RESEARCH ARTICLE

Abundance and chromosomal distribution of six Drosophilabuzzatii transposons: BuT1, BuT2, BuT3, BuT4, BuT5,and BuT6

Ferran Casals & Josefa González & Alfredo Ruiz

Received: 17 February 2006 /Revised: 3 May 2006 /Accepted: 4 May 2006# Springer-Verlag 2006

Abstract The abundance and chromosomal distribution ofsix class-II transposable elements (TEs) of Drosophilabuzzatii have been analyzed by Southern blotting and insitu hybridization. These six transposons had been previous-ly found at the breakpoints of inversions 2j and 2q7 of D.buzzatii. These two polymorphic inversions were generatedby an ectopic recombination event between two copies ofGalileo, a Foldback element. The four breakpoints becamehotspots for TE insertions after the generation of theinversion and the transposons analyzed in this work areconsidered to be secondary invaders of these regions.Insertions of the six transposons are present in theeuchromatin but show an increased density in the peri-

centromeric euchromatin–heterochromatin transition regionand the dot chromosome. They are also more abundant inthe inverted segments of chromosome 2 rearrangements. Wefurther observed that the accumulation of TE insertionsvaries between elements and is correlated between dot,proximal regions, and inverted segments. These observationsfully agree with previous data in Drosophila melanogasterand support recombination rate as the chief force explainingthe chromosomal distribution of TEs.

Introduction

Transposable elements (TEs) make up a substantial fractionof all eukaryotic genomes, e.g., ∼44% for the humangenome and an even higher fraction for some plant genomes.Kaminker et al. (2002) reported an exhaustive analysis of theTE composition of the sequenced Drosophila melanogastergenome. This analysis was mainly based in the euchromaticfraction of the genome, where TE density is lower(Bartolomé et al. 2002). A total of 1,572 copies (31% ofthem were full length) belonging to 93 TE families werefound. These TEs represent 3.86% of the genome, retro-transposons with long terminal repeat (LTR) being the mostabundant elements (2.65% of the genome), followed byLINE elements (0.87%), transposons (0.31%), and foldbackelements (0.04%). Recently, improved bioinformatic toolshave led Quesneville et al. (2005) to annotate a substantiallyhigher number of TEs, which now represent up to 5.3% ofthe euchromatic portion of the D. melanogaster genome. Theinformation currently available for other Drosophila species,besides D. melanogaster, is very limited (see review byBiemont and Cizeron 1999) although it is expected toincrease substantially as a consequence of the sequencing

ChromosomaDOI 10.1007/s00412-006-0071-7

Communicated by C. Lehner

Electronic Supplementary Material Supplementary material isavailable in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0071-7 and is accessible for authorized users.

Sequence data from this article have been deposited in the EMBL/GenBank Data Libraries under accession number DQ402469.

F. Casals : J. González :A. Ruiz (*)Departament de Genètica i de Microbiologia,Universitat Autònoma de Barcelona,08193 Bellaterra, Barcelona, Spaine-mail: Alfredo.Ruiz@uab.es

F. CasalsUnitat de Biologia Evolutiva, Facultat de Ciències de la Saluti de la Vida, Universitat Pompeu Fabra,08003 Barcelona, Spain

J. GonzálezDepartment of Biological Sciences, Stanford University,Stanford, CA 94305, USA

Present address:

Present address:

and annotation projects for 11 other Drosophila species(http://rana.lbl.gov/drosophila/multipleflies.html).

The chromosomal distribution of TEs in D. melanogasteris clearly nonrandom with a significant excess of insertionsin the heterochromatin and the dot and the Y chromosomes(Charlesworth et al. 1994; Carmena and González 1995;Pimpinelli et al. 1995; Dimitri 1997; Junakovic et al. 1998;Dimitri et al. 2003). For instance, TEs contribute >50% ofthe ∼20 Mb of assembled heterochromatin sequence(Hoskins et al. 2002) and >10% of the dot chromosomesequence (Locke et al. 1999; Kaminker et al. 2002). Acommon feature of these chromosomal regions is theabsence of recombination. Within the sequenced euchromat-ic portion of the genome, the evidence also suggests thatrecombination rate is the main factor determining thechromosomal distribution of TEs, with an inverse correlationbetween recombination rate and number of TE insertions(Bartolomé et al. 2002; Rizzon et al. 2002). This observationcan be explained if the number of TE copies within a givengenome is the result of an equilibrium between transpositionthat increases the number of copies and natural selection thateliminates them (Charlesworth and Langley 1989; Charles-worth et al. 1994). Selection will eliminate TE insertions ifthey disrupt genes or affect their coding capacity and theefficiency of this selection will decrease with recombinationrate (Hill and Robertson 1966; Gordo and Charlesworth2001). Selection will also eliminate the products of ectopicrecombination and the efficiency of this process will alsodecrease with recombination rate if ectopic exchange isreduced in parallel with regular meiotic exchange (Langleyet al. 1988; Montgomery et al. 1991; Goldman and Lichten1996).

TEs have been shown to mediate the generation ofnatural chromosomal rearrangements in a wide range oforganisms (Daveran-Mingot et al. 1998; Kim et al. 1998;Schwartz et al. 1998; Cáceres et al. 1999b). In particular,previous work in our lab has shown that Galileo, a foldbacktransposable element (Casals et al. 2005), was responsiblefor the generation of two polymorphic inversions ofDrosophila buzzatii: 2j (Cáceres et al. 1999b, 2001) and2q7 (Casals et al. 2003). In each case, the two breakpointshave accumulated insertions of different TEs after thegeneration of the inversion, becoming hotspots for geneticinstability and TE insertions (Cáceres et al. 2001; Casals etal. 2003). Among the 11 TEs found at the inversionbreakpoints, three (Galileo, Kepler and Newton) sharingsequence similarities have been tentatively classified asfoldback elements due to their structural characteristics(Cáceres et al. 2001; Casals et al. 2005). Six other TEs(BuT1, BuT2, BuT3, BuT4, BuT5, and BuT6) have beenclassified as class II elements due to their structure andsequence similarities (Cáceres et al. 2001; Casals et al.2003). Finally, another two TEs (ISBu1 and ISBu2)

seemingly belong to a new type of class I elements namedmini-me (Wilder and Hollocher 2001). The abundance andchromosomal distribution of Galileo, Kepler, and Newtonhave been recently analyzed (Casals et al. 2005). Thesethree elements seem to be ancient components of thegenome in the buzzatii species complex. With regard totheir chromosomal distribution, these elements showed avery significant accumulation in the low recombinationregions of chromosomes, such as the dot chromosome, theproximal regions of the chromosomes, and the regionsincluded in polymorphic chromosomal inversions (Casalset al. 2005).

In this work, we investigated the abundance andchromosomal distribution of the six transposons BuT1,BuT2, BuT3, BuT4, BuT5, and BuT6 in the genome ofD. buzzatii. The results come from 15 chromosomal linesanalyzed by Southern blot hybridization and nine linesanalyzed by in situ hybridization to the salivary glandchromosomes. The chromosomal distribution analysisprovides valuable information about the main forcesdetermining the location of the TEs in the genome. Inaddition, we tested for associations between differenttransposons and between the transposons and the break-points of those inversions described in the buzzatii speciescomplex to shed light on the nature of the inversionbreakpoint hotspots.

Materials and methods

Drosophila stocks

The following 15 D. buzzatii lines were used: st-1, st-3, st-7,and st-10 (homokaryotypic for the standard arrangement);j-2, j-9, and j-19 (homokaryotypic for the 2j arrangement);j-23 (homokaryotypic for the 2j arrangement and the 5Iinversion); j-24 [homokaryotypic for the 2j arrangementand the translocation t(5,1)]; jq7-1 and jq7-4 (homokaryo-typic for the 2jq7 arrangement); jz3-6 and jz3-7 (homokar-yotypic for the 2jz3 arrangement); y3-1 (homokaryotypic forthe 2y3 arrangement); s-1 (homokaryotypic for the 2y3

arrangement and segregating for the 4 standard (4st) and 4schromosomal arrangements). All these are natural chromo-somal arrangements except the 5I inversion and the t(5,1)translocation, which were induced by introgressive hybrid-ization (Naveira and Fontdevila 1985). The 15 lines werederived from different natural populations that cover mostof the species geographical distribution: st-1, j-2, jq7-1, andjz3-6 are derived from Carboneras (Spain); st-3 from Vipos(Argentina); st-10 from Termas de Río Hondo (Argentina);and st-11 from Trinkey (Australia); j-23, j-24, and y3-1 arefrom San Luis (Argentina); j-9 from Quilmes (Argentina);j-19 from Ticucho (Argentina); jq7-4 from Otamendi

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(Argentina); and jz3-7 and s-1 from Pingado, CanaryIslands (Spain).

DNA probes

Probes containing complete copies of the six TEs analyzedin this work were produced by PCR (Table 1). In all cases,except the BuT4 element, the probes were obtained frompreviously described copies of the elements. These copiesare located at the hotspots for TE insertions described at thebreakpoints of the chromosomal inversions 2j and 2q7,which have been cloned in several lines (Cáceres et al.2001; Casals et al. 2003). The only described copy of theBuT4 element (BuT4-1) contains an insertion of another TE(Cáceres et al. 2001). A new copy (BuT4-2) has beenobtained through PCR amplification from genomic DNA.Sequences were obtained on an ABI 373 A (Perkin–Elmer)automated DNA sequencer.

PCR was carried out in a volume of 50 μl, including100–200 ng of genomic DNA, 20 pmol of each primer,200 μM dNTPs, 1.5 mM MgCl2, and 1–1.5 U of TaqDNA polymerase. To generate probes of the D. buzzatiiTEs, primers located at their ends were designed. PCRswith different primer pairs and cycling conditions of30 rounds of 30 s at 94°C; 30 s at 55–65°C, and 1 min30 s–2 min at 72°C were carried out from genomic DNA ofline st-1 for the BuT4 probe and from DNA clonescontaining the breakpoints of the chromosomal inversions2j and 2q7 (Cáceres et al. 2001; Casals et al. 2003) for theBuT1, BuT2, BuT3, BuT5, and BuT6 probes. Products weregel-purified and cloned into the pGEM-T vector (Promega).

Southern analysis

Southern hybridizations were performed according tostandard procedures (Sambrook et al. 1989). Probes were

labeled by random primer with digoxygenin-11-dUTPunder the conditions specified by the supplier (Roche).Hybridization was performed overnight in standard bufferwith 50% formamide at 42°C. Stringency washes wereperformed with 0.1×SSC 0.1% SDS solution at 68°C.Genomic DNA of the 15 D. buzzatii lines was digested withthe following restriction enzyme pairs: EcoRI/HindIII(BuT1), SalI/XbaI (BuT2), BamHI/EcoRI (BuT3 andBuT4), BamHI/ClaI (BuT5), and BamHI/HindIII (BuT6).

In situ hybridization

Hybridization to the larval salivary gland polytene chromo-somes was carried out according to the procedure describedby Montgomery et al. (1987) using the same probes as in theSouthern hybridizations. Probes were labeled with biotin-16-dUTP (Roche) by nick translation. In situ hybridizationswith D. buzzatii lines (see above) were carried out at 37°C.Detection was carried out using the ABC-Elite kit fromVector Laboratories. Chromosomal localization of thehybridization signals was determined using the cytologicalmaps of D. buzzatii (Ruiz and Wasserman 1993). Therelative length of chromosomes and different chromosomalregions was estimated according to the number of bandsincluded in them in Drosophila repleta, the reference speciesof the group (Wharton 1942). The numbers of bands are 234,286, 240, 223, 231, and 18 for chromosomes X, 2, 3, 4, 5,and 6 (dot), respectively. The portions of chromosome 2represented by inversions 2j, 2z3, 2q7 and 2y3 are 0.37, 0.43,0.26, and 0.44, respectively (including 10 additional bandsadjacent to each breakpoint to take into account therecombination reduction in these regions; see Andolfatto etal. 2001). Coincidence of the different TE insertion sites andbetween the TE insertion sites and the cytological localiza-tion of chromosomal inversion breakpoints described in D.buzzatii and the buzzatii species complex (Casals et al. 2005)

Table 1 Characteristics of the TE probes used in this study

Elementcopy

Size(bp)

Breakpointof origin

Strain Clone Accessionnumber

Primers

BuT1a 769 Proximal, inversion 2j j-1 pGPE109 AY551075 E1 (5′-CAGTGATGCCAACTTGTTCT-3′)/E2(5′-TAGGCAAAACTGTCCGTAA-3′)

BuT2a 2,775 Proximal, inversion 2j j-8 pGPE156 AF368884 E3 (5′-CAGTGCTGCCAACAATTTGT-3′)/E4(5′-CACTGCTGCCAATTTAGCTA-3′)

BuT3-4a 795 Distal, inversion 2j jq7-2 PGPE142 AF368870 E6 (5′-TAGAGCTGGCAAACTATCGA-3′)BuT4-2b 720 – st-1 – DQ402469 E8 (5′-TAGTGCTGGGAAAAGATCGA-3′)BuT5-1a 1,039 Proximal, inversion 2j jz3-1 PGPE129 AF368894 E9 (5′-CACGATTGAGTAACACTAGG-3′)/E10

(5′-CACTGTTAAAAGACTCAGTAG-3′)BuT6c 387 Distal, inversion 2q7 jq7-4 PGPE209 AY187768 E11 (5′-CAGTGTTTGAAAAGCAAAGC-3′)/E12

(5′-CAGTGTTTAAAAAACTGCGA-3′)

aCáceres et al. 2001bThis workcCasals et al. 2003

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was tested with the statistical method described byZelentsova et al. (1999). Significance levels were adjustedfor multiple tests using the sequential Bonferroni procedure(Rice 1989).

Results

Abundance

Southern blot analyses indicated that the six transposons arepresent in all chromosomal lines of D. buzzatii (Fig. S1 inthe Appendix). The number of bands varied significantlybetween elements (χ2=137; df=5; P<0.001) BuT2 being theleast abundant element with 6.93 copies per line and BuT6being the most abundant element with 18.40 copies per line

(Table 2). On the other hand, the total number of bands perline varied between 62 and 87 but no significant differencesbetween lines were observed for any of the elements or forthe total (results not shown).

The abundance of the six TEs in nine D. buzzatii lineswas also analyzed using in situ hybridization, and thecytological localization of all signals is given in Table S1.The number of hybridization signals varied significantlybetween elements (χ2=359; df=5; P<0.001) with a mini-mum for BuT2 of 1.67 signals per line and a maximum forBuT5 of 23 signals per line (Table 2). The total number ofsignals per line varied between 10 and 72, and thedifferences were significant when all six TEs wereconsidered together (χ2=45,89; df=8; P<0.001). The chi-square test cannot be applied for each TE individuallybecause of the low number of insertions in some TEs.

Table 2 Number of observed insertions of six TEs in D. buzzatii detected by Southern blot hybridization (15 chromosomal lines) and in situhybridization (nine chromosomal lines)

Southern blot hybridization In situ hybridization

TE Mean (SD) st-1 j-2 j-23 j-24 jq7-4 jz3-6 jz3-7 y3-1 s-1 Mean (SD)

BuT1 9.53 (3.11) 11 1 2 4 3 5 2 5 0 3.67 (3.24)BuT2 6.93 (2.63) 0 0 1 1 1 2 10 0 0 1.67 (3.20)BuT3 10.87 (2.92) 18 5 5 8 13 6 5 8 4 8.00 (4.64)BuT4 9.67 (3.33) 8 4 3 4 5 7 9 6 4 5.56 (2.07)BuT5 17.67 (1.83) 29 11 31 22 46 27 20 19 2 23.00 (12.53)BuT6 18.40 (1.50) 1 5 8 9 4 0 4 5 0 4.00 (3.24)Total 73.07 (7.93) 67 26 50 48 72 47 50 43 10 45.89 (18.90)

Fig. 1 In situ hybridization of the six TEs included in this work to thesalivary gland chromosomes of different D. buzzatii lines. a BuT1(line st-1), b BuT2 (line j-23), c BuT3 (line jq7-4), d BuT4 (line jz3-7),

e BuT5 (line jz3-6), and f BuT6 (line j-24). Arrows indicate TEinsertions at the breakpoints of polymorphic inversions

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However, when it has been possible (BuT3, BuT4, andBuT5), significant differences between lines have also beendetected for BuT3 (χ2=21.50; df=8; P=0.006) and BuT5(χ2=54.61; df=8; P<0.001).

Chromosomal distribution

In situ hybridization also provides information about thechromosomal distribution of the six TEs in the D. buzzatiilines (Fig. 1). TE insertions are not randomly distributedover chromosomes (Table 3). TEs clearly tend to accumulatein the dot-like chromosome 6 both when the elements areconsidered together and separately. When the dot chromo-some is excluded from the analysis, there is still anonrandom distribution between chromosomes. There is alsoan excess of TE insertions in the X chromosome whencompared with the autosomes (Table 3). However, all thedifferences between chromosomes, or between chromosome

X and the autosomes, disappear when the proximal regions ofthe chromosomes are excluded from the analysis (see below).

To analyze the intrachromosomal distribution of theelements, we divided each chromosome into three parts,proximal, central, and distal, containing the 10, 80, and 10%of the euchromatin, respectively. This loosely followsvariation in recombination rate along the D. melanogasterchromosomes (Bartolomé et al. 2002). TEs clearly tend toaccumulate in the proximal regions of the chromosomes butnot in the telomeric regions (Table 4). When TE insertionslocated in the proximal regions are excluded from theanalysis (see above), there are no significant differences inthe number of insertions between the chromosomes (exceptin the dot chromosome) both when the six TEs areconsidered together (χ2=11.02; df=4; P=0.97) and separately.

We also compared the number of insertions locatedwithin inverted segments with the number of insertionslocated outside inverted segments of the same chromosome,excluding those insertions located in the proximal region(Table 5). To carry out this comparison, only chromosomallines fixed for inversions were considered (see “Materialsand methods” section). Inversion 4s was not included in theanalysis because no TE insertion outside the proximalregion was observed in the chromosomes carrying thisinversion. It is interesting to note that TE density issignificantly higher in the inverted segments than innoninverted segments of the second chromosome (Table 5).

Association between TEs and chromosomal inversionbreakpoints

The chromosomal localization of the TEs has also beencompared with the cytological localization of the breakpointsof chromosomal inversions described in D. buzzatii and in thebuzzatii species complex (see “Materials and methods”section). In addition to the previously reported localization

Table 3 Number of observed TE insertions in the six chromosomes of D. buzzatii

Chromosome Chi-square valuesa

TE X 2 3 4 5 6 (dot) H0 H1 H2

BuT1 15 5 7 1 0 5 64.67*** 26.48*** 21.13***BuT2 0 2 5 4 2 2 19.80** 6.34 3.10BuT3 14 11 18 12 8 9 63.50*** 5.06 0.35BuT4 14 8 6 2 2 18 412.76*** 15.26** 12.30***BuT5 43 47 40 20 42 15 54.32*** 8.61 1.20BuT6 0 7 10 11 1 7 93.61*** 18.46** 6.92**Total 86 80 86 50 55 56 418.91*** 13.95** 5.30*

*P<0.05**P<0.01***P<0.001aH0 assumes a random distribution of TE insertions in which the number of insertions per chromosome is proportional to chromosomesize (df=5). H1 assumes a random distribution, excluding chromosome 6 (df=4). H2 assumes a random distribution between X chromosome andautosomes, excluding chromosome 6 (df=1)

Table 4 Number of observed TE insertions in the distal, central, andproximal regions of chromosomes X, 2, 3, 4, and 5 of D. buzzatii

Chromosomal region Chi-square valuea

TE Distal Central Proximal H0

BuT1 0 6 22 146.46***BuT2 0 5 8 38.63***BuT3 1 35 27 77.18***BuT4 1 23 8 8.98*BuT5 5 98 89 284.38***BuT6 0 2 27 222.55***Total 7 169 181 662.05***

*P<0.05***P<0.001aH0 assumes a random distribution of TE insertions in which thenumber of insertions per region is proportional to its size (df=2)

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of the TEs analyzed in this work at the breakpoints ofchromosomal inversions 2j (Cáceres et al. 2001) and 2q7

(Casals et al. 2003), BuT5 has been precisely located at thedistal breakpoint of chromosomal inversion 2z3, in a linecarrying this inversion (Fig. 1). We have also considered thecytological localization of all the inversion breakpoints in the16 polymorphic inversions described in D. buzzatii, represent-ing 27 breakpoints (Casals et al. 2005), due to the fact thatsome of the breakpoints are shared by two inversions. To testwhether there is an association between TE insertions and theinversion breakpoints of those polymorphic inversions de-scribed in D. buzzatii, we calculated the expected number ofrandom coincidences and the probability of the observednumber of coincidences (see “Materials and methods”section). The dot-like chromosome 6 and the proximal regionsof the chromosomes were excluded from the analysis becauseof the significant accumulation of TE insertions in theseregions (see above). In addition to the six transposonsanalyzed in this study, we considered also the chromosomallocalization of the Galileo element analyzed in previous work(Casals et al. 2005). Overall, there are 1,094 bands, 26inversion breakpoints (excluding one of the breakpoints of the2d9 inversion located in band 2G3d that is included in theproximal region) and 185 bands where at least one TE hasbeen localized. The associations of BuT1, BuT5, and BuT6with the breakpoints of polymorphic inversions were statisti-cally significant, but only BuT5 remained significant when thesequential Bonferroni correction was applied (Table S2).Overall, the association of TE insertions with the breakpointsof D. buzzatii polymorphic inversions was only marginallysignificant (P=0.057). Similarly, we also tested for anassociation of the TE insertions with the inversion breakpointsof all the chromosomal inversions (fixed and polymorphic)described in the buzzatii species complex (Ruiz and Wasser-man 1993; Casals et al. 2005). The association of BuT3 andGalileo was significant but none of them remained significant

when the sequential Bonferroni correction was applied.Overall, the association of all TE insertions with the break-points of inversions present in the buzzatii species complexwas only marginally significant (P=0.059; Table S2).

Association between TEs

Finally, we also tested for an association between theinsertions of the different TEs. We again included the sixTEs and the Galileo elements described in D. buzzatii(Casals et al. 2005). Table S3 shows the 71 chromosomalbands where two or more TEs have been located. Thisanalysis has been done including the same regions as above(excluding the proximal regions of the chromosomes andthe dot chromosome) and including/excluding the bandswhere the breakpoints of inversions 2j and 2q7 are located,which may serve to elucidate the cause of the accumulationof TEs at the inversion breakpoints. Table S4 shows thesignificance of the association between elements. Becausewe have seven TEs, we must consider 21 pairs of twoelements. We also considered the associations between eachelement and other elements (designed collectively in Table S4as TE). Including the breakpoints of inversions 2j and 2q7,four TE pairs (BuT1–BuT2, BuT1–Galileo, BuT3–BuT5,and BuT5–Galileo) showed a significant association(P<0.05). However, when the sequential Bonferroni cor-rection was applied, only one of them (BuT3–BuT5)remained significant. In addition, four associations betweenan element (BuT1, BuT3, BuT5, and Galileo) and theremaining TEs were significant; all except one (BuT1)remained significant with Bonferroni correction.

When the breakpoints of 2j and 2q7 inversions wereexcluded from the analysis, two element pairs (BuT3–BuT5and BuT5–Galileo) showed a significant association(P<0.05) but none of them was significant after Bonferronicorrection. Also, three associations between an element

Table 5 Distribution of TEs over chromosome 2 with respect to inversions

2j 2z3 2q7 2y3 Total Chi-square a

TE Inside Outside Inside Outside Inside Outside Inside Outside Inside Outside H0

BuT1 3 0 1 0 1 0 0 0 5 0 7.96**BuT2 2 0 0 0 0 0 0 0 2 0 3.19BuT3 1 2 0 1 3 0 0 0 4 3 1.02BuT4 0 0 3 3 0 0 0 1 3 4 0.05BuT5 2 6 3 4 5 2 2 5 12 17 0.10BuT6 0 0 0 0 1 0 0 0 1 0 1.59Total 8 8 7 8 10 2 2 6 27 24 5.35*Chi-squarea 1.16 0.08 20.50*** 1.17 5.35*

*P<0.05**P<0.01***P<0.001aNull hypothesis assumes a random distribution in which the number of insertions in each region is proportional to its size (df=1)

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(BuT3, BuT5, and Galileo) and the remaining TEs weresignificant, but again the significance disappeared whenBonferroni correction was applied.

Discussion

The abundance of six transposons, BuT1, BuT2, BuT3,BuT4, BuT5, and BuT6, in the genome of D. buzzatii wasinvestigated by Southern blotting and in situ hybridizationto salivary gland chromosomes. The number of insertionsdetected by Southern blotting was somewhat higher thanthe number observed by in situ hybridization in all casesbut BuT5. Three reasons might contribute to these differ-ences: (1) the greater sensitivity of Southern blotting; (2)the fact that in situ hybridization only detects insertionslocated in the euchromatin (that is polytenized in thesalivary gland chromosomes), whereas Southern hybridiza-tion detects all insertions in the genome (including those inthe heterochromatin); and (3) the possibility that in somecases Southern blotting produces more than one band froma single insertion due to the presence of internal restrictionsites. We tried to minimize the latter effect by choosingthose restriction enzymes that do not cut within the knowncopies of each element. However, such restriction sites mayexist in other, unknown copies of the elements. The lowernumber of insertions detected by Southern blotting for theabundant BuT5 is probably due to the generation of equallength fragments that are only computed as one insertion. A

similar pattern has been previously described in D.melanogaster, where a saturation with bands in agarosegels was invoked to explain the deficit of insertions inSouthern blots when compared to in situ hybridization inthe more abundant TE families (Maside et al. 2001).

The inter- and intrachromosomal distribution of the sixD. buzzatii transposons is clearly nonrandom (Tables 3, 4,and 5). These elements tend to accumulate in particularchromosomal regions, although to different extent forindividual elements. We have estimated TE density in thedifferent chromosomal regions as 1,000 times the numberof TE insertions per genome (chromosomal line) divided bythe number of chromosomal bands in the region. A basalTE density was estimated using the central and distalportions of the five major chromosomes after removing theinverted chromosomal segments. These basal densities are0.11, 0.33, 3.53, 2.32, 10.04, and 0.11 for BuT1, BuT2,BuT3, BuT4, BuT5, and BuT6, respectively. Similarly, thebasal density for Galileo estimated using data from Casalset al. (2005) is 7.72. Thus, BuT5 is the most abundantelement in the central and distal chromosomal regions,followed by Galileo, BuT3, and BuT4. TE insertionsaccumulate in the dot chromosome and the proximalregions of all major chromosomes. On average, theinsertion density in the dot chromosome was ∼21 timesthe basal density, whereas that in the proximal regions was∼10 times the basal density. However, there were differ-ences between individual elements, with BuT1, BuT2, andBuT6 being the elements with the highest relative accumu-lation (Fig. 2). For comparison, the density of Galileo in thedot chromosome and the proximal regions was ∼76 timesand ∼36 times the basal density, respectively (Fig. 2). TEinsertions tend to accumulate also in the inverted segmentsof chromosomal rearrangements. Insertion density of all sixtransposons was higher in the inverted segments than in the(noninverted) central–distal regions, with an averagedensity ∼2 times the basal density (Fig. 2). Galileo showsalso an increased density in the inverted segments, which isnearly six times the basal density. Figure 2 shows that theaccumulation of TE insertions varies between elements andis correlated between dot, proximal regions, and invertedchromosomal segments. With the exception of BuT4, whichexhibits a somewhat anomalous behavior, those elementswith the highest TE density in the dot chromosome (relativeto the basal density) are also those with the highest densityin the proximal regions and inverted segments. Thisobservation points to a common mechanism.

The chromosomal distribution of the six transposons inD. buzzatii is consistent with the distribution of TEs in D.melanogaster (see “Introduction” section). The accumula-tion of TEs in the dot and pericentromeric regions has beenattributed to preferential targeting into the heterochromatin(Pimpinelli et al. 1995). Alternatively, the accumulation of

Fig. 2 TE insertion density (relative to central–distal regions) in thedot chromosome, the proximal regions of the five major chromo-somes, and the segments included in chromosomal inversions of D.buzzatii. TE density was calculated as 1,000 times the number ofinsertions divided by the number of chromosomal bands in theconsidered region. Note the logarithmic scale of the y-axis

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TEs in these regions can be a consequence of the reducedrecombination decreasing their elimination rate (Charlesworthand Langley 1989; Charlesworth et al. 1994). Our resultsstrongly support the latter hypothesis. In D. melanogaster,recombination rate in the dot chromosome and thepericentromeric regions of the major chromosome is greatlyreduced or suppressed (Ashburner 1989; Charlesworth1996; Bartolomé et al. 2002). In D. buzzatii, the averagerecombination rate is more than twice that in D. mela-nogaster (Cáceres et al. 1999a), but it is reasonable toassume that it is similarly reduced or suppressed in thepericentromeric regions and the dot chromosome (althoughwe lack detailed studies). Recombination is also reduced ininversion heterokaryotypes (Navarro et al. 1997), whichexplains the accumulation of TE insertions in the invertedsegments of rearranged chromosomes, particularly thosewith low frequencies (Eanes et al. 1992). The accumulationof TE insertions in inverted segments of D. buzzatii and thecorrelation across elements between dot, proximal regions,and inverted segments (Fig. 2) strongly support the role ofrecombination rate as a major force determining thedistribution of TEs.

Low recombination regions show usually a low genedensity, which should also favor the insertion of TEswithout a deleterious effect in the host genome. However,the dot chromosome shows a similar gene density to thatdescribed in the high recombination regions of autosomes(Charlesworth et al. 1992; Bartolomé and Maside 2004),suggesting that gene density is not a major factordetermining the chromosomal distribution of TEs. Theaccumulation of TE insertions in inverted segments pointsto the same conclusion because there is no reason to assumethat inverted segments have a lower gene density thannoninverted segments. Therefore, ectopic recombinationrather than gene disruption is the likely mechanism for thedecreased efficiency of TE elimination in low recombina-tion regions. The relatively moderate accumulation of TEinsertions in inverted segments compared with the dotchromosome and proximal regions is consistent with therelatively young age of the inversions. For instance, the ageof inversion 2j has been estimated as ∼1 Myr, while that ofinversion 2q7 is 0.49–0.67 Myr (Cáceres et al. 2001; Casalset al. 2003). Perhaps the surprising observation is that thecolonization of inverted segments proceeds so fast. Asimilar phenomenon of fast colonization by TEs of newlyarisen low recombination regions has been described in theneo-Y chromosome of Drosophila miranda (Steinemannand Steinemann 1991, 1997; Bachtrog 2003).

The variation between individual TEs in the rate ofaccumulation in low recombination regions (Fig. 2) isexpected because they are likely to differ in rate oftransposition, number and size of the copies, as well asage and evolutionary history. Previously, differences be-

tween class I and class II elements have been observed. Thenumber of class II TE insertions is negatively correlatedwith recombination rate in the euchromatin of D. mela-nogaster, whereas the number of class I TE insertions is not(Rizzon et al. 2002). However, four LTR elements showsignificant associations with rare inversions of D. mela-nogaster chromosome 3R, suggesting that at least someclass I elements are also preferably located at lowrecombination regions. Petrov et al. (2003) argued thatunder the ectopic recombination model, the strength ofselection should vary across TE families because thefrequency of ectopic recombination increases with copynumber and length of elements. Their observations on fournon-LTR class I elements were entirely consistent with thisprediction.

The six TEs analyzed in this work were shown to besecondary insertions of the breakpoints of the polymorphicinversions 2j and 2q7 of D. buzzatii, produced after thegeneration of the inversions (Cáceres et al. 2001; Casals etal. 2003). These elements are located exactly at theinversion breakpoints but did not originate the inversions.The fact that all but one of the elements (BuT6) found at the2q7 inversion breakpoints are also present at the 2jinversion breakpoints is very intriguing. Also remarkableis the fact that all these TEs are class II elements, the lessabundant class in the D. melanogaster euchromatin(Kaminker et al. 2002). There is no available data aboutthe relative abundance of the different TE classes in the D.buzzatii genome, but several examples of class I elementshave been reported (Francino et al. 1993; Labrador andFontdevila 1994; Lathe et al. 1995). None of these elementswas found at the inversion breakpoints. The accumulationof TEs at the breakpoints of chromosomal inversions maybe due to the drastic reduction of the recombination rate atthese regions (Navarro et al. 1997). Alternatively, the factthat the four inversion breakpoints appear to have becomegenetically unstable regions and hotspots for the accumu-lation of TE insertions and other structural changes mightbe the result of preferential insertion (Craig 1997). In D.melanogaster, 21% of TEs are inserted into other TEs or inan adjacent position (Kaminker et al. 2002), and associa-tions between TEs have been previously reported forPenelope and Ulysses elements in Drosophila virilis(Zelentsova et al. 1999; Evgen’ev et al. 2000) and forLTR retrotransposons in maize (SanMiguel et al. 1996,1998). If the accumulation of TEs at the inversion break-points were due to preferential insertion, other associationsbetween these TEs might be found in other chromosomalsites. The comparison of the cytological positions of the sixTEs included in this study and the three foldback elementspreviously described (Casals et al. 2005) indicated severalcases of associations between TEs (Table S4). However,when the inversion breakpoints were excluded from the

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analysis, none of the associations remained significant. Thisobservation suggests that preferential insertion cannot bethe sole explanation. As a matter of fact, the reduction ofrecombination rate and the preferential insertion are notexclusive hypotheses. A molecular investigation of thetarget insertion sequences of the six transposons analyzedin this study might help to elucidate the relative role of thetwo hypotheses.

The precise location of TEs at the breakpoints ofchromosomal inversions is sometimes interpreted as evi-dence for a causal role of TEs in the generation ofinversions (Evgen’ev et al. 2000). Besides its presence atthe breakpoints of inversions 2j and 2q7, the element BuT5has been located at the distal breakpoint of inversion 2jz3

(Fig. 2) and shows a significant association with thebreakpoints of the polymorphic inversions described in D.buzzatii (Table S2). These results are interesting but mustbe interpreted with caution because no molecular evidencesupports them. The mere presence of a TE in an inversionbreakpoint does not imply the participation of the TE in theorigin of the inversion, as has been shown with thesecondary TE invasions at the 2j and 2q7 inversionbreakpoints (Cáceres et al. 2001; Casals et al. 2003).

Acknowledgement This work was supported by grant BMC2002-01708 from the Dirección General de Enseñanza Superior eInvestigación Científica (Ministerio de Educación y Cultura, Spain),which was awarded to A.R.

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