d) has already been included in (a to c). Indeed, there should be contraction, because the value of the expansion term declines over longer distances. The published expansion plots (I, 44) show no points that might relate to such a situation. Mousseau's (29) data for the B and C clusters in series 19 in A. immersus show many examples of expansion when already expanded intervals are added. Some of these data are shown in Figure 3. Mousseau (29) also reported that the amount of expansion depends on the number of subintervals added to obtain d, so that rid is less than 2 when only two subintervals are used, but increases to 17 or 18 when five or six subintervals Mop

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(29) and illustrate the point that expansion does not come in a single burst, because an already expanded region may be used as a subinterval to show further expansion.


136

HASTINGS

are used. Again, such an effect is not predicted by the Fincham & Holliday algebra, because the expansion term contributes to r only once and declines with increasing distance. The implication of this observation is that expansion does not corne as a single burst of extra recombinants when d exceeds x, but is more strongly seen in longer intervals than in short intervals. Hence, only the very shortest intervals show no expansion. When d is computed from any but the shortest intervals, it is already partly expanded, so that d is overestimated, and expansion is underestimated. The Fincham & Holliday algebra can be modified to account for all these observations by allowing x to show considerable variation. The sharp inflection in plots of r against d implies that there is a definite minimum length for x, but above that minimum, x must be allowed to vary to cover distances as great as h. Strong support for the idea of Fincham & Holliday that expansion is caused by the recombination that is due to independent conversion is found in Mousseau's (29) data. In series 19, the A cluster has no independent conversion and no expansion, while the B and C clusters show evidence of independent conversion and strong expansion.

Other Possible Causes of Expansion Several other explanations of the phenomenon are possible. Ahmad & Leupold (45) recently offered one in which they proposed that crossovers can be included in long intervals but not in short intervals and that this could be a cause of expansion if one assumes that there is a marker effect that reduces conversion, but has no effect on included reciprocal events. Moore (25) and Hawthorne & Leupold (40) have noted that a major cause of map expansion is the inclusion in crosses of alleles that show strong marker effects. Such crosses contribute to expansion because the enhancement of recom bination caused by marker effect alleles is weak over short distances and strongest in crosses with alleles at a distance which includes one third to one half of the gene. This is best seen in the data of Gutz (28) and Stadler & Kariya (39). This distance dependence of the enhancement was attributed above to the length of co-conversion of conversion events originating at the marker effect allele. Type II marker effect, that is, allele:specific variation in use of the opportunity to convert, appears to be a general phenomenon, applicable not only to marker effect alleles. If this ability of a site of heterozygosity to cause free convcrsion hctcroduplcx to cover it is distance dependent, this may be a general cause of map expansion. A close allele would more often bring free conversion het eroduplex within reach than would a more distant allele, so that short intervals would be relatively more contracted by co-conversion. I have found no evidence of this distance dependence, unless it is the basis for polarity, as discussed below. POLARITY

Polarity in recombination is seen in three distinct ways: (a) as a preferential conversion of the marker on one side rather than the marker on the other



137

' side in recombinant tetrads for any cross throughout a region of a gene (47), (b) as a gradient in conversion frequencies of markers in one-point crosses (47), or (c) as an asymmetry between the two classes of prototrophs that have parental combinations of outside markers (48). Polarity has been explained by postulating that events originate between genes and extend a variable length of heteroduplex, in which correction can occur, into genes (43, 49). Hence, only recombination that results from heteroduplex ends contributes to the appearance of polarity; recombination that results from independent conversion of two markers will not show polarity. Leblon & Rossignol ( 10) argue that their results show that the correction lProcess is not polarized either at the level of recognition of heterozygosity, or at the level of excision in a preferential direction from the site. Their evidence is, first, that in a cross involving two mutants that have different conversion characteristics, each modifies the behavior of the other. and. second. that conver sion of one allele under the influence of another seems to act hath on a strand that was originally mutant and on one which was originally wild type. Study �f Gutz's data concerning the marker effect allele M26 in ade-6 of S. pombe (28) leads to the conclusion that conversion that begins at M26 does. in fact, extend in both directions in the same event. Gutz found that M26 has a conversion frequency of 5% in a one-point cross. About 90% of these conver sions are from mutant to wild type. This frequency is about one order of magnitude higher than the conversion frequencies of nearby markers. Alleles throughout the gene show an enhanced frequency of prototrophs when crossed with M26 in comparison with the equivalent crosses involving an allele near M26. This enhancement is in the range of one order of magnitude except in a region extending in both directions from M26, where the enhancement is threefold for alleles nearest M26, with a gradient of increasing enhancement as distance from M26 increases. The prototroph frequencies in crosses with M26 vary from 10-6 for the closest markers to 10-3 for crosses showing maximum enhancement.

Tetrad analysis shows that, in the crosses showing only 10-:; or 10-6 proto trophs, M26 is still converted at more than 10-2, but recombination does not result from the conversion, because the other allele is co-converted. The gradient of enhancement would therefore appear to reflect the length of co-conversion resulting from conversion of M26. If a conversion length induced by the heterozygosity at M26 were equally likely to extend in one or the other direction, co-conversion should result only half the time. The other half would lead to prototroph production or not, according to what occurred independently at the other allele. So the prototroph frequency should be lower than the frequency of conversion to wild type of M26 by less than one order of magnitude. That the prototroph frequencies are three to four orders of magnitude less implies that conversion does not extend in only one direction from the heterozygosity that induced it.

The same conclusion can be made from the results of a three-point cross with M26 in the middle. In this cross, 13 of the 2 1 conversion tetrads identified


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HASTINGS

showed co-conversion of all three alleles. The more extensive three-point cross data of Goldman (41) show the same trend. Because these conversions do not occur when M26 is not included in a cross, it seems that they are induced by the presence of M26. It is then seen that most conversions extend in both directions on one chromatid from the position of the allele that caused them. Both Gutz and Goldman explain their results with a model that involves excision simultaneously in both directions from M26, but they do not attribute this process to correction of a heteroduplex. If, however, co-conversion from M26 does occur by a process of excision from the point of heterozygosity, the enzyme system involved appears to have properties quite unlike those of dimer excision after ultraviolet irradiation (50). This interpretation of the M26 data supports the conclusion of Leblon & Rossignol (10) that it is not the repair process itself that is polarized. This leaves the question of whether polarity is a property of the opportunity to convert, of the free conversion heteroduplex, or of both. Rossignol (37) took it to be the opportunity to convert which was polarized, but he was not distinguishing the precondition from the free conversion heteroduplex. The loss of polarity in the presence of a silent allele shown by Stadler & Kariya (39) for the mtr locus of Neurospora implies that the opportunity to convert is present to a constant extent throughout that locus. If marker effect alleles reflect high efficiency of the use of the opportunity to convert, then the precondition would seem to be about ten times more common than is the occurrence of conversion for most alleles. It seems unneces sary on the present evidence, therefore, to postulate polarity in the frequency of occurrence of the opportunity to convert. An obvious extension of the ideas expressed here is that all markers show a type II marker effect. Thus, it is the presence of heterozygosity that causes the occurrence of free conversion heteroduplex. It is as though a site of heterozygosity draws a length of free conversion heteroduplex out from the ends of the gene. The frequency with which this occurs depends both on the distance from the end of the gene and on the nature of the mismatching for different alleles. MAPPABILITY

On the model of the origin of intragenic recombinants used by Fincham & Holliday (44), recombination can result either from the ending of a length of heteroduplex between the markers, or from independent conversion within a length of heteroduplex. If one assumes, as they did, that the length of co-conver sion is constant, the only distance-dependent variable is the frequency of ending of a heteroduplex. Thus, it will be possible to construct a prototroph frequency map only if there is more recombination resulting from heteroduplex endings than will be masked by the amount of variability in observed frequencies of conversion caused by allele-specific aspects of the conversion process. If the Fincham & Holliday model is modified, as suggested above, by allowing



139

extensive vanatIOn in the lengths of co-conversion above a minimal length, it is to be expected that some ability to map by recombination frequency will be found, particularly over long distances, in situations that lack heteroduplex endings. The ability to map by the asymmetry between the two classes of prototrophs that are recombinant for outside markers also depends on heteroduplex endings, since, if both alleles are converted, the proportion of the two recombinant outside marker classes depends on the frequency of independent conversion to wild type of the two alleles, and not on their relative positions. It is, therefore, not surprising to find that a locus that gives no consistent prototroph frequency map is also unmappable by outside marker combinations. Examples of this are seen in mlr (39) and in am-J (51), both in N. crassa. Polarity is also explained by heteroduplex endings and is not expected when such endings are rare. It is found in SUP6 in yeast (L. L. DiCaprio, personal communication) that inconsistent mapping by these two criteria is correlated with an absence of polarity. It is, therefore, surprising to find that a locus that shows strong polarity is unmappable (51, 52). Smyth (53) found that this locus was mappable when the crosses were made in a background that gave tenfold higher recombination. One explanation for this paradox is that, while crossover events show few or no heteroduplex endings within the locus, giving unmappability, noncrossover events do have such endings, giving polarity (52, 53). There is a precedent for believing that it is possible for the two types of events to have different het eroduplex lengths. This has been used (54) to explain systematic changes in the proportion of crossover to noncrossover prototrophs from crosses in different regions of a gene, seen, for example, in me-2 in N. crassa (48). Fincham (52), however, prefers an explanation based on the idea of Pritchard (55) that negative interference is caused by a clustering of events, rather than an explanation based entirely on the occurrence of conversion with and without crossing-over. Pas zewski, Prazmo & laszczuk (56) also found it necessary to return to a cluster model to explain their results in series 84 W of A. immersus. CROSSOVER AND NONCROSSOVER EVENTS

It is found that, on the average, half of the chromatids showing intragenic recombination are also recombinant for outside markers. It has been said that for yeast, from tetrad data (57), the proportion is actually half, implying that a random decision is being made. Sigal & Alberts (58) have shown that it is possible for scission to involve either the exchanged or the nonexchanged nu cleotide chains with equal probability in the resolution of a half-chromatid chiasma of the sort postulated by Holliday (43). A survey of the data available in 1965 (54) showed that the proportion is rarely half for any specific locus. Stadler (5) reviewed the subject and concluded that the process described by Sigal & Alberts does not conform with what has been observed. The data of L. L. DiCaprio in this laboratory show that noncross-


140

HASTINGS

over convertants are in excess in tetrads, and noncrossover intragenic recom binants are in excess in random products from crosses involving SUP6 in yeast. Although it is possible to argue that many factors may change the apparent ratio of outside marker combinations when it is determined from selected random meiotic products, it is difficult to believe that the broad range of observed ratios can represent perturbations of a 50:50 ratio. The values reported range from 300/0 (53) to 60% (59) of the prototrophs recombinant for outside markers. An even more extreme case is seen in the data of Murray (60). Her data are compatible with her interpretation that all the events that originate between me-7 and me-9 in N. crassa are parental for outside markers. The ratios of parental or recombinant outside marker combinations are characteristic of a locus, implying that the decision is under locus-specific control. Random resolution of a half chromatid chiasma would also imply that cross over and noncrossover events do not differ in any other respect. There are, however, several reports in the literature of differences between the two types of event. I. Not only is the proportion of crossover to noncrossover events characteristic of a locus, but also it can be seen in some loci to vary according to position in the locus. This is particularly true for me-2 (48) and pan-2 (61) in N. crassa. This was interpreted (54) as evidence that the heteroduplex lengths are different in the two types of event. 2. The relative amount of conversion and postmeiotic segregation was found to be different in crossover and noncrossover events at the buff locus in Sordaria brevicollis (62). It was reported in the same paper that this is also true in the data of Kitani & Olive (63) for g locus in S. fimicola. 3. Stadler & Towe (64) found differences in the proportion of different types of asci for a spore color locus of A. immersus, which they interpreted as showing that noncrossover events have conversion on only one chromatid while crossover events concern two chromatids.

Because the ratio of crossover to noncrossover events is locus-specific and they seem to differ from each other in respects other than the combination of outside markers, it is difficult to believe that the difference between them is in the resolution of the half chromatid chiasma. Stadler & Towe (64) gave a modified version of the Holliday model in which a half chromatid chiasma is always resolved to give a crossover, while noncrossover events arise by forming heteroduplex on one chromatid only. Sobell (65, 66), in his modification of Holliday's model, uses the same mechanism as Holliday (43) and Sigal & Alberts (58) to make the decision, but he proposes an intermediate configuration which is a bubble of heteroduplex on two chromatids bounded on each end by a half chromatid chiasma. This bubble reminds one of the explanation for the crossover or noncrossover decision on the Whitehouse model (49, 54) in which a noncrossover event is two crossover events back to back. Murray (60) failed to find evidence that noncrossover events cause simultaneous conversion in two adjacent genes, but one may assume that there is a silent region between the two genes she studied.


141


It would, therefore, be worth considering the possibility that the bubble is not always resolved into a single half chromatid chiasma as Sobell proposed. but that in a proportion of events the complete bubble survives until the half chromatid chiasmata are resolved into crossovers. at which time they become noncrossover events. All such speculations may be upset by the recent findings of Catcheside & Angel (67). Using an interchange heterozygote in N. crassa, they showed that a recombination event is unable to cross the discontinuity in the interchanged chromosome. but that it can travel along the wild-type homologue to involve chromatids of both homologues after passing the interchange breakpoint. CONCLUSIONS

Marker Effects The study of marker effects has made it necessary to complicate the heterodu plex-excision repair explanation for intragenic recombination by postulating that the heteroduplex in which conversion occurs freely has a precursor. There appears to be no evidence on the nature of the precursor. It is described here as an "opportunity to convert." One possibility is that it is a heteroduplex in which conversion is directed to restore parental linkage relationships. Another idea is that it represents a region crossed by a bubble of heteroduplex of the type postulated by Sobell (65). One might visualize such bubbles crossing consid erable lengths of chromatids, only causing recombination if they encounter heterozygosity or a region predetermined to have a recombination event. The studies of intragenic recombination during the past two decades have been concerned with the distribution of free conversion heteroduplex and the associated recombination of outside markers. The idea that free conversion heteroduplex is not the primary condition of a chromatid involved in recombina tion, but occurs as a response to heterozygosity. leads to the realization that we do not know what the primary event looks like. Consequently, the conven tional understanding of many phenomena, such as mapping and polarity, needs to be reconsidered.

Map Expansion It is shown that several situations should contribute to the expansion of maps. The model of Fincham & Holliday (44) is modified by introducing considerable variation in the lengths of co-conversion so that the excision lengths vary as much as, and independently of, the heteroduplex lengths. Polarity Polarity appears to be a property of the distribution of free conversion het eroduplex, and not of the conversion process, which extends in both directions after it has been induced by heterozygosity, nor of the opportunity to convert. which is much more common than free conversion heteroduplex.

142

HASTINGS

Recombination Between Outside Markers Resolution of a half chromatid chiasma to make a recombination event into a crossover or a noncrossover does not seem to be a satisfactory explanation of the differences between the two types of event. A noncrossover event may be a unidirectional transfer of information, or it may be caused by the occurrence of two half chromatid chiasmata close together, both of which become crossovers.


Perspective Although the subject of eukaryote recombination is still in the stage of becoming more complicated with each new observation, it is still possible to find an explanation for all data on the same basic model. The key seems to be to recognize and separate species-, locus-, region-, and allele-specific effects, and to allow the parameters of heteroduplex occurrence and length, the occurrence and length of excision repair, and the recombination of outside markers to vary independently. I have tried to show that the study of extreme situations, in which the variation in some parameters is negligible, yields insights that are obscured in more conventional situations. The descriptive study of eukaryote recombination does not seem to be con cerned primarily with the properti es of enzyme systems, which, apart from allele-specific effects, would give uniform results for an organism. It seems, rather, to be concerned with the structure and informational content of the genetic material of the specific locus at which a recombinational event occurs. ACKNOWLEDG MENTS

I am grateful to the many students and colleagues who have read and criticized the manuscript, and especially to L. L. DiCaprio and D. P. Morrison for their extensive help throughout the work. This work is supported by grant number A5735 from the National Research Council of Canada. Literature Cited

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