1980; WORTON et al. 1988), raising the question of how genes ..... ucts were lethal over Df(I)bb158 because they lacked sufficient rDNA to allow survival without ...
Copyright 0 1989 by the Genetics Society of America
Reciprocal Recombination and the Evolutionof the Ribosomal Gene Family of Drosophila melanogaster Scott M. Williams,**'James A. Kennison,t'2Leonard G. Robbins* and Curtis Strobeck* *Department ofZoology and tDepartment of Genetics, University ofAlberta, Edmonton, Alberta T6G 2E9; and 'Departmento f Zoology and Genetics Program, Michigan State University, East Lansing, Michigan 48824 Manuscript received August 19, 1988 Accepted for publication March 15, 1989 ABSTRACT T h e role of reciprocal recombination in the coevolution of the ribosomal RNA gene family on the X and Y chromosomes of Drosophila melanogaster was assessed by determining the frequency and nature of such exchange. In order to detect exchange events within the ribosomal RNA gene family, both flanking markers and restriction fragment length polymorphisms within the tandemly repeated gene family were used. T h e vast majority of crossovers between flanking markers were within the ribosomal RNA gene region,indicating that this region is a hotspot for heterochromatic recombination. T h e frequency of crossovers within the ribosomal RNA gene region was approximately 10-4 in both X / X and X / Y individuals. In conjunction with published X chromosome-specific and Y chromosome-specific sequences and restriction patterns, the dataindicate that reciprocal recombination alone cannot be responsible for the observed variation in natural populations.
ULTIGENE families in eukaryotic organisms are known to bemuch more homogeneous within a species than would be expected if the members of the family were evolving independently (HOOD,CAMPBELL and ELGIN, 1975; ZIMMER et al. 1980; DOVER1982;ARNHEIM1983). The greater than expected homogeneity often includes members of a gene family on different chromosomes-homologous and nonhomologous (TARTOF and DAWID 1976;ARNHEIMet al. 1980; WORTONet al. 1988), raising the question of how genes on different chromosomes evolve in concert when the transferof information is presumably limited. Homogeneity of the members of amultigene family, however,requires that some information be transferred among all chromosomal locations of the family. At least two mechanisms may account for this transfer of information: (1) unequal homologous exchange (or exchange between homologous regions of different chromosomes) (OHTA 1980; ARNHEIM et al. 1980; DOVERet al. 1982; DOVER1982; COEN and DOVER1983; ARNHEIM 1983; GILLINGS et al. 1987) and (2)interchromosomal gene conversion (FOGEL and MORTIMER 1969; HOOD, CAMPBELL and ELGIN1975; FOGELet al. 1978; DOVER 1982; DOVERet al. 1982). T o determine how each of these mechanisms might be involved in gene family evolution, it is necessary t o experimentally test the role of each mechanism. The ribosomal RNA (rDNA) gene family of Dro-
M
' Present address: Department of Biology, Boston University, 2 Cummington Street, Boston, Massachusetts 02215. Present address: Laboratory of Molecular Genetics, Building 6, Room 31 1. NICHD, NIH, Bethesda, Maryland 20892. Genetics 122: 617-624 (July, 1989)
sophila melanogaster offers an excellent system for the study of gene family evolution on different chromosomes. It consists of approximately 250 tandemly repeated genes located in the heterochromatin of the X and Y chromosomes (RITOSSA1976). The X-linked and Y-linked rDNA arrays are in general similar in sequence, yet have some discernible differences(TARTOF and DAWID1976; YAGURA, YAGURA and MURAMATSU 1979; INDIK and TARTOF 1980; COEN, THODAY and DOVER1982; WILLIAMS et al. 1987). This paper addresses the role of reciprocal recombination in the evolution of therDNAgene familyof D. melanogaster. Using geneticmarkersflanking the rDNA regions of both chromosomes and molecular markers within therDNA, we have estimated the frequencies of both X-X and X-Y exchange within the rDNAtandemarrays. The dataindicatethat the patterns of variation within and between the rDNA arrays on the two sex chromosomes cannot be explained solely as a product of reciprocal recombination. At least one other mechanism must, therefore, be responsible for the pattern of rDNA variation on the two chromosomes. However, it is possible that XY recombination is important in transferring genetic information from the Y chromosome to the X chromosome array. MATERIALSANDMETHODS
Drosophila stocks and crosses: Unless otherwise noted, mutations and chromosome aberrations used in this study are described in LINDSLEYand GRELL(1968). wha2 is a spontaneous derivative of w' with a phenotype similar to wh. Flies were reared ona yeast-sucrose medium at 25 . O
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Because recombination between rDNA regions is known to be relatively rare in Drosophila, putative recombinants were detected by the exchange of flanking visible markers. Restriction fragment length polymorphisms between chromosomes were then used to identify exchange events within the rDNA regions. X-Xrecombination: The rDNA region of the X chromosome is in the proximal heterochromatin of the left arm (RITOSSA 1976) and has been named the bobbed (bb) locus becauseof the phenotype of partial deficienciesof the region. The nearest known conventional genetic locus distal to the rDNA is the suppressor of forked [sum] locus. The right arm of the X chromosome, and all of the left arm proximal to the s u m locus, are not necessary for viability or fertility in the presence of another bb locus. In order to mark the right arm of the X , we used a rearrangement in which the extreme distal portion of the left arm of the X carrying they+ allele has been duplicated on the right arm. Females heterozygous for this Dp(l;l)sc'" du lication chromosome (carrying the marker mutations y wh 'fear s u m in the left arm) and a Canton S X chromosome (carrying the marker mutations y wha2f) were crossed to y a c v f s u m males. Two differentCanton S X chromosomes both marked withy whRZfwere used. Each X chromosome stock used was derived from a single male shortly before the start of the experiments. All copiesofeach parental X chromosome were therefore isogenic. On subsequent analysis it was determined that the two Canton S X chromosomes had identical rDNA restriction patterns. Recombinant chromosomes were detected as y y ( t h e y phenotype results from the s u o genotype) or y + J males or females (Figure la). All recombinants were made into balanced stocks and subsequently crossed toa Df(l)bb158 chromosome with the marker mutation y for the molecular analyses.Df(l)bb158is deficient for 82% of the X heterochromatin, and is deleted for all of the ribosomal genes (LINDSLEY and ZIMM 1987). Thus, flies bearing any recombinant chromosome and Df(l)bbl58contain ribosomal DNA only from the recombinant chromosome. DNA was extracted from 50-150 mg of adult flies by theprocedure of ISH-HOROWICZ et al. (1979), except that the DNA was pelleted in a centrifuge at 10,000 x g for 10-20 min instead of being spooled out. The DNA was then digested with either Hind111 or Hind111 and EcoRI. Reaction conditions were as prescribed by the manufacturer (Bethesda ResearchLabs).Digested DNA samples werethen run on 0.6% agarose gels, transferred to nitrocellulose and hybridized with 92P-labeledpDmrY22c DNAas previously described (WILLIAMS, DESALLEand STRORECK 1985). Since the DNA was extracted from adult flies, restriction pattern differences reflect genomic differences and should be unaffected by stage of tissue specific 1979). replication of the rDNA (ENDOWand GLOVER The clone of a complete ribosomal gene repeat, pDmrY22c, was kindly provided by I. DAWID.The rDNA from the parental X chromosomes differed with respect to their banding patterns after digestion with these restriction enzymes.In addition to somecommon bands seen in both chromosomes, there are bands diagnostic of each chromosome (Figure 2a). We emphasize that the number of rDNA crossovers as determined by molecular analysisis only a minimal estimate of exchange in the rDNA. Notall exchanges within the rDNA can be resolvedby the molecular markers used. Only exchange products that have rDNA variants unique to both parental chromosomes can bedetected by molecular analysis as recombinants, whereas all single crossovers will be detected by the exchange of flanking markers. The actual frequency of within-rDNA crossovers must lie betweenthe
r
Y
f
+
I M
Y -I
f
+
I
Y'
XW
b
C
FIGURE1 .-Schematic representation of crosses and recombinant chromosomes. X-X recombination, (a) shows the parental female genotype diagrammed on the left. On the right are the two reciprocal products of an exchange event in the rDNA region. The wild-type (+) us. mutant allele for the s u m locus and the presence (shown as y+) or absence of Dp(I;I)scv' were used to detect the exchange events. X-Y recombination, (b and c) show the parental males and exchange products for crosses I and 11, respectively. The parental males in panel b carry a Y chromosome with the markers Byon the long (YL.)arm and y+ on the short (YS)arm. In panel c, the parental males bear an X chromosome with the mutant alleley' on the left arm, andDp(l;l)scv' (shown asy+) on theright arm. For all X and Y chromosomes in this figure, the rDNA regions are shaded.
frequency estimated by exchange between the visible outside markers and the frequency of crossovers between diagnostic molecular markers within the rDNA itself. X-Y recombination: Males of twogenotypes were used to study X-Y recombination. One cross (designated cross I) used males witha normal X chromosome that carried the markers y whRZf and a marked Y chromosome that carried the dominant markers BS and y+ on the long and short arms respectively (Figure 1b). The other cross (designated cross 11) used males with an unmarked Y chromosome from an Oregon R strain and an X chromosome bearing the Dp(1;I)mV'duplication already described. The duplication chromosome carried the recessive marker mutations yz su(w') w' cu vfin the left arm and y+ on the duplicated segment (Figure IC). The second cross was done to determinewhether the appended euchromatic markers, BS and y+, have any effect on recombination frequencies. All of the males in each cross carried the same X and Y chromosomes that were derived from single flies just prior to the experiment. Both genotypes were crossed to YSX.YL, I n ( l ) E N , y virgin females. Recombinants from cross I were detected as y+ B+ males and y BS females. In cross I1 the recombinant chromosomeswere recovered in y+ males and y2 females. All putative recombi-
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-C
FIGURE2.-Autoradiographs of DNA cut with various restriction enzymes, run on agarose gels. and hybridized with the ribosomal gene probe pDmrY22c. a, An example of the data from the X-X recombination experiments. The two outer lanes are DNA from the two parental X chromosomes and the middle lane is DNA from one of the recombinant chromosomes. The DNA was digested with the restriction enzymes EcoRI and HindllI. The parental chromosome on the left [labeled s u o ] has the unique bands A and C, while the parental chromosome on the right [labeled Canton S] has the unique bands B and D. The recombinant chromosome has all four bands (A, B, C and D), demonstrating that it is the result of recombination between rDNA cistrons. The approximate sizes of the diagnostic fragments are as follows: A = 8.4 kb, B = 8.2 kb, C = 6.6 kb and D = 6.1 kb. b, An example of the data from the X-Y cross I experiment. Again the DNA was digested with both EcoRI and HindllI. The lanes marked X and Y are DNA from the parental chromosomes; the other three lanes are DNA from three different recombinant chromosomes. Band A is unique to the parental Y chromosome and band B is unique to the parental X chromosome. Recombinant I contains both bands A and B, showing that it results from recombination within the rDNA region of both parental chromosomes. Recombinants I I and 111 are not resolved as rDNA recombinants using these particular restriction enzymes. The approximate sizes of the diagnostic fragments are as follows: A = 8.5 kb and B = 7.8 kb. c, X-Y cross I 1 experiment. The DNA was digested with HindlII. The lanes from the parental chromosomes are labeled X and Y, and have the unique bands A and B, respectively. Band C is common to both parental chromosomes. The otherlanes are DNA from three different recombinant chromosomes. Recombinants in lanes I and I 1 have both bands A and B, showing that they are the products of recombination between both parental rDNA regions. Note that band C is lacking in the recombinant in lane I , suggesting that either the exchange event was unequal or that band C is in different positions in the rDNA regions of the two parental chromosomes. The sizes of the labeled fragments are as follows: A = 1 1.5 kb, B = 10.5 kb and C = 9.5 kb.
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Heterochromatic exchange between sex chromosomes X-Y recombination
x-x Exchange
recombination
Total scored Flanking marker exchange Exchange within rDNA
56,256 16
42,184 40,411 5 (3 X.YL)
12 (6 X.YL)
13
4 (3 X.YL)
7 (4 X.YL)
Cross 1
Cross 11
nant chromosomes were balanced for subsequent genetic and molecular analyses. The X-Y recombinant chromosomes were genetically analyzed for their Y material (KENNISON 1983). The molecularidentification of exchange events within the rDNA regions was performed as described above for the X-X exchange events (Figures 2b and 2c). of the X and Y Exchange between the rDNA cistrons chromosomes yields two types of recombinantchromosomes. The first type contains the X euchromatin and the long arm of the Y chromosome (designatedX.YL) and behaves as a normal X chromosome in terms of sex determination and other genetic properties.It should be recoverable from rDNA exchangesin natural populations. The other type of recombinant chromosome (designated YSX.) contains the short arm of the Y chromosome appendedto the centric heterochromatin (includingthe centromere) of the X chromosome. YSX. lacks the genes located on the long arm of the Y chromosome that are required formale fertility (STERN 1929). In natural populations, YSX. recombinants would be found only in sterile males. Thus, the YSX. chromosome is an evolutionary dead end. The frequency of X-Y exchange important for the evolution of the rDNA should, therefore, only be calculated from the X. YLchromosomes recovered.
RESULTS
X-X recombination: Of a total of 56,256 progeny recovered, only 16 were recombinants between the s u o locus and the duplication marking the right arm (Table 1). Of the 16 recombinantsbetween the flanking markers, 13 could be shown to result from exchange between the rDNA of the two parental chromosomes using restriction fragment length polymorphisms. T h e frequency of X-X exchange in the rDNA region is, therefore, between 2.3 X and2.8 X lop4 (the estimates from the molecular and genetic analyses, respectively). X-Y recombination: T h e total number of progeny scored and the number ofexchangesbetween the genetic markers flanking the rDNA in the Y chromosome are shown in Table 1. Of a total 82,595 progeny scored, 17 recombinants were crossovers between klI and ks-I onthe Y chromosomeandthe centric heterochromatin of the X chromosome. No significant difference was found in the frequencies of exchange in cross I and cross 11 ( x 2= 0.8765, 1 d.f., 0.1 < P < 0.5). Thus, presence of the transposed X chromosome fragmentsonthe BsYyC chromosome oronthe Dp(1 ;I)sc"' chromosome does not affect recombination in the rDNA region. Twoof the exchange prod-
ucts were lethal over Df(I)bb158because they lacked sufficient rDNA toallow survival without the presence of rDNA elsewhere in the genome. Only the 15 exchange products that wereviable in combination with Df(I)bbI58 were analyzed for exchange within the rDNA by molecular techniques. Ten of these 15 were clearly fromexchanges within therDNA regions. Another of the flanking markerrecombinants was subsequently found to be a product of exchange in therDNA region using thenontranscribed spacer probe, pDmr103HH2 (S. M. WILLIAMS and P. CLUSTER, unpublished results). Of the 17 X-Y crossovers, nine were recovered as X.YL chromosomes. Eight ofthesewere analyzed using molecular techniques. Seven of the eight were shown to beexchanges betweenrDNA cistrons (Table 1). T h e frequency of all X-Y exchanges is, therefore, between 1.3 X 10-4 and 2.1 x 10-4 and thefrequency of X-Y products that are recoverable in natural populations (X.YL chromosomes) is between 8.5 X and 1.1 X In addition to the recombinantsshown in Table 1, three otherexceptional progeny were recovered (two from cross I andonefrom cross 11). One of the exceptionaloffspringfrom cross Icarrieda BSYyf chromosome that had lost the Bs marker. The other exception from cross I carried an element resulting from exchange betweenkl-I and the bb locus of the Y chromosome and an unknown chromosome (the element is BSKL.bb-). If the exchange that produced this element was between the rDNA regions on the X and Y chromosomes, this product would be a dicentric chromosome. It is more likely that the exchange involved the small fourth chromosome (PARKER 1967). T h e exceptionaloffspring from cross I1 carrieda chromosome lacking the majority of the paternal X chromosome, but still bearing bb+ and Dp(1 ;I)sc". No evidence of Y chromosome fertility factors could be detected in this chromosome. In neither the X-X nor the X-Y exchange experiments was there any indication of clustering of crossovers. Thus, the recombination events are probably meiotic. DISCUSSION T h e X-linked and Y-linked rDNA of D.melunoguster are, in general, homogeneous at the level of DNA restriction enzyme site variation within an array as well as very similar betweenarrays(TARTOF and DAWID 1976; COEN, THODAY and DOVER1982; WILLIAMS et al. 1987). There exist, however, some diagnostic differences between the arrays on the two types of chromosomes.Nucleotidesequence of the 18 S genes appear to be different between the two arrays (YAGURA, YACURA and MURAMATSU1979), as do somespacerlengthvariants(INDIK and TARTOF
Recombination and 1980), as well as the distribution of moderately-repetitive DNA inserted in the28 S genes(WELLAUER, DAWID and TARTOF 1978). One class of inserts, called Type 1, are found only in the X-linked rDNA genes (WELLAUER, DAWID and TARTOF 1978). In addition, X-linked rDNA spacers are significantly more similar to each other than Y-linked rDNA spacers are toeach other (WILLIAMS et al. 1987). The pattern of gross similarity in restriction site maps, with diagnostic differences between the X and Y chromosome rDNA arrays, implies that, although some isolation between the two rDNA arrays exists, some mechanism must keep the genes on the X and Y chromosomes at least marginally similar. Natural selection may be partially responsible for this observation (TARTOF and DAWID 1976), butit is unlikely to be the sole mechanism since natural selection appears to be operating much more strongly on X-linked rDNA arrays (WILLIAMS et al. 1987). This leaves the mechanisms included in the term molecular drive (DOVER1982). One of the mechanisms proposed to homogenize tandemly repeatedsequences is unequal reciprocal recombination (SMITH 1976, PERELSON AND BELL 1977; HOOD,CAMPBELL and ELCIN1975; DOVERet al. 1982; COEN and DOVER1983; GILLINCSet al. 1987). In order toassess the importance of reciprocal recombination for the evolution of rDNA sequences in Drosophila, we estimated the frequency of recombination between rDNA regions in both X-X and X-Y individuals. Previous estimates of rDNA exchange in Drosophila have not been designed to recover all of the recombinant types (STERN1929; SCHALET1969; MADDERN1981; HAWLEY andTARTOF 1983), have often used chromosomes of inverted sequence or unknown rDNA provenance (NEUHAUS 1937; LINDSLEY 1955), andhave failed to directly compare X-X and XY recombination frequencies. Our experiments were designed to recover all recombinant types from both X-X and X-Y events (even those that are not recoverable in natural populations) and to compare,as directly as possible, recombination in XlXand XIY individuals. T h e use of visiblegenetic markers flankingthe rDNA regions on both the X and Y chromosomes allows us to detect and recover all single crossovers within the rDNAregions. Because the exchange of flanking markers could also result from exchange in the heterochromatinflankingtherDNAregions,thefrequency of exchange of flanking markersis a maximum estimate of rDNA recombination frequency. T h e use of molecular markers within the rDNA regionsallows us to differentiate most crossovers in the rDNA from crossovers proximal or distal to the rDNA. T h e frequency of crossovers between the restriction fragment length polymorphisms within therDNAregion is, however,a minimum estimate of the frequency of rDNArecombination. As the majority of flanking
rDNA Evolution
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markerexchangecouldbe shown to be between rDNA cistrons, there is little difference between the minimum and maximum estimates of rDNA recombination. These experiments also map the regions of heterochromaticexchange in the X and Y chromosomes more accurately than previous studies. From the size of the rDNA repeat and the number of copies per chromosome it can be estimated thatthe rDNA region comprises approximately 5% of the Y chromosome APPELS and 25%of the X heterochromatin (HILLIKER, and SCHALET1980). The distribution of radiationinduced X-Y translocations agrees with these physical estimates (KENNISON1981). Seven percent of the radiation-induced exchanges were between the closest loci flanking the Y chromosome rDNA, and approximately 17% were within the X chromosome rDNA. T h e location of spontaneous exchanges between the X and Y chromosomes are decidedly nonrandom. The vast majority, and possibly all, of the X-Y and proximal X-X exchanges are within therDNA regions. Although much of the non-rDNA Y and X heterochromatin are not homologous (HILLIKERand APPELS 1982), which would indicate that recombination would have to be between the rDNA of these two chromosomes, the non-rDNA of the two X chromosomes are homologous. This suggests that rDNA exchange is not random breakage and rejoining in a translocation-like event, but is the result of either a specific recombination system forthe exchange of rDNA or a specific response of the rDNA to the generalrecombination machinery. For the spontaneous X-Y exchanges reported here, 100% (17117) were between the Y chromosome loci that flank the rDNA region,significantly different from the 7% seen for radiation-induced exchanges. At least 11 out of the 17 were within the X chromosome rDNA itself. The distribution of spontaneous X-X exchanges is also significantly different from the 17% within the rDNA seen for the induced exchanges.At least 13 of the 16 crossovers were within therDNA.Takentogether these results clearly indicate that the rDNA is a hotspot for heterochromatic recombination, as suggested by MADDERN(1 981). Although the frequency of X-X crossovers between the rDNA cistrons is not significantly different from the total frequency of X-Y crossovers in the same region ( x 2= 0.8699, 1 d.f., 0.2 < P < 0.5, from the flankingmarkerdata), the frequency of recovered X.YL chromosomes was significantly lower ( x 2 = 5.722, 1d.f., 0.01 < P < 0.025). The two- to threefold difference in recovered X vs. X . YL recombinant chromosomes has implications for the transfer of genetic information between the two chromosomes as well as for the homogenization of their rDNA arrays. These
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S. M. Williams et al.
two phenomena, although related, should be considered separately. Our data suggest that the movement of an X chromosome rDNA sequence toanother X chromosome is more probable than the movement of an X derived sequence to a Y chromosome. This is based on the difference in frequency of the two types of events and the necessity ofa second recombination eventto make an X.YL into a normal Y chromosome. Assuming the second recombination event occursat about thesame frequency as the original X-Yexchange, the difference in the frequency of X to X transfer vs. X to Y will be approximately 4-9 x lo4. With one modification, the same difference in frequency holds for Y chromosome to X chromosome transfer.The difference is that X.YL chromosomes can spontaneously lose the YL arm, yielding cytologically normal X chromosomes. This has been observed in some X . YL chromosomes keptin the laboratory (GILLINGS et al. 1987). Therefore, the ease of transfer of rDNA sequences is ordered (in decreasing likelihood) fromone X chromosome to another, from a Y chromosome to an X chromosome, and froman X chromosome to a Y chromosome. Transfer from a Y chromosome to another Y chromosome via reciprocal recombination will be about as difficult as movement from an X array to a Y array because this can be accomplished only via an X.YL intermediate. This is consistent with the presence of type I inserts in 28 S genes of X chromosomes but not Y chromosomes. The inference would be that type I inserts arose in the X chromosome and rarely, if ever, were transferred to the Y chromosome, whereas the type I1 inserts originated on the Y chromosome and were able to spread to the X linked arrays. Unlike informationtransfer,homogenizationrequires multiple recombination events amongall chromosomes. The model of OHTA andDOVER(1983) can be used as a framework for interpreting our data in terms of these repeated events. Their model analyzes the effects of rate differences of asymmetric gene conversion within one chromosome and asymmetric gene conversion between homologous and nonhomologous chromosomes. It mayalso be used to approximate symmetric events and reciprocal recombination with minor modification (OHTAand DOVER 1983). With this in mind, the frequencies we measured can beinterpreted using their model in the following way: (1) X-X recombination can be viewed as analogous to one of their gene conversion events. Using OHTA and DOVER’S terminology, X-X reciprocal recombinants are symmetrical and terminal with respect to the rDNA; (2) each X-Y crossover canbe viewed as one half of one of their conversion events because, as discussed above, it takes two events to transfera block of informationfrom one intact X chromosome to an intact Y chromosome (a product
similar to their geneconversion product) or vice versa. The second event can be either another recombination event or a spontaneous deletion of the YL arm from an X.YL chromosome. In the case of X-Y recombination, the events are effectively asymmetric because reciprocal products do not survive in natural populations. If the second event can only be reciprocal recombination, the frequency of “conversion-like” events will be approximately fourorders of magnitude higher for X-X recombination than for X-Y recombination. The predictedoutcomefrom this ratio of frequencies is little or nosimilarity between rDNA on the X and Y chromosomes. This predicted divergence should be even greater because of the selective disadvantage of the X.YL chromosome (STERN1929; R. FRANKHAM, personal communication). Only if the YL arm is deleted at a very high frequencycould the observed similarity of rDNA variation on the X and Y chromosomes be explainedas a consequenceof reciprocal recombination. This does not seem likely because after almost 10 yr only 5 of 17 X.YL chromosome containing lines lost all or part of the YL (GILLINGS et al. 1987). We would, therefore, expect very different patternsof variation in X and Y chromosome rDNA arrays if recombination of the type we measured were the major mechanism for the evolution of Drosophila rDNA. Since reciprocal recombination by itself cannot yield the observed pattern of Drosophila rDNA variation, another mechanism must be considered. T h e mechanism most commonly invoked is gene conversion (asymmetrical exchange of information). However, depending on the exact mechanism of conversion, it alone may also be insufficient to explain natural variation.Forexample, ifwe assume: (1)thatthe recombinantchromosomes we recoveredwere the products of conversion events that were resolved as crossovers; and (2) that the probability of resolving conversion events as crossovers is the same for both X-X and X-Y events, then we can reach the following conclusions about gene conversion and rDNA evolution. If flanking marker crossovers occur in approximately 50% of conversion events (FOGELand MORTIMER 1969; FOGEL,MORTIMERand LUSNAK 198l), the effective frequency of X-X exchange would be twice that of X-Y exchange. This is based on the fact that XY conversion with crossing-over requires a second rare event to produce normal X and Y chromosomes, but conversion without crossing-over does not.If a smaller fraction of the conversion events yield crossovers (KLEIN 1984; KLAR andSTRATHERN1984; JINKSROBERTSON and PETES1985), the frequencies of effective exchange will be even closer. In either case, the model of OHTA andDOVER(1 983)would predict virtually indistinguishable identity coefficients with no more differences between X and Y rDNA arrays than
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DOVER, G . A., S. BROWN,E. S. COEN, J. DALLAS, T . STRACHAN and between different X arrays. This is clearly not the M. TRICK, 1982 T h e dynamicsofgenomeevolutionand case. 343-372 in Genome Evolution, edited species differentiation, pp. It is unlikely that either gene conversion or recipby G. A. DOVERand R. B. FLAVELL.AcademicPress,New rocal recombination aloneis responsible for molecular York. ENDOW,S. A,, and D. M. GLOVER,1979 Differentialreplication structure of the rDNA cluster. Geneconversion even of Drosophila of ribosomal gene repeats in the polytene nuclei with accompanying crossing over would yield homoCell 17: 597-605. melanogaster. geneity, and crossing-over alone would yield diverFOGEL,S., and R. K. MORTIMER,1969 Informationtransfer in gence. However,acombination of the two could meiotic gene conversion. Proc. Natl. Acad. Sci. USA 62: 96produce the level and type of variation observed. The 103. FOGEL,S., R.MORTIMERand K. LUSNAK,1981 Mechanismsof ratio of conversions to crossovers needed to produce meitotic gene conversion,or “wandering on a foreign strand,” the observed pattern of variation depends on how pp. 289-339 in The Molecular Biology ofthe Yeast Saccharomyces: often recombination complexes are resolved as crossE. W. L$e Cycle and Inheritance, edited by J. N. STRATHERN, overs with no conversion. It may also be worth considJONES and J. R. BROACH. Cold Spring Harbor Laboratory Press, ering that heteroduplexes are certainly much shorter Cold Spring Harbor, N. Y . FOGEL, S., R. K. MORTIMER, K. LUSNAKand F. TAVARES, than the length of the rDNA cluster and conversion 1978 Meiotic gene conversion: a signal of the basic recombiwill only transfer information within these short segnation event in yeast. Cold Spring Harbor Symp. Quant. Biol. ments while crossovers will transfer much larger por43: 1325-1 342. tions of the rDNA. Alternatively, a high frequencyof GILLINGS,M. R., R. FRANKHAM,J.SPEIRSand M. WHALLEY, double crossovers (high negative interference) may be 1987 X-Y exchange and coevolution of the X and Y rDNA arrays in Drosophila melanogaster. Genetics 116 24 1-25 1 . important. Since our data demonstrate that the rDNA HAWLEY, R.S., and K. TARTOF,1983 T h e effectofmei-41 on is a recombinational hotspot in a recombination-less rDNA redundancy in Drosophilamelanogaster. Genetics 104: region the possibility exists that rDNA recombination 63-80. is not conventional. Double recombinantsmay, thereHILLIKER, A.J., and R. APPELS, 1982 Pleiotropic effects associfore, occur relatively frequently. Because we could ated with the deletion of heterochromatin surrounding rDNA not detect these events in our study, we cannot address on the X chromosome of Drosophila. Chromosoma 8 6 469490. this possibility directly. HILLIKER, A. J., R. APPEIS and A. SCHALET,1980 T h e genetic It is clear that knowing the frequency of events is analysis of Drosophila melanogaster heterochromatin. Cell 21: not sufficient (although it is necessary) for understand607-619. ing the importance of a given mechanism in the evoHOOD, L., J. H. CAMPBELL andS. C. R. ELGIN,1975 The orgalution of a gene family. It is also necessary to know nization, expression,and evolution of antibody genes and other multigene families. Annu. Rev. Genet. 9 305-353. the exact mechanism thatunderlies the observed INDIK, 2. K., and K. D.TARTOF, 1980 Longspacersamong events. How heteroduplexes are resolved will cerribosomal genes of Drosophila melanogaster. Nature 284 477tainly affect how a family evolves. 479. We thank J. CORREIA for technical assistance and P.CLUSTER, G . DOVER,G . B. GOLDING, JOHNSON D. and R.RASOOLY for reading and commenting on earlier versions of this paper. I. DAWIDprovided the rDNA clone, pDmrY22c. M. DRYGAS-WILLIAMS helped with the graphics. This work was supported by Alberta Heritage FoundationforMedicalResearch Fellowships (toS.M.W.and J.A.K.), Natural Science and Engineering Council (Canada) grants to C.S. and to M. A. RUSSELL.
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