RECOMBINATION AT THE BAR LOCUS IN AN INVERTED ATTACHED-X SYSTEM IN DROSOPHILA MELANOGASTER'

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1 RECOMBINATION AT THE BAR LOCUS IN AN INVERTED ATTACHED-X SYSTEM IN DROSOPHILA MELANOGASTER' SUSAN J. GABAY AND JOHN R. LAUGHNAN School of Life Sciences and Department of Botany, University of Illinois, Urbana, Illinois Manuscript received March 26, 1973 Revised copy received July 26, 1973 ABSTRACT Recombination at the Bar locus in Drosophila melanogaster was investigated in an inverted attached-x system which enhanced the frequency of homozygosis for the Bar region. Females among the progeny of homozygous B mothers were searched for changes of B to BB and to B+. Marker genes were followed and exceptional half-tetrads were analyzed in regard to two hypotheses: that of exchange between obliquely synapsed members of the duplication, which is associated with exchange of outside markers, and that of intrachromosomal exchange, which does not involve recombination of markers.-recombinant exceptions of B + /BB genotype, carrying the outside marker combinations predicted on the hypothesis of exchange between obliquely synapsed duplication members, were encountered repeatedly. It is established that B+ and BB strands are reciprocal products of the same event.--twelve nonrecombinant exceptional strands were isolated; ten of these were B+ and two were BB. Only one of the nonrecombinant half-tetrads offered the opportunity to test the prediction of reciprocity of the intrachromosomal event. Analysis showed the exceptional female to be of the constitution BB/B, a type not expected on the hypothesis. While it could have arisen through some kind of copy error in the repair of a chromatid break, a valid test of the hypothesis of intrachromosomal exchange must rest on the isolation and analysis of more cases of the appropriate exceptional genotype.-in several cases Bar changes were found to be associated with aberrations; all but one of these involved spontaneous, cytologically identifiable deletions. DUPLICATIONS have been considered to play a role in the origin of new genes through functional diversification of duplicate members. Mechanisms have been postulated by which duplications may give rise to higher levels of repetition. In particular, much attention has been focused on the behavior of tandem repeats which arise when a chromosome segment is duplicated in situ. The Bar duplication in Drosophila melanogaster has been particularly wellstudied. STURTEVANT (1925) observed that changes of Bar to wild-type and to double-bar are correlated with crossing over. The hypothesis of unequal crossing over was formulated predicting B+ and BB to be reciprocal crossovers. T. H. MORGAN (1927) attempted unsuccessfully to recover females of the B+/BB This research was supported by National Science Foundation grants GB-7635 and GB This report is based on a thesis submitted by SJG in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Botany, University of Illinois. Genetics 75: November, 1973.

2 486 S. J. GABAY AND J. R. LAUGHNAN reciprocal type using a strain of homozygous Bar females giving 22% nondisjunction. L. V. MORGAN (1931) used attached-x Bar females in mass matings and recovered one exceptional narrow-eyed female whose constitution was found to be B+/BB; no outside markers were followed. STURTEVANT S data included one case of a nonrecombinant B+ exception, but it was not possible to rule out contamination. MULLER and WEINSTEIN (1933) and LINDSLEY ( 1953) also attempted to recover nonrecombinant exceptions. PETERSON and LAUGHNAN (1961) reported observing such exceptions and their further studies ( 1963) supported the interpretation that the nonrecombinants resulted from exchanges between intrachromosomally paired members of the Bar duplication. According to this model (LAUGHNAN 1955, 1961), isochromatid and sister strand exchanges, although not associated with outside marker recombination, result in the loss of exactly one member of the duplication. In addition, sister chromatid exchange produces a triplication strand as the complement of the single-membered strand. The studies reported here are concerned with the precision and frequencies of the two types of unequal crossing over within the Bar duplication. Both exchange between obliquely synapsed homologs, and intrachromosomal exchange within a proposed double-loop configuration, were investigated. The reciprocity of these events was of particular interest. An attached-x system was employed because half-tetrad analysis permits the study of reciprocity by allowing the joint recovery of the strands involved in exceptional events. A system in which the X chromosomes are attached at their yellow ends increases the likelihood of the joint recovery of the reciprocal products of exceptional events and also facilitates the ensuing analysis of exceptional females by increasing the frequency of homozygosis for the Bar region. Such an Ynverted attached-x system was employed in this investigation. MATERIALS AND METHODS The modified attached-x stock was synthesized and kindly provided for our use by DR. E. NOVITSKI. The females carried a compound reversed metacentric X chromosome. They were homozygous for y (yellow body color) and B (Bar eye), and heterozygous for U (vermilion eye color), f (forked bristle) and od (outstretched wing; synonym oso) (Table 1). The stock females were of the constitution In(f)EN od B f+ U y (YL?).(YL?) y U+ f B od+ Ys In(1)EN. This genotype will be referred to simply as U f+ B od/u+ f B od+ (Figure la), keeping the genes in their usual order, it being understood that the position of the centromere is reversed. In the course of this study, females having the marker constitution U f B od+/u+ f+ B od arose through crossing over (Figure lb). These, being suitably marked, were also used as parental females in this study. The X-chromosome rearrangement employed here will be referred to as inverted attached-x. Single-female matings were used throughout the experiment, both in the search for exceptional females and in their later analysis. Each female was mated with two XYs.YL males having the constitution yz SU-wa wa Ys.YL y+. The total number of female progeny in vials being searched for exceptions was recorded only if the appropriate marker constitution was verified by the appearance of homozygosis products which were f od+ and f+ od, but not f od. Female siblings of exceptional females were mated in turn for the number of generations required to verify the constitution of marker genes by homozygosis. If the appropriate constitution of marker genes was not confirmed, the exception was excluded from further consideration. The constitution of the attached-x chromosomes of the

3 RECOMBINATION AT THE BAR LOCUS TABLE 1 X-chromosome mutants employed as marker genes in inverted attacbd-x females. Map posifions given for a normal X chromasome; after LINDSLEY and GRELL 1967, except as noted 48 7 Symbol Y U f B od (synonym 08) Description yellow body color vermilion eye color forked bristle Bar eye outstretched wing Distance from centromere in inverted attached-x Cytological position A in or near 10Ai F1-2* A A-f * DEMEREC and HOOVER f J. R. LAUGENAN, unpublished. Y V f+b od V f+b od Y V f B odf / f B od+ + f B od + f B odt b f+b ftb od od FIGURE 1.-Marker constitution of females employed in these studies. (a) U f+ B &/U+ f B od+. (b) v f B od+/u+ f+ B od. exceptional female was determined by examining the homozygosis products among her progeny or their descendants. The matings carried out in order to verify and analyze the exceptional females provide ample opportunity to observe aberrant genetic behavior, especially that indicative of a deficiency. The fact that these studies were undertaken in attached-x females makes the recovery of deficient strands possible since the second X chromosome is available to cover the deficiency. Such deficiencies are detected as exceptional strands which are homozygous inviable. RESULTS The search for exceptional females: A total of 59,836 female offspring was searched for changes of B to B+ or to BB. Of this total, 31,517 females were daughters of U f B od*/v+ f+ B od mothers and the remaining 28,319 were daughters of v f + B od/v+ f B od+ mothers. The recovered exceptions are expected to fall into one of three classes: B+/B, BB/B or B+/BB. The B+/B class, being half-bar, is usually easily distinguished from the B/B parental type and from other exceptional types. The two doublebar phenotypes, BB/B and B+/BB, could not be distinguished from one another by microscopic examination (20~). They could, however, usually be distinguished from B/B, although some overlap between B+/BB and the parental type did occur. Therefore, any female with eyes judged to be smaller than the average Bar eye of its sibs was tested to determine whether it was in fact double-bar.

4 488 S. J. GABAY AND J. R. LAUGHNAN A total of 329 females with presumptive exceptional eye phenotype was found. The results of tests of these presumptive exceptions are presented in Table 2. Of the 329, 207 were confirmed and these will be considered in subsequent sections. Analysis of recombinant B+ and BB exceptions: Recombination within the 16A duplication, between obliquely synapsed members, should result in a B+ strand and a BB strand as reciprocal types, leaving the remaining two strands unchanged (Figure 2). A population of tetrads having such an event, and no other exchanges between Bar and the centromere, should produce egg nuclei of the four genotypes BIB, B+/B, BB/B and B+/BB with equal frequencies. The three exceptional genotypes would occur in the ratio 1 : 1 : 1. Given sufficient genetic distance between the centromere and the Bar locus, the latter would be assorted at random with respect to the centromere. In this case the expected ratio of the exceptional genotypes would be 2 B+/B : 2 BB/B : 1 B+JBB. Of the confirmed Bar changes, 240 X chromosomes, carried by 193 individuals, proved to be recombinant for outside markers, a rate of one per 480 X chromosomes. The numbers of exceptional females in each of the three genotypic classes were 100 B+/B, 36 BB/B and 57 B+JBB. The ratio of B+JB to BB/B is expected to be 1 : 1 regardless of the frequency of coincidental exchange between the centromere and the Bar locus. That this expectation was not realized is probably due to the lower viability of the double-bar females. Since the double-bar phenotype is not always distinguishable from the Bar phenotype, it is also possible that some double-bar exceptions were overlooked. The observed numbers of B+/B and B+/BB exceptions provide a better fit to a 2 : 1 ratio than to a 1 : 1. Again it must be considered, however, that the lowered viability of females carrying BB could have caused a reduction in the B+JBB class. While the Bar locus in this inverted attached-x system lies far enough from the centromere to permit coincidental exchanges, their frequency is not sufficiently high to simulate random assortment. Thus the relative frequencies of the exceptional classes are difficult to predict but TABLE 2 Strand analyses of 329 presumptive exceptional females Confirmed exceptions Recombinant for outside markers Nonrecombinant for outside markers One strand nmrecombinant and one aberrant Aberrant No progeny Double-Bar Half-Bar Nonexcep tions Double-Bar Half-Bar Lost before complete analysis Double-Bar Total

5 RECOMBINATION AT THE BAR LOCUS 489 f,, odf a n fl; I od f B od+ FIGURE 2.-Constitution of half-tetrads derived from tetrads with an unequal crossover event. (a) Both exceptional strands recovered in the same half-tetrad. (b) The two exceptional strands recovered in different half-tetrads. they should certainly be intermediate between the 1 : 1 : 1 ratio expected if there are no coincidental exchanges, and the 2 : 2 : 1 ratio expected if the frequency of exchanges were high enough to randomly assort the Bar locus. Analysis of nonrecombinant B+ and BB exceptions: Nine of the 207 exceptional females carried Bar changes which proved to be nonrecombinant for outside markers; in each case, only one of the two X chromosomes was exceptional. Another exceptional female, B+ in phenotype, carried two nonrecombinant changes of B to B+. Still another carried two exceptional strands; one of these was a nonrecombinant BB type, while the other was aberrant. Thus excluding the single aberrant strand, 12 X chromosomes were found to carry nonrecombinant Bar changes. This is a rate slightly higher than one nonrecombinant Bar change per 10,000 X chromosomes. The phenotypes, genotypes and cytological analyses of the eleven females carrying nonrecombinant exceptional strands are given in Table 3. The nonrecombinants designated intra-1 through intra-8 each carried a B+ exceptional strand along with an unchanged, parental B strand. In two cases, intra-i and intra-8, the nonrecombinant B+ strands were recovered along with their sister chromatids, at

6 490 S. J. GABAY AND J. R. LAUGHNAN TABLE 3 Exceptional females carrying nonrecombinant B+ and BB strands Intra Chromosome constitution event Phenotype Genotype 15F-16A region 1 f half-bar od+ 2 f+ half-bar od+ 3 f+ half-bar od+ 4 f+ half-bar od+ 5 f+ half-bar od+ 6 f+ half-bar od+ 7 f+ half-bar od+ 8 f half-bar od+ 9 f+ double-bar od 10 f+ normal eye od+ 11 f+ double-bar od+ f B+ od+/ f B od+ Noanalysis f Bf od+/ f+ B od 15F 16A/15F 16A 16A f+ B+od / f B od+ 15F 16A/15F 16A 16A f B+ od+/ f+ B od 15F 16A/15F 16A 16A f+b+od / f B od+ 15F 16A/15F 16A 16A f+ B+od / f B od+ 15F 16A/15F 16A 16A f B+ od+/ f+ B od 15F 16A/15F 16A 16A f B+od+/ f B od+ 15F 16A/15F 16A 16A f+ BB od / f + B od 15F 16A 16A 16A/15F 16A 16A f B+ od+/ f+ B+ od 15F 16A/15F 16A f+ BB od / (f)*-b+ od+ 15F 16A 16A 16A/-16A * Deletion extends into the forked cistron but does not include the f mutant site. See text for explanation. least for the f-od region. According to the model of intrachromosomal recombination, intra-1 and intra-8 involved isochromatid events because in each case the sister strand of the B+ chromatid remained unchanged at the B locus. The two X chromosomes carried by each of the exceptions intra-i through intra-8 were homozygous viable. There was the possibility, however, that the observed nonrecombinant changes from B to B+ could have been associated with mutation, rather than with the physical loss of one member of the duplication. Accordingly, the salivary gland chromosomes of the nonrecombinant exceptional stocks which were maintained (intra-2 through intra-8) were analyzed. In all seven cases, the change from B to B+ was associated with loss of one 16A member of the Bar duplication (Table 3). Intra-9 involved a nonrecombinant BB strand recovered along with its sister strand for the f-od region. On the basis of the model of intrachromosomal recombination, nonrecombinant BB strands should involve sister chromatid, rather than isochromatid, events. The sister chromatid of the BB strand is expected to carry B+ as a result of the same event which produced BB. The sister strand of the nonrecombinant BB strand in the case of intra-9 was B, not B+ as predicted. Both the BB and B strands were homozygous viable. It is possible that the B strand, while producing a B phenotypic effect, carried only one representation of the 16A region. However, cytological analysis (Table 3) indicated that one of the X chromosomes of the exceptional daughters carried three 16A members, while the other carried two. Therefore, in this case, the gain of a 16A member in one strand was not associated with the loss of a 16A member in its sister strand. The nonrecombinant exception designated intra-10 arose as a female which was B+ in phenotype. Exceptions with B+ phenotype are not expected except by coincidental or aberrational events. Both B+ strands proved to be homozygous viable and nonrecombinant for outside markers and they arose from nonsister

7 RECOMBINATION AT THE BAR LOCUS 49 1 chromatids. Indications are that two coincidental intrachromosomal events were involved. Cytological analysis indicated that only one 16A member was carried in each of the nonrecombinant strands. Intra-I1 exceptional females were phenotypically f+ BB od+ and, upon attempts to determine the marker constitution of the X chromosomes which they carried, it was found that the strand carrying BB was viable when homozygous, and that the other strand, most probably bearing a deletion, was not. From females of the homozygous BB phenotype, it was determined that the BB strand carried the markers f+ and od and was, therefore, nonrecombinant for outside markers. Since the BB strand carried od, and the phenotype of the exceptional females was od+, the aberrant strand must have carried od+. Further genetic analysis of the intra-i 1 strain indicated that the aberrant chromosome had the constitution f B+ od+, and that it did in fact carry a deletion extending to the left from within the Bar duplication and terminating within the forked cistron. The details of the genetic analysis oe this unique aberration will be presented in a separate publication. Cytological examination of intra-i 1 females showed that one strand carried 15F and three 16A members, and that the other was deficient for 15F and carried one 16A1-7 segment. Thus, the nonrecombinant event resulting in the loss of a 16A member involved the simultaneous loss of the 15F region, including the 15F1-2 doublet. The nonrecombinant event which gave rise to BB on the other strand involved the gain of one 16A member. Analysis of aberrant exceptions: Aside from intra-i 1, three other exceptional females, each with half-bar eye phenotype, exhibited aberrant genetic behavior involving lethality of one or both of the X chromosomes when homozygous for the 16A region. The phenotypes, genotypes and results of cytological analyses of these three exceptions are given in Table 4. Females of aberrant strain I had half-bar eyes and an exaggerated forked phenotype, indicating that they were hemizygous for forked. They gave forked, Bar daughters by homozygous but there were no B+ or od daughters. It was considered that this strain carried a chromosome deficient for part or all of the forked cistron and for both of the parental 16A segments. Cytological analysis supported this interpretation as aberrant strain I females were found to carry an X chromosome deficient for 15F and both 16A members. TABLE 4 Exceptional females carrying Bar changes associated with aberrations Aberrant Chromosome constitution Strain Phenotype Genotype 15F-16A region I f! half-bar od+* - B+ (od+)/f B od+ - - /15F 16A 16A I1 f! half-barod+* -B+ (od+)/f B od+ - 16A/15F 16A 16A I11 f + half-bar od no homozygosis 15F 16A/15F 16A 16A * f! refers to exaggerated phenotype associated with hemizygous forked genotype.

8 492 S. J. GABAY AND J. R. LAUGHNAN Aberrant strain I1 was phenotypically identical to aberrant strain I. The genetic behavior was different, however, as females of this strain produced rare exaggerated forked, B+ daughters, presumably by crossing over. It was considered that this strain carried a strand deficient for part or all of the forked region and for at least one, but not both, 16A members. This was confirmed by cytological analysis which indicated that aberrant strain I1 females carried a strand deficient for 15F and for bands 16A14, and possibly all, of the left 16A member of the Bar duplication. In both aberrations I and 11, the event which produced the Bar change also involved loss of the forked locus. As a result of the physical loss of this marker, it was not possible to determine whether the deleted segment carried the ff or f allele and hence, whether these deletions involved recombinational events; most likely they did not. The exceptional females of aberrant strain I11 had half-bar eyes, normal bristles and outstretched wings. They were propagated for nine generations without the appearance of eye phenotypes that would suggest homozygosis events. The absence of both Bar-eyed and wild-type offspring suggests that both X chromosomes of aberrant strain I11 females are homozygous lethal. Moreover, no forkedbristle offspring were ever observed in this pedigree so that it is reasonable to conclude that the event leading to the original aberrant I11 exception involved ff od sister strands. In later generations of this pedigree there were two instances of females that produced B+ and BB offspring; however, the detailed analysis of sublines established from these females was hampered by the fact that both X chromosomes of the aberrant I11 strain carried ff and od, and it was therefore not possible to determine whether these events were associated with recombination of marker loci. Cytological analysis of salivary gland nuclei indicated that one of the X chromosomes of aberrant I11 females carried a single 16A member, and that the other had two 16A segments. This is consistent with the half-bar phenotype of these individuals but, unfortunately, provides no clue to their aberrant genetic behavior. To arrive at an estimate of the incidence of Bar-locus exceptions associated with chromosomal aberration we note that the intra-i 1 strain, and aberrant strains I and 11, each carried an X chromosome that was shown by homozygosis analysis to be homozygous lethal, and by cytological analysis to carry a deletion. In addition, aberrant strain I11 carried two exceptional X chromosomes; both were shown to be homozygous lethal but neither exhibited a cytologically identifiable aberration. Thus, five of the exceptional strands encountered in this study were aberrant, a rate of about one per 25,000 X chromosomes. This, no doubt, represents a minimal estimate as it does not take into account those cases of chromosomal aberration in or near the Bar locus which are not associated with changes in Bar phenotype and which, therefore, would not be identified. DISCUSSION A total of almost 60,000 inverted attached-x females, equivalent to about 120,000 X chromosomes, were examined for changes at the Bar locus. Among the progeny of these homozygous B females, changes of Bar (B) to double-bar

9 RECOMBINATION AT THE BAR LOCUS 493 (BB) and to wild type (B+) were identified. Most of the Bar-locus changes were recombinant for outside markers (1 per 500 X chromosomes), but some were nonrecombinant (1 per 10,000 X chromosomes). Five of the Bar-locus changes were associated with spontaneous chromosome aberrations (1 per 25,000 X chromosomes). The studies reported here provide an opportunity to determine the precision of crossover events occurring between obliquely synapsed members of a duplication. To the extent that such exchanges are imprecise, some of the resulting chromosomes should carry deletions, and at least some of these should have recessive lethal effects. In the ordinary mating system, however, sons receiving such a chromosome would not be viable and would, of course, not be identifiable as exceptions. In the attached-x system employed here, however, the lethal effect of such a chromosome is covered, and progeny analysis of exceptional females should reveal its presence. In this study there were 250 crossover chromosomes carrying Bar-locus changes identifiable as exceptions. Of these, 157 were B+ and 93 were BB. Progeny tests,of the 193 exceptional females which carried these strands indicated that in every case the exceptional chromosome was homozygous viable. It appears, therefore, that exchanges Setween obliquely synapsed members of the Bar duplication rarely if ever result in aberrant chromosome products, and that they have a precision which approaches or equals that of ordinary crossover events. Use of the attached-x system also affords the opportunity to test the reciprocity of unequal crossover events associated with the exchange of outside markers. Among approximately 120,000 X chromosomes involved in this study, 157 were recombinant B+, and 93 were recombinant BB strands; these correspond to frequencies of 13.1 x and 7.7 x respectively, The probability that an attached-x female offspring would carry both a B+ and a BB chromosome, assuming that these changes arise independently from the parental B strands, is one per million. Yet, among the total population of about 60,000 female offspring observed for such changes, there were 57 B+/BB exceptions in which both chromosomes were recombinant for outside markers. Moreover, in every case the constituent crossover strands carried the marker combinations predicted on the basis of reciprocal products of crosslng over between obliquely paired members of the Bar duplication. This clearly demonstrates that recombinant B+ and BB strands are reciprocal products of the same event, and adds more substantial evidence to L. V. MORGAN S observation (1931) of a single B+/BB attached-x daughter from a mass mating of homozygous Bar, attached-x females. Another, though less stringent, criterion for reciprocity is based on the expectation that if a single exchange in homozygous Bar females gives rise to both B+ and BB crossover products, these should occur with equal frequency. As it turns out, this expectation is not satisfied; the ratio of B+ to BB crossover products was approximately 1.7 : 1. In view of the more critical evidence in support of reciprocity presented above, it seems most reasonable to attribute the inequality of the two crossover products to the lower viability of females heterozygous for BB. It is also likely that, owing to some amount of phenotypic overlap involving B/B

10 494 S. J. GABAY AND J. R. LAUGHNAN and BB/B, some individuals in the latter category escaped recognition as exceptions. Recombinant Bar-locus changes occurred with an overall frequency of one per 480 X chromosomes in the inverted attached-x system. This is somewhat higher than rates observed by ZELENY (1921, 1923), RASMUSON (1957, 1961) and PETERSON and LAUGHNAN (1963), in studies involving the standard mating system, and is more than four times the frequency reported by STURTEVANT (1925). It may be that the higher rates encountered in our studies are characteristic of the particular strain we employed. It is also possible that the crossover event is favored in attached-x chromosomes, or that it is enhanced as a result of the changed position of the Bar duplication, relative to the centromere, in the inverted attached-x chromosomes. In any case, it is apparent that the relatively high rate of crossing over between obliquely synapsed duplication members, along with the precision we have shown for that event, provides an efficient basis for addition of chromosomal material and hence for the establishment of potentially new genes. Genetic and cytological analyses of the nonrecombinant exceptional strands provide evidence on the precision of intrachromosomal crossover events. Ten of the 13 exceptional strands (Table 3) lost one member of the 16A duplication, two gained one member to become triplications, and one, the B+ chromosome of intra-11, lost a segment extending from within the forked cistron through the left member of the 16A duplication. The deleted segment of intra-11, and those of aberrant exceptions I and I1 (Table 4) extend to the left of and well beyond the Bar duplication and it must be concluded that they arose as a result of spontaneous deletion rather than from imprecise intrachromosomal crossovers. Hence, the twelve nonrecombinant nonaberrant exceptional strands, ten of which lost a 16A member, and two of which gained a 16A member, provide strong evidence for an intrachromosomal event which has the precision attributed to standard crossover events. The frequency of intrachromosomal exchanges leading to loss or gain of one member of the 16A duplication is estimated from these data to be about one per 10,000 X chromosomes, or about one for every 20 recombinant exceptions. According to the model for intrachromosomal exchanges proposed by LAUGH- NAN (1955, 1961), if isochromatid and sister chromatid events are equally frequent, nonrecombinant B+ exceptional strands should, on the average, be twice as frequent as nonrecombinant BB exceptional strands. Unfortunately, the number of nonrecombinant exceptional strands identified in this study is not sufficient to afford a critical test of this prediction but, in any case, the finding of ten B+ and two BB chromosomes in this category is not in disagreement with the above prediction. Three of the nonrecombinant exceptions consisted of sister strands recovered in the same half-tetrad and thus afforded the opportunity to test the reciprocity of proposed exchanges within a double-loop configuration. In two of these cases, intra-1 and intra-8, the exceptional females had the genotype B+/B and, since they could have resulted from isochromatid events, they provide no basis to test

11 RECOMBINATION AT THE BAR LOCUS 495 for reciprocity. Intra-9 females, however, had the genotype f + BB od/f $- B od and were cytologically 16A 16A 16A/16A 16A. Females of this type are not expected on the hypothesis of intrachromosomal exchange according to which the nonrecombinant gain of a 16A member by one chromatid is proposed to involve the loss of a 16A member from its sister chromatid. While this single case is not in agreement with the hypothesis, it is obvious that more cases in which the nonrecombinant BB exceptional strand is recovered with its sister strand are needed before any final conclusions concerning the model can be drawn. It is possible that the intra-9 exception, involving apparent nonreciprocal exchange, arose via a copy error in the repair of a chromatid break. Such an error might resemble the copy switches discussed by MESELSON (1967) and used to explain the apparent nonreciprocal exchanges involved in gene conversion. While it is apparent from these studies that intrachromosomal exchanges occur with precision, so far as the balanced nature of crossover products is concerned, and that they lead to gain or loss of duplication members, the question concerning the involvement of iso- and sister chromatid events remains unresolved. LITERATURE CITED DEMEREC, M. and M. E. HOOVER, 1936 Deficiencies in the forked region of the X-chromosome of Drosophila melanogaster. Amer. Nat. 70: 47. LAUGHNAN, J. R., 1955 Intrachromosomal association between members of an adjacent serial duplication as a possible basis for the presumed gene mutations from Ab complexes. (Abstr.) Genetics 40: , 1961 The nature of mutations in terms of gene and chromosome changes. In: Mutation and Plant Breeding, NAS-NRC, 891: LINDSLEY, D. L., 1953 Failure to demonstrate sister-strand crossing over. Drosophila Inform. Serv. 27: MESELSON, M., 1967 The molecular basis of genetic recombination. Edited by R. A. BRINK. pp. 81-1M. In: Heritage from Mendel. University of Wisconsin Press, Madison. MORGAN, L. V., 1931 Proof that bar changes to not-bar by unequal crossing over. Proc. Natl. Acad. Sci. (Wash.) 17: MORGAN, T. H., 1927 Exceptional classes of individuals in an experiment involving the bar locus of Drosophila. Hereditas 9: (Festskrift for W. Johannsen) MULLER, H. 5. and A. WEINSTEIN, 1933 Evidence against the occurrence of crossing over between sister chromatids. (Abstr.) Am. Naturalist 67: PETERSON, H. M. and J. R. LAUGHNAN, 1961 Nonrecombinant derivatives at the bar locus in Drosophila melanogasier. (Abstr.) Genetics 46 : , 1963 Intrachromosomal exchange at the bar locus in Drosophila. Proc. Natl. Acad. Sci. U. S. 50: RASMUSON. M., 1957 Unequal crossing over in the bar region of Drosophila melanogaster. I. High temperature as a means of increasing the frequency. Hereditas. 43: , 1961 Unequal crossing over in the bar region of Drosophila melanogaster 11. Influence of temperature, X-rays and EDTA. Hereditas 47: STURTEVANT, A. H., 1925 The effects of unequal crossing over at the bar locus in Drosophila. Genetics 10: ZELENY, C., 1921 The direction and frequency of mutation in the bar-eye series of multiple allelomorphs of Drosophila. J. Exptl. Zool. 34: , 1923 Further studies of the rate of mutation in the bar series of Drosophila (Abstr.). Anat. Rec. 24: Corresponding editor: G. LEFEVRF