population genetics of tandem repeats in …population genetics of responder 479 w 250- 200- 150 -...

Copyright 0 1993 by the Genetics Society of America

Population Genetics of Tandem Repeats in Centromeric Heterochromatin: Unequal Crossing Over and Chromosomal Divergence at the Responder

Locus of Drosophila melanogaster

Eric L. Cabot,* Pam1 Doshi? Mao-Lien Wu* and Chung-I Wu*”

*Department ofEcology and Evolution, University of Chicago, Chicago, Illinois 60637, and ?Department of Biology, University of Rochester, Rochester, New York 14627 Manuscript received March 23, 1993

Accepted for publication June 12, 1993

ABSTRACT The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion

and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts ofany single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.

T HE Responder (Rsp ) locus is the target of the segregation distortion meiotic drive system in

Drosophila melanogaster (GANETZKY 1977; LYTTLE 199 1; TEMIN et al. 199 1 ; Wu and HAMMER 199 1). It is localized next to the centromere of the right arm of the second chromosome (BRITTNACHER and GA- NETZKY 1989; LYTTLE 199 1 ; PIMPINELLJ and DIMITRI 1989). Rsp has been shown to be comprised of a localized array of tandem repeats. Chromosomes car- rying large Rsp repeat arrays are sensitive to the action of the Segregation distorter locus, which render Rsp- bearing sperm nonfunctional, whereas chromosomes that have only a small number of Rsp repeats are insensitive to segregation distortion (Wu et al. 1988).

This study does not directly address the properties of segregation distortion. Rather, it focuses on the evolution of tandem repeats and the evolution of the centromeric heterochromatin. Both subjects are of considerable interest in population genetics (STEPHAN 1989; BERRY, AJIOKA and KREITMAN 1991 ; BEGUN and AQUADRO 1992). The Rsp repeat array is a good model system for such inquiries for the following

I To whom correspondence should be addressed.

Genetics 135: 477-487 (October, 1993)

reasons. First, the copy number of Rsp repeats varies greatly among different naturally occurring second chromosomes, as determined by the chromosomes’ sensitivity to segregation distortion (TEMIN and MAR- T H A ~ 1984) and by molecular means (WU et al. 1988). To account for this large spectrum of repeat lengths, Wu and HAMMER (1 991) suggested unequal crossing over as the mechanism that generates copy number variation. Sequence variation among Rsp repeats per- mits us to study the evolution of the tandem array and the recombination mechanisms underlying it. Sec- ond, the Rsp repeat array is localized in the centromeric heterochromatin of the second chromosome and is perhaps closer to the centromere than any other known locus in Drosophila (BRITTNACHER and GA- NETZKY 1989; LYTTLE 1991 ; PIMPINELLI and DIMJTRJ 1989). Since it is in a region where recombination between homologous chromosomes is low, much of the unequal crossing over is likely to be between sister chromatids, as other studies of unequal exchange have shown (ENDOW, KOMMA and ATWOOD 1984; KADYK and HARTWELL 1992). In the extreme case when exchanges between homologous chromosomes are

478 E. L. Cabot et al.

sufficiently infrequent relative to exchanges between sister chromatids, repeats within the same chromosome might be more similar than repeats from different chromosomes. This would be analogous to the proposal of concerted evolution in which multigene sequences are more similar within species than between species [see ARNHEIM (1983) for a review].

Third, the cytological location and the repeat structure of Rsp are interesting with respect to the population genetics of regions of reduced recombination, notably centromeric and telomeric regions (BERRY, AJIOKA and KREITMAN 199 1 ; BEGUN and AQUADRO 1992). The prevailing view is that the substitution of a newly arisen allele favored by natural selection would carry nearby regions along with it to fixation and temporarily reduce the level of polymorphisms in the vicinity. This process is often referred to as a “selective sweep.” The higher the density of genes per unit of recombination distance, the greater the prob- ability of reduced polymorphism.

The centromeric and telomeric regions are conspic- uously associated with recombination reduction and indeed appear to have much reduced polymorphism (BEGUN and AQUADRO 1992). Interestingly, these hap- pen to be two regions of special chromatin properties. For example, MCKEE and LINDSLEY (1 987) reported that heterochromatic pairing and the segregation ratio between homologs are inseparable properties. Moreover, the bias in segregation appears to favor the one with less heterochromatin. There is indeed an extensive body of literature on pairing and segregation (BAKER and CARPENTER 1972; YAMAMOTO 1979; HILLIKER, HOLM and APPELS 1982) which we will address in Discussion. Since unequal segregation is expected to drive a chromosome to fixation very rapidly (SANDLER and NOVITSKI 1957), polymorphisms near the centromere could also be reduced by meiotic aberration associated with the centromeric heterochromatin. We shall refer to this hypothetical process as “meiotic sweep.” Selective sweep is due to positive Darwinian selection that drags a piece of chromatin to fixation whereas meiotic sweep is a consequence of aberration in chromosome mechanics. [Meiotic sweep referred to in this report should not be confused with the known systems of segregation distortion, which are the manifestation of genic inter- actions rather than chromosomal mechanics (LYTTLE 1991; WU and HAMMER 1991).]

It is possible to look for the “footprint” of selective sweep (ie., region of reduced polymorphism) by ex- amining regions away from the centromere or telo- mere. Nevertheless, it is desirable to be able to rule out meiotic sweep as an explanation for the observed reduction in polymorphism in the centromeric region, which consists of proximal euchromatin and heterochromatin. Although the two types of sweep appear

to generate similar predictions for the proximal euchromatin, the “selective sweep” model may, under certain conditions, yield a different prediction for centromeric heterochromatin. This is because the chance of being selectively swept depends on the density of genes per unit of recombination distance and centromeric heterochromatin probably has a low gene density judging from the marked paucity of lethal complementation groups (HILLIKER, APPELS and SCHALET 1980). Thus, the “footprint” of selective sweep is expected to cover proximal euchromatin more often than it covers the gene-poor heterochromatin. Depending on the strength of positive selection and the location of the favored mutation in relation to the region of recombination suppression, the age of alleles in heterochromatin (as reflected in nucleotide polymorphism) could be older than those at proximal euchromatin. While such a pattern may or may not be the prediction of a selective sweep model, it should never be expected if meiotic sweep is the predominant force governing polymorphism in the centromeric region. Therefore, it may be possible to reject the meiotic sweep model. Unfortunately, sequence variation at single gene loci in the centromeric region is usually too small to reveal any age differences between different subregions (BERRY, AJIOKA and KREITMAN 1991; BEGUN and AQUADRO 1992). Be- cause tandem repeats in heterochromatin are highly variable in both array length and DNA sequence, [for example, each of 40 chromosomes surveyed has a different Rsp allele as revealed by genomic Southern hybridization (Wu et al. 1988)], they may provide the resolution for detecting age differences between proximal euchromatin and heterochromatin.

Fourth, the size of the Rsp tandem array has been shown to have a fitness consequence, in addition to the segregation distortion phenotype. Short arrays, which normally confer insensitivity to distortion, are at a selective disadvantage in the absence of segregation distortion (Wu, TRUE JOHNSON SON 1989). Some of the molecular properties of Rsp have also been determined, including the sequence-directed curva- ture, the unusual nucleosome spacing and the presence of Rsp-binding proteins (DOSHI et al. 199 1). Such detailed knowledge may be valuable for studying the organization and evolution of the heterochromatin and tandem repeats. Before addressing the questions discussed above, it was necessary to determine the large-scale molecular structure of the Rsp array. This was done by isolating large intact DNA segments (on the order of >1 Mb) from nuclei embedded in agarose blocks and by fractionating DNA in a field inversion gel.

MATERIALS AND METHODS Chromosomes: Four different chromosomes were used

in this study. A description of them, including their sources

Population Genetics of Responder 479

w 250- 200-

150 -

100 -

48-

23.1 -

FIGURE 1.-Southern blot of high molecular weight genomic DNA from cn b w , It pk cn b w , and R31 strains digested with restriction enzymes EcoRl (RI). and BamHI (HI) and hybridized to a RsP probe. The sizes (in kb) shown on the left are based on concatemers of bacteriophage X which were used as size markers. See MATERIALS AND METHODS for more detail.

and properties, can be found in LYTTLE, BRITTNACHER and GANETZKY (1 986). They are: (1) It p k cn bw (abbreviated Ip), a supersensitive chromosome with about 2500 copies of RsP repeats. (2) cn bw (abbreviated cb), a sensitive chromosome with about 700-800 copies. Both chromosomes are standard in the genetic analysis of segregation distortion. (3) Canton- S (cs) is a semisensitive chromosome with about 300 copies of Rsp repeats. (4) cn b d " (R31) was an insensitive chromosome, created from cn bw by X-ray irradiation (GA- NETZKY 1977). It is homozygous inviable and is maintained over the Cy0 balancer, which has only a few copies of Rsp repeats (WIJ et al. 1988).

Preparation of high molecular weight DNA: High molecular weight DNA was prepared from third instar larvae. Brains from 20 larvae were dissected in Ringer's solution and homogenized in 250 pl of buffer A (30 mM Tris, pH 8.0, 100 mM sodium chloride, 50 mM EDTA, 0.5% Triton X-100, 7.7 mM j3-mercaptoethanol; the last component was added just before use). Then 1 % low melting agarose (Seap

laque, FMC Corp.) was prepared in buffer A without p- mercaptoethanol and equilibrated at 37". An equal volume of agarose was added to the homogenate, mixed gently to avoid air bubbles and 100 pl of this suspension was poured into the wells of a block-maker (Bio-Rad Laboratories). The agarose was allowed to solidify on ice for 30 min. The blocks were then dropped into the NDS lysis buffer (10 mM Tris. pH 9.5, 0.5 M EDTA, 1% sodium Sarkosyl, 2 mg/ml pro- teinase K) and incubated at 37" for 30 min followed by incubation at 50" for 48 hr. The blocks were then washed in TEP (10 mM Tris, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride for 12-16 hr at room temperature on a shaker. The phenylmethylsulfonyl fluoride was washed off in multiple volumes of T E before restriction digest. For restriction digestion of high molecular weight DNA immobilized in agarose, the blocks were first equilibrated in appropriate restriction buffer for 6 hr. The DNA was then digested overnight in fresh restriction buffer containing 100 pg/ml acetylated bovine serum albumin and 100 units of the appropriate restriction enzyme at 37".

Field inversion gel electrophoresis (FIGE): A 0.8% agarose gel was cast and agarose blocks containing restriction digested DNA were inserted into the wells. Insertion was carefully done to avoid trapping air bubbles. The blocks were immobilized by pouring 0.5% agarose into the wells. The gel was electrophoresed in the cold-room in 0.25 X T B E at 170 V for 17 hr. We used program 4 of the PPI- 100 power inverter of M. J. Research (forward time ranging from 0.03 to 9 sec and reverse time from 0.01 to 3 sec in 300 steps of increment). The high molecular weight markers were in multiples of X phage genome (48 kb), up to the 250- kb range. Southern blotting and hybridization were carried out as described in WIJ et al. (1988).

Cloning and sequencing of Rsp repeats: The experiment was performed in a single step by directly cloning the digested genomic fragments into the M 13 sequencing vector and then screening the plaques with the Rsp sequence probe. Genomic DNAs of the cb and Ip stocks were digested to completion with the XbaI restriction enzyme. M 13mp( 19) circular RF DNA (BRL) was digested with Xbal and then dephosphorylated with calf intestine alkaline phosphatase according to the specifications of the supplier (Promega). The concentration of dephosphorylated vector DNA was about 40 ng/pl and the concentration of the genomic DNA was about 200 ng/pI in the ligation mixture. Ligation reactions were incubated at room temperature for 3 hr and 0.2 volume of ligation mixture was used in the subsequent transfection reactions with freshly prepared competent JM 10 1 cells. Plaque hybridization was carried out and positive clones were picked. The "C" test was done on all positive clones against a pair of standard Rsp clones prior to

M 1 2 T M " A T 6 T ~ T 6 C U M M G U M M U A C A " M C T C C M 5777 Rsp-412 --C-T-6------T----------T6T----------6-~-----6.----C--------T-~--A-T---C---AC-A------------l---- 398

FIGURE 2.-Alignment of the 5"flanking sequence of Rsp clone Ho (Wu et al. 1988) to the pol region of the retrotransposon 412 reported by YUKI et al. (1986) reveals that this Rsp clone is flanked by a 4 1 2 element (designated Rsp-412). Dashes represent positions in Rsp-412 that are identical to the published 4 1 2 sequence. The region of homology begins at position 5380 of Dm412. The putative start of the 412 integrase domain is indicated by an arrowhead (V). The presence of gaps 0, numerous substitutions and the truncation of its 3' terminus suggest that the heterochromatic Rsp-412 element is not functional.

E. L. Cabot et al. 480

LEFT cb3-1 cb5-1 cb6-1 cb9-1 CblO-1

lp13-1 1017-1

lpZ3-1 lp27-1 lp60-1 lp61-1 lp68-1 lp72-1 lp74-1

csl-1 ~ 5 3 - 1 C S ~ - 1 cs7-1

lp18-1

TCTAGAGATTCTGT TCA AC TGG TAAG CAAAAACAG TAAATTGCCTAAG TTTTA CA TTATA AGCGGTCAAA ATTGGTG ATTTT CCGATTTCAAG TA CCAGA CAAACAGAAGATACCT _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ """"-----A----- _ _ -----C--------G-TAC------- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ --G-----C---------------- """"""" "_ " "_ "" """"_ """"""_ _"" " ""_ """"" """_ ""_ """""- " ""_ """"""""

""""""" "_ " "_ "" """"_ """"""_ ""_ " ""_ """"" """_ ""- """""- " ""- """""""" _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __-" -__-------- -- ----- ----T----------- """"""" "_ " "_ "" """"_ """"""_ ""_ " ""_ """"" """_ ""- """""- " ""- """"""""

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116

lp13-2 lp17-2 B*vf lp18-2

' lp22-2 * .

lp23-2 lp27-2 1060-2 lp61-2 lp68-2 lp72-2 1~74-2

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sequencing according to the supplier's manual (BRL). This test ensures that the positive clones indeed contain Rsp sequences. Sequencing was done with the U.S. Biochemical Sequenase kit. The cs clones from the Canton-S stock were described in Wu et al. ( 1 988).

Sequence nomenclature: The designation, for example, cb l , is for the first clone from the chromosome cb. It is a monomer of 120 bp. A dimer is designated by, for example, cb3-1 for the left repeat and cb3-2 for the right repeat. A dash followed by a number indicates that the clone is either a dimer or a trimer.

Computer analysis: Sequences were manually aligned using the program ESEE (CABOT and BECKENBACH 1989). Computation of sequence divergence was based on the two- parameter method of KIMURA (1980). For each pairwise comparison, positions containing gaps in either sequence were excluded from analysis.

RESULTS

The large scale molecular structure of the Rsp locus: The Southern blot of genomic DNAs from three different chromosomes digested with two different enzymes were hybridized to the Rsp sequences. The digested DNAs were run on a field inversion gel to separate the large molecules and the results are

shown in Figure 1. I t turned out that chromosomes R31 and cn bw are nearly identical (6 lanes 2 and 3 us. lanes 8 and 9) and will thus be considered dupli- cates.

We shall concentrate on the cn bw (=R3 1) chromosome. The four largest EcoRI bands, with lengths equal to 270, 180, 70 and 40 kb, are equivalent to the single large band in Figure la of Wu et al. (1 988), accounting for more than 95% of the total Rsp sequences on this chromosome. These four bands were all absent in four different X-ray induced Rsp deficiencies. Two of these deficiencies are in fact homozygous viable and fertile, suggesting that the deficiencies are small enough not to include any lethal complementation groups (GANETZKY 1977). The cytological boundaries of these deletions have been de- fined by PIMPINELLI and DIMITRI ( I 989).

The minimal size of the region occupied by the Rsp array on a cn bw chromosome is about 600 kb, which is the sum of all the Rsp fragments in Figure 1. Since the chromosome is estimated to have 700-800 equiv- alents of Rsp monomers (Wu et al. 1988), only about

Population Genetics of Responder

LEFT

cb3-1 cb5-1 cb6-1 cb9-1 cblO-1 lp13-1 lp17-1 lp18-1 lp23-1 lp27-1 lp60-1 lp61-1 lp68-1 lp72-1 lp74-I c s l - 1 C S ~ - 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

48 1

2 cb5-1

5 cblO-1 0 C : 8 0.w9 4 cb9-1 0.M 6.m 3 cb6-1 0 .M

0.w4 0.W 0.000 8.w 6 lp13-1 0.035 0.026 0.026 0.035 0.026 7 lp17-1 0.054 0.044 0.044 0.054 0.044 8 l p l 8 - 1 0.046 0.036 0.036 0.046 0.036 9 lp23-1 0.026 0.017 0.017 0.026 0.017

10 lp27-1 0.026 0.017 0.017 0.026 0.017 11 lp60-1 0.026 0 . h O&l8 0.018 BtO@ 12 lp61-1 0.037 0.C28 0.028 0.037 0 028 13 lp68-1 0.065 0.055 0.055 0.065 0.055 14 lp72-1 0.035 0.026 0.026 0.035 0.026 15 lp74-1 0.027 0.018 0.018 0.027 0.018

16 c s l - 1 0.063 0.054 0.054 0.063 0.054 0.082 0.101 0.036 0.035 0.063 0.063 0.047 0.115 0.044 0.073

18 cs5-1 0.037 0.028 0.028 0,037 0,028 0,056 0.076 0,028 Q.m 0,047 0,037 0,029 0.088 0.0:8 0.047 17 CS3-1 0.044 0.035 0.035 0.044 0.035 0.045 0.063 0,036 0,,017 0.054 0.045 0.037 0.094 0.026 0.036 -

cb3-1 cb5-1 cb6-1 cb9-1 cblO-1 lp13-1 lp17-1 lpl8-1 lp23-1 lp27-1 lp60-1 lp61-1 lp68-1 lp72-1 lp74-1 csl-1 csj-1

RIGHT

cb3-2 cb5-2 cb6-2 cb9-2 cb10-2 lp13-2 lp17-2 lp l8 -2 lp22-1 lp22-2 lp23-2 lp27-2 lp60-2 lp61-2 lp68-2 lp72-2 lp74-2 ct3-2 C S ~ - 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

2 3 4 5

6

8 7

9 10 11 12 13 14 15 16 17

18

20 19

cb5-2 cb6-2

cb10-2 cb9-2

lp13-2 lp17-2 lp18-2 lp22-1 lp22-2 lp23-2 lp27-2 lp60-2 lp61-2 lp68-2 lp72-2 1 ~ 7 4 - 2

CS3-2

cs7-2 CS5-2

0.069 0.069 0.051 0.051 0.069

0.026 0.026 0.034 0.051 0.017 0.025 0.025 0.042 0.051 0.025 0.068 0.068 & 0.060 0.067 0.083 0.083 0.102 0.037 0.082 0.034 0.034 0.051 0.069 0.034 0.057 0.057 0.080 0.062 0.056 0.051 0.051 0.061 0.051 0.040

0.079 0.079 0.105 0.106 0.087 0.026 0.026 0.053 0.069 0.035

0.055 0.055 0.035 0.069 0.026 0.070 0.070 0.095 0,103 0.070 0.055 0.055 0.045 o.090 0.036

0.045 0.045 0.063 0.036 0.044

0.039 0.039 0.051 0.048 0.025 0.036 0,036 0.054 0.018 0.036

0 023 0.033 0.075 0.082 0.W #io811 0.045 0.083 0 023 0.022 0.034 0.033 0.030 0.030 0.085 0.065 0.030 0.042 O.Oi8 0.017 0.060 0.067 0.026 0.035 0.041 0.062 0.060 0.080 0.069 0.069 0.082 0.027 0.026 0.061 0.087 0.035 0.036 0.052 0.045 0.083 0.036 0.034 0.046 0.046 0.023 0.086 0.061 0.083 0.062

&&38 $.WS 0.035 0.018 0.024 0.043 0.028 0.055 0.038 0.037 0.018 0.017 0.060 0.027 0.048 0.054 0.037 0.083 0.047 0.062

B . W B.cKO 0,040 OYW 0.020 0.051 0.027 0.052 0.014 Kw E:: 0.013 0.012 0.013

cb3-2 cb5-2 cb6-2 cb9-2 cb10-2 lp l3-2 lp17-2 lp18-2 lp22-1 1~22-2 lp23-2 lp27-2 lp60-2 lp61-2 lps8-2 lp72-2 lp74-2 CS~-2 c~5-2

FIGURE 4.-Pairwise estimates of genetic divergence ( K ) for left (upper) and right (lower) repeats of Rsp dimers. Within chromosome comparisons are indicated by boxes. \'slues of K less than 1.3% or greater then 9% are shaded or underlined, respectively.

100 kb (1 20 bp X 800) of the entire 600-kb region is occupied by the Rsp repeats. We have also found that phage and cosmid clones containing several tandem copies of the Rsp sequence always harbor dispersed repetitive sequences that are not related to Rsp. In one instance, the interspersed sequence is the 412 retrotransposon of Drosophila as shown in Figure 2. In this comparison, the Rsp-412 sequence is the first 400 bp of a 2.5-kb EcoRI fragment that contains about 1.3 kb of Rsp tandem repeats (see Figure 2b of Wu et al. 1988). When this 400-bp fragment is compared with a full-length Drosophila melanogaster 412 sequence, Dm412 (YUKI et al. 1986), there is no ambi- guity that it is a 412 element. An interesting observation is that this Rsp-412 sequence resembles a 412 element from Drosophila pseudoobscura even more than it resembles Dm412 (E. L. CABOT and A. T. BECKENBACH, unpublished results). D. SMOLLER and D. HARTL (personal communication) have also isolated a YAC clone that hybridizes to Rsp. The Rsp arrays in their YAC clone are organized into islands of tandem repeats, interspersed with long stretches of

non-Rsp sequences. Thus, genomic Southern blots and molecular cloning both suggest that the RsP repeat array is interrupted by other dispersed repetitive sequences with Rsp repeats accounting for no more than 10-20% of the region. [Recently, CAIZZI, CAGCESE and PIMPINELLI (1 993) also described a tandemly arranged array of the transposon, Bari-l, which is adjacent to the Rsp array but does not appear to interdigitate with it.]

What then is the maximal size of the RsP locus? Are all the major Rsp-containing fragments of Figure 1 contiguous (hence, Rsp repeats are highly localized) or are these fragments far apart, occupying a region much larger than the sum total of their physical sizes? We already mentioned that 95% of the Rsp repeats on the cn bw chromosome, represented by four different EcoRI fragments, are contained within either of two small deficiencies. We can further interpret from Figure 1 that at least 80% of Rsp repeats are restricted to a region of no more than 450 kb. This is because the two largest EcoRI fragments, 270 and 180 kb in length and containing more than 80% of total


Left Repeats 50 I

v) c :r 35 n rn 0 Within

Between

K

Right Repeats 5 0 , I

- I Within rl Between 1

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 K

FIGURE 5.-Frequency distributions of genetic divergence ( K ) in within- (solid bars) and between- (open bars) chromosome comparisons for left (upper) and right (lower) repeats of the Rsp dimers shown in Figure 3. The K values from Figure 4 were grouped into intervals of 0.02 and frequencies were standardized to the percent of the total number of comparisons for each category.

Rsp signal, are likely to be adjacent to each other. Comparing the restriction patterns between the lanes of EcoRI and EcoRIIBamHI, we can see that none of the largest four EcoRI bands contains a BamHI site. If the 270 kb and 180 kb EcoRI fragments are not adjacent and are on two different BamHI fragments, we expect to see two BamHI bands that are larger. There are indeed two BamHI bands around 200 kb in size but both have much weaker signals than the corresponding EcoRI bands, indicating that the putative BamHI band, or bands, containing the 270- and 180-kb EcoRI fragments must be larger than the resolution of FIGE in this experiment. We can observe

strong hybridization signals in the BamHI lane at the position of the well (not shown). The most straight- forward interpretation is that the two large EcoRI bands are contiguous, creating a BamHI band greater than 450 (= 270 + 180) kb in size. We thus tentatively estimate the actual size of the Rsp region on the cn bw chromosome to be the sum of all the major EcoRI fragments, which is about 600 kb.

The similarity between cn bw and R3 1 (i.e., cn b d ” ) is interesting for another reason. R31 was created from cn bw by X-ray irradiation (GANETZKY 1977) and was genetically insensitive to segregation distortion when last tested in the early 1980s. However, as reported in Wu et al. (1 988), it later became a sensitive chromosome as determined by both the genetic and molecular means. This is the case for three R31 cultures maintained in three different laboratories since the mid-l980s, but all were derived from the original stock of GANETZKY (1 977). Wu et al. (1 988) suggested that the reversion could be the result of Rsp sequence amplification, similar to the observations on the ribosomal gene repeats (TARTOF 1974; ENDOW, KOMMA and ATWOOD 1984). They did not favor the simple interpretation of contamination because R3 1 is not identical to other standard cn bw chromosomes (including subtle changes in molecular structure and the presence of a recessive lethal). However, consid- ering the similarity between cn bw and R31 in their large-scale structure, we now suggest that contamination by a cn bw chromosome, followed by some changes in the new line, is a more plausible explanation for the present properties of the R31 chromosome than sequence amplification.

The divergence of Rsp repeats within and between chromosomes: The most abundant class of Rsp repeats is that of 240 bp dimers. When a Southern blot of D. melanogaster genomic DNA, digested with the XbaI restriction enzyme, is probed with Rsp DNA, a characteristic 120-bp ladder appears with a very prom- inent 240-bp band. Bands of decreasing intensity are 480, 360 and 120 bp in length (see Figure Ib of WU et al. 1988). We have cloned and sequenced 19 dimers, 10 monomers (120 bp) and 3 trimers (360 bp) from three different chromosomes. Monomers and trimers were apparently derived from dimers and are ana- lyzed in the next section.

The sequences of dimers are presented in Figure 3 with left repeats on the top and right repeats in the bottom. The junction between the repeats is TCTACA which differs from a XbaI restriction site (TCTAGA) by a single base pair. It is evident that left and right repeats form two distinct monphyletic clades with an average divergence of 16% between them. The divergence between the two types of repeats is much higher than the average divergence in euchromatic sequences between D. melanogaster and its clos-

483 Population Genetics of Responder

TABLE 1

Mean divergence between repeats on the same and different chromosomes

Left repeats Right repeats Left us. right

n E SEE n K SEE n R SEE

Within cb-cb 10 0.0070 0.0017 cb-cb 10 0.0495 0.0075 cb-cb 5 0.1513 0.0046 lPlP 45 0.0491 0.0034 Ip-lp 66 0.0546 0.0039 Ip-lp 10 0.1661 0.0053 cs-cs 3 0.0364 0.0138 CS-cs 3 0,0174 0.0048 CS-cs 4 0.1462 0.0057

Pooled data: 58 0.0412 0.0034 79 0.0525 0.0035 19 0.1580 0.0037 Between

cb-lp 50 0.0316 0.0020 cb-lp 60 0.0574 0.0030

Ip-cs 30 0.0517 0.0047 Ip-cs 36 0.0376 0.0046 cb-cs 5 0.0425 0.0032 cb-cs 15 0.0410 0.0029

Pooleddata: 85 0.0397 0.0021 111 0.0488 0.0024

est sibling species, Drosophila simulans, which ranges from 6-1 1 % (WERMAN, DAVIDSON and BRITTEN 1990). In other words, the age of the dimeric structure is greater than the age of these two species, if we assume equivalent rates of divergence for euchromatic sequences and Rsp repeats. In a subsequent paper we will present data on Rsp-like repeats in D. simulans which are highly divergent from the Rsp repeats shown here (our unpublished results).

Pairwise divergence estimates within each clade are given in Figure 4. The average level of divergence within either clade is only one-fourth of the divergence between the two clades. In Figure 4, clones from the same chromosome (cb, lp and cs) are grouped and comparisons between repeats of the same chromosome are indicated by boxes. Because the Rsp array is localized near the centromere of chromosome ZZ (BRITTNACHER and GANETZKY 1989; LYTTLE 1991; PIMPINELLI and DIMITRI 1989) where homologous recombination is nearly absent, exchanges of Rsp sequences between homologous chromosomes may be so rare that repeats from different chromosomes could have diverged independently, like members of two reproductively isolated populations.

The distributions of within-chromosome divergence and between-chromosome divergence for the left and right repeats respectively are shown in Figure 5. Surprisingly, the between- and within-chromosome distributions are nearly identical. The only possible exception is the divergence between the left repeats of the clones from the cb chromosome, which are considerably smaller than the overall average. How- ever, this observation is perhaps due to the small sample size. In fact, the right halves of those same clones are quite different, as can be seen from the boxes in the upper left corner of Figure 4. Moreover, when all the within-chromosome comparisons are pooled together, the data show no evidence for concerted evolution within the same chromosome. A more detailed analysis which is given in Table 1 con-

firms that the levels of within- and between- chromo- some divergence are the same.

Tandem repeats are not phylogenetic units: The discrepancy in relatedness between the left and right halves of the cb clones raises an interesting question- is the dimeric repeat of a tandem array a phylogenetic unit? If unequal crossing over or gene conversion occurred frequently (in an evolutionary time scale) between different dimer copies, then different parts of the dimer repeat may have different evolutionary histories. For convenience, we may simply ask if the left halves and the right halves of a set of repeats have congruent phylogenies. For this data set, the answer appears to be “no” and this can be most easily seen, not by constructing two unwieldy phylogenies, but by highlighting the most and least divergent pairs of the left and right repeats, respectively. In Figure 4, shaded numbers are less than 1% and underlined numbers are greater than 9%. It is clear that there is no correspondence between the two halves of the same repeat. Thus, the same pair of repeats are never most (or least) divergent in both halves simultane- ously, indicating different phylogenetic histories for different parts of the same clone. Because of the low level of divergence between dimers, however, this inference has limited statistical power. In this regard, observations on monomers and trimers presented in the next section clearly demonstrate the “hybrid” (or chimeric) nature of repeats. The absence of identifi- able phylogenetic units should be taken into consid- eration whenever an attempt is made to construct phylogenetic trees of tandem repetitive sequences.

Evidence for unequal crossover: That the left and right halves of the dimers do not have congruent phylogenetic history is indirect evidence that either gene conversion and/or unequal exchange is taking place. Direct evidence comes from exchanges between left and right repeats as illustrated in Figure 6A. Because the left and right repeats are characterized by 16 diagnostic nucleotide sites (out of 120 bp), it is easy to recognize most hybrid repeats that contain

E. L. Cabot et al . 484

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FIGURE 6.-(A) Model for the formation of Rsp monomers and trimers by unequal exchange between two dimers. Mispairing between the right repeat (solid bar) of the upper dimer and the left repeat (open bar) of the lower, followed by crossover is expected to produce the two hybrid products illustrated. Monomers cb-1 and lp20 are probably generated from normal left repeats by the gain of an Xbal restriction site. Similarly, the atypical dimer lp22 was probably derived from a trimer by the gain of an XbaI site between the first and second repeats. (B) Comparisons of Rsp monomers and trimers to the left repeat consensus sequence illustrating agreement between sequence data and the model described in A. Monomers and the first repeats of trimers are shown in the upper panel; the second and third repeats of trimers are shown in the lower panel. Shading denotes nucleotide positions that are similar to right repeats of dimers. The 29-bp element that is perfectly conserved between left and right repeats is indicated by a horizontal bracket shown above the left consensus. See Figure 3 legend for more detail.

characters of both. According to the model of Figure 6A, XbaI monomers of 120 bp and trimers of 360 bp could be generated by unequal crossing over between a left and a right repeat of dimers. The sequence in the monomer and that of the second repeat of a trimer would have opposite patterns. Ten monomer and three trimer sequences are presented in Figure 6B. Sequence characteristics of a right repeat are shaded. The mechanism presented in Figure 6A is strongly supported by the sequence data shown in Figure 6B. While the first and third repeats of trimers clearly belong in the left and right repeat clade, respectively, the second repeats as well as all but two of the mono-

mers are all "hybrids." Eight of the ten monomers resemble the left repeat on the left side and the right repeat on the right side. The second repeats of trimers have the opposite pattern with respect to parts origi- nating from left or right repeats. This again shows that each repeat is often not a phylogenetic unit.

The most striking feature of Figure 6B is the narrow region within which crossovers seem to have taken place. Ten of the 11 detectable crossover events apparently occurred in the interval between the two shaded areas, a distance of only 29 bp. This is the region where the left and right repeats diverge least. In fact, there are no diagnostic differences between


A Sister Exchange only B Sister + Homologous Exchange

7 t

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d l a p e &I 1 i,h FIGURE 7.-A schematic representation of divergence between

repeats. This figure incorporates three parameters-divergence time and the rates of exchange between homologous chromosomes and between sister chromatids. The observed level of interchromosomal divergence, d , is the ratio of divergence between repeats from different chromosomes over divergence between repeats from the same chromosome. When the d values from two repeat arrays are different, the smaller d value could be due to either a short divergence time, T , as shown on the left or higher homologous exchange rate as shown on the right. (We cannot distinguish between the two possibilities based on the sequence information alone as we are comparing two different arrays.) The scaled divergence time, T , is the actual divergence time multiplied by the rate of sister chromatid exchange. A large value of 7 means a long divergence time meas- ured in the unit of occurrences of sister chromatid exchange.

the left and right repeats within these 29 bp although there are polymorphic sites within the clades of left and right repeats in this region. We also interpret the sequence of Lp16-2 to be the product of the same kind of exchange but the crossover point was further to the right than other events. It was likely between the end of the shaded area and the end of the Lp16- 2 sequence. The implication of these sequence data on the mechanisms of recombination will be discussed.

There are three sequences, also shown in Figure 6A, that cannot be explained by the simple model of exchange between dimers. cbl and lp20 (sequences shown in Figure 6B) are bounded by XbaI sites but show the characteristics of the left repeat in their entirety and lp22-1 (sequence in Figure 3) appears to have two adjacent right repeats bounded by XbaI sites. In all three cases, the origin can be explained by the gain of an XbaI site ( i e . , a change from TCTACA to TCTAGA). In the cases of cbl and lp20, it appears that the left repeat of a dimer has gained an XbaI site on the right, resulting in the exclusion of the adjacent right repeats during cloning. On the other hand, the lp22-1 and 22-2 sequences (these are contiguous repeats from the same clone but presented separately for the purpose of analysis; see Sequence nomenclature) were perhaps derived from a sequence like Ipl6-2 and -3, after the junctional sequence between the leftmost repeat and the middle repeat changed from TCTACA to TCTAGA. In fact, lp22-1 and lp16-2 share 4 unique nucleotides in a short stretch of 12 bp (shaded region of lp22-1, Figure 3) and are probably quite closely related.

DISCUSSION

The absence of betweenshromosome differences in Rsp sequences: In this report, the term interchro-

mosomal differences refers to the divergence in Rsp sequences rather than variations in copy numbers; the latter is subject to strong balancing selection such as the kind modeled by CHARLESWORTH and HARTL (1 978). The extent of interchromosomal differences in Rsp sequences, relative to intrachromosomal differences, depends on the relative rates of unequal exchanges within ( i e . , sister chromatid exchanges) and between chromosomes (Le . , homologous exchanges) as well as the coalescence time of chromosomes. Long coalescent times among chromosomes and frequent sister chromatid exchanges (relative to homologous exchanges) should lead to a greater similarity among repeats of the same chromosomes as illustrated in Figure 7A. A priori, the Rsp repeat array is expected to be one of the best places in the Drosophila genome to find such a phenomenon for the following reason. Unequal exchanges between either sister or homologous chromosomes have to be frequent to account for the high variability in copy number (WU and HAMMER 1991) and yet Rsp is located at a position where homologous exchanges, which could homogenize sequences between chromosomes, are expected to be rare. A direct study of homologous exchange rate in the X chromosome heterochromatin is that of WIL- LIAMS et al. (1 989). The rate was estimated to be 1 0-4 events per chromosome, with the majority of them occurring at the hot spot of the ribosomal RNA locus. If we assume that the Rsp repeats are scattered within a region of 600 kb (Figure l), the Rsp region accounts for only 2% of the centromeric heterochromatin of the second chromosome. Thus, the rate of homologous exchange at Rsp could be as low as

The surprisingly low level of interchromosomal vs. intrachromosomal differences in Rsp sequences (Fig- ure 5 and Table 1) suggests either that the chromosome coalescence time is short in the Rsp region ( i e . , all Rsp arrays were derived from a common ancestral array in the very recent past) or that homologous exchanges, albeit infrequent, are sufficient to obliter- ate between-chromosome differences. The latter explanation, as shown in Figure 7B, is less convincing in light of the contrast with those of the histone array, which is in the proximal euchromatin of the D. melanogaster second chromosome (MATSUO and YAMAZAKI 1989). These authors reported that the divergence among histone repeats of different chromosomes is nearly twice as great as that among repeats of the same chromosome. Since there is no reason to believe that the exchange rate between homologous chromosomes is higher at Rsp than at the histone locus (if anything, it should be lower), the Rsp array was expected to show at least as much interchromosomal divergence as the histone array. We thus suggest that the scenario of Figure 7A is more plausible. A smaller T means that the product of the chromosome coales-


cent time and the rate of sister chromatid exchange at Rsp is smaller. Although direct measurements of the rate of sister chromatid exchange at either locus are unavailable, it is not unreasonable to expect the rate for histones to be more selectively constrained than that for Rsp because of the possible fitness con- sequences of variation in histone copy number. There- fore, the more reasonable explanation for the contrast between Rsp and the histone array is that the actual age of alleles is younger at the Rsp locus. The Bari-1 repeat array, which is adjacent to the Rsp locus also shows a surprisingly low level of structural variation between chromosomes (CAIZZI, CACGESE and PIMPI- NELLI 1993). In other words, the immediate vicinity of the centromere may have experienced a more recent (selective or meiotic) sweep than the histone region within the proximal euchromatin.

Our observation is thus consistent with either a meiotic or selective sweep model. The latter cannot be rejected by our approach because its prediction can be similar or opposite to our observation. The meiotic sweep model predicts a lower level of interchromosomal divergence at Rsp than at loci of proximal euchromatin which is what we observed. We are thus unable to reject this model either. Although our study does not resolve the issue, further studies on tandem repeats in the centromeric regions may provide useful information on the population genetics of regions of reduced recombination mainly because of their high variability in both copy number and DNA sequence. There are many other studies on the population genetics of tandem repeats; for example, human alphoid satellite DNA (DURFY and WILLARD 1989) or rDNA in Drosophila (WILLIAMS et al. 1989). In the context of the meiotic vs. selective sweep models, we need information on two different repeat arrays, like the Rsp and histone arrays, from the exact same chromosomes.

Ruling out the meiotic sweep model would provide further support for the selective sweep interpretation. However, it is difficult to eliminate the meiotic sweep model based on studies of chromosome mechanics. The correlation between X:Y pairing and segregation in Drosophila (MCKEE and LINDSLEY 1987) appears to be due to the presence of pairing sites between the X and Y chromosome. Pairing of autosomes was shown not to depend on heterochromatin (YAMAMOTO 1979) but HILLIKER, HOLM and APPEU (1982), observing the segregation of compound chromosomes, suggested proximal euchromatin to play such a role. Since the suppression in recombination and the correlated reduction in polymorphism are associated with proximal euchromatin, we cannot rule out meiotic sweep on the basis of mechanistic studies. Moreover, observations on males and females can be different and

weak segregation distortion in the order of a few percent would not have been detected.

Evidence and mechanisms of unequal exchange: The existence of a large number of monomers and trimers poses another interesting question. The align- ments between the left and right repeats are rather imperfect, with 16% differences between them. Since the extent of DNA sequence match is important for DNA crossing over, the abundance of trimers and monomers seems surprising [see Figure 1 b of Wu et at. (1 988)-the 120-bp band did not show strongly because small DNA molecules did not bind to the membrane very well. Its abundance is evident in the cloning experiment]. Recent studies of recombination mechanisms in bacteria, yeast, Drosophila, and mam- mals (WALDMAN and LISKAY 1987; RESNICK, SKAAN- ILD and NILSSON-TILLCREN 1989; METZENBERC et al. 1991; NASSIF and ENGELS 1993), however, suggest that short stretches of DNA with nearly perfect match are what is needed for intermolecular exchanges. The number appears to be no greater than 1 15 bp in Drosophila (NASSIF and ENGELS 1993) and 25 bp in mammalian cells (AYARES et al. 1986). These studies provide a very reasonable explanation for the results of Figure 6B in which the majority of the crossover breakpoints apparently fall in a region of 29 bp (between the two shaded areas). This happens to be the only stretch of nucleotides where the left and right repeats are nearly identical. Although these monomers and trimers may not always represent independ- ent crossover events, their abundance on the chromosomes from which they were extracted suggests that such events occur regularly. Thus, our data support the idea that sequence identity, rather than general homology, is the prerequisite for recombination (WALDMAN and LISKAY 1987; METZENBERC et al.

Finally, the frequent occurrence of unequal crossing over (both between left and right repeats and espe- cially between two left or two right repeats) prohibits the treatment of Rsp repeats as phylogenetic units. Reconstruction of phylogenetic trees for tandem repeats, such as the one by BACHMANN and SPERLICH (1993), is interpretable only if such a confounding effect is addressed.

199 1).

We thank SUNJAY KAUSHAL for technical assistance, GARY KAR- PEN for the protocol of preparing large molecular weight DNA and MIKE PALOPOLI for stimulating discussions. Comments from BRIAN CHARLESWORTH, NORMAN JOHNSON and MICHAEL WADE are greatly appreciated. E.L.C. and P.D. were supported by postdoctoral fei- lowships from the Alfred P. Sloan Foundation, and C.I.W. is s u p ported by National Institutes of Health grants (GM39902 and HG00005) and a National Down Syndrome Society Fellowship.

LITERATURE CITED

ARNHEIM, N., 1983 Concerted evolution of multigene families, pp. 38-6 1, in Evolution of Genes ond Proteins, edited by M. NEI


and R. K. KOEHN. Sinauer Press, Sunderland, Mass. AYARES, D., L. CHEKURI, K.-Y. SONG and R. KUCHERLAPATI,

1986 Sequence homology requirements for intermolecular recombination in mammalian cells. Proc. Natl. Acad. Sci. USA

BACHMAN, L. D., and D. SPERLICH, 1993 Gradual evolution of a specfic satellite DNA family in Drosophila ambigua, D . tristis and D. obscura. Mol. Biol. Evol. 10: 6447-659.

BAKER, B. S., and A. T. CARPENTER, 1972 Genetic analysis of sec chromosome meiotic mutants in Drosophila melanogaster. Ge- netics 71: 255-286.

BEGUN, D. J., and C. F. AQUADRO, 1992 Levels of naturally occurring DNA polymorphism correlate with recombination rates in D. melanogaster. Nature 356: 519-520.

BERRY, A. J., J. W. AJIOKA and M. KREITMAN, 1991 Lack of polymorphism on the Drosophila fourth chromsome resulting from selection. Genetics 129 11 11-1 117.

BRITTNACHER, J. G., and B. GANETZKY, 1989 On the components of segregation distortion in Drosophila melanogaster. IV. Con- struction and analysis of free duplications for the Rsp locus. Genetics 121: 739-750.

CABOT, E. L., and A. T. BECKENBACH, 1989 Simultaneous editing of multiple nucleic acid and protein sequences using ESEE. Comp. Appl. Biosci. 5: 233-234.

CAIZZI, R., C. CAGCESE and S. PIMPINELLI, 1993 Bari-I: a new transposon-like family in Drosophila melanogaster with a unique heterochromatic organization. Genetics 133: 335-345.

CHARLESWORTH, B., and D. L. HARTL, 1978 Population dynamics of the Segregation Distorter polymorphism of Drosophila melanogaster. Genetics 8 9 171-192.

DOSHI, P., S. KAUSHAL, C. BENYAJATI and C.-I. Wu, 1991 Molecular analysis of the Responder satellite DNA in Drosophila melanogaster: DNA bending, nucleosome structure and Rsp-binding proteins. Mol. Biol. Evol 8: 72 1-74 1.

DURFY, S. J., and H. F. WILLARD, 1989 Patterns of intra and interarray sequence variation in alpha satellite from the human X chromosome: evidence for short range homogenization of tandemly repeated DNA sequences. Genomics 5: 8 10-82 1.

ENDOW, S. A., D. J. KOMMA and K. C. ATWOOD, 1984 Ring chromosomes and rDNA magnification in Drosophila. Genetics

GANETZKY, B., 1977 On the components of Segregation Distor- tion in Drosophila melanogaster. Genetics 8 6 321-355.

HILLIKER, A. J., R. APPELS and A. SCHALET, 1980 The genetic analysis of D. melanogaster heterochromatin. Cell 21: 607-619.

HILLIKER, A. J., D. G. HOLM and R. APPEIS, 1982 The relation- ship between heterochromatic homology and meiotic segregation of compound second autosomes during spermatogenesis in Drosophila melanogaster. Genet. Res. 3 9 157-168.

KADYK, L. C., and L. H. HARTWELL, 1992 Sister chromatids are preferred over homologs as substrates for recombination repair in Saccharomyces cerevisiue. Genetics 132: 387-402.

KIMURA, M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1 6 l 11-1 20.

LYTTLE, T . W., 1991 Segregation distorters. Annu. Rev. Genet.

LYTTLE, T. W., J. G. BRITTNACHER and B. GANETZKY, 1986 Detection of Rsp and modifier variation in the meitotic drive system Segregation Distorter of Drosophila melanogaster. Genetics 114: 183-202.

83: 51 99-5203.

108: 969-983.

25: 51 1-557.

MATSUO, Y., and T. YAMAZAKI, 1989 Nucleotide variation and

divergence in the histone multigene family in Drosophila melanogaster. Genetics 122: 87-97.

MCKEE, B., and D. L. LINDSLEY, 1987 Inseparability of X-heterochromatic functions responsible for X:Y pairing, meiotic drive and male fertility in Drosophila melanogaster. Genetics 116

METZENBERG, A. B., G. WURZER, T. H. J. HUISMAN and 0. SMI- TIHES, 1991 Homology requirements for unequal crossing over in humans. Genetics 128: 143-161.

NASSIF, N., and W. ENGELS, 1993 DNA homology requirements for mitotic gap repair in Drosophila. Proc. Natl. Acad. Sci. USA

PIMPINELLI, S., and P. DIMITRI, 1989 Cytogenetic analysis of the Responder (Rsp) locus in Drosophila melanogaster. Genetics 121: 765-772.

RESNICK, M. A., M. SKAANILD and T . NILSSON-TILLGREN, 1989 Lack of DNA homology in a pair of divergent chromosomes greatly sensitizes them to loss by DNA damage. Proc. Natl. Acad. Sci. USA 86: 2276-2280.

SANDLER, L., and E. NOVITSKI, 1957 Meiotic drive as an evolutionary force. Am. Nat. 41: 105-1 10.

STEPHAN, W., 1989 Tandem-repetitive noncoding DNA: forms and forces. Mol. Biol. Evol. 6 198-212.

TARTOF, K. D., 1974 Unequal mitotic sister chromatid exchange as the mechanism of ribosomal RNA gene magnification. Proc. Natl. Acad. Sci. USA 71: 1272-1276.

TEMIN, R. G., and M. MARTHAS, 1984 Factors influencing the effect of segregation distortion in natural populations of Dro- sophila melanogaster. Genetics 107: 375-393.

TEMIN, R. G., B. GANETZKY, P. A. POWERS, T. W. LYTTLE and S. PIMPINELLI. 199 1 Segregation distorter (SD) in Drosophila melanogaster: genetic and molecular analyses. Am. Nat. 137:

WALDMAN, A. S., and R. M. LISKAY, 1987 Differential effects of base-pair mismatch on intra-chromosomal versus extrachro- mosomal recombination in mouse cells. Proc. Natl. Acad. Sci.

WERMAN, S. D., E. H. DAVIDSON and R. J. BRITTEN, 1990 Rapid evolution in a fraction of the Drosophila nuclear genome. J.

WILLIAMS, S. M., J. A. KENNISON, L. G. ROBBINS and C. STROBECK, 1989 Reciprocal recombination and the evolution of the ribosomal gene family of Drosophila melanogaster. Genetics 122:

WU, C.-I., and M. F. HAMMER, 1991 Molecular evolution of ultraselfish genes of meiotic drive systems, pp. in Evolution at the Molecular Level, edited by R. K. SELANDER, A. G. CLARK and T . S. WHITTAM. Sinauer, Sunderland, Mass.

WU, C.-I., J. R. TRUE and N. A. JOHNSON, 1989 Fitness reduction associated with the deletion of a satellite DNA array. Nature

WU, C.-I., T. W. LYTTLE, M.-L. WU and G.-F. LIN, 1988 Association between a satellite DNA sequence and the Responder of Segregation Distorter in D. melanogaster. Cell 54:

YAMAMOTO, M., 1979 Cytological studies of heterochromatin function in D. melanogaster males: autosomal meiotic pairing. Chromosoma 72: 293-328.

YUKI, s., s. INOUYE, S. ISHIMARU and K. SAIGO, 1986 Nucleotide sequence characterization ofa Drosophila retrotransposon, 412. Eur. J. Biochem. 158: 403-410.

399-407.

9 0 1262-1266.

287-33 1.

USA 84: 5340-5344.

Mol. EvoI. 3 0 281-289.

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341: 248-251.

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