Structural Heterogeneity and Genomic Distribution of Drosophila ...

Structural Heterogeneity and Genomic Distribution of Drosophila melanogaster LTR-Retrotransposons Lucıá Alonso-Gonza´lez, Ana Domıńguez, and Jesu´s Albornoz Area de Gene´tica, Departamento de Biologıá Funcional, Universidad de Oviedo, Oviedo, Spain Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, b-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.

Introduction A large fraction of the eukaryotic DNA is composed of transposable elements that can cause mutations when they transpose to novel sites. Due to their significant influence on genomic diversity, the nature of forces affecting the transpositional spread of transposons and their distribution throughout the genome is a major problem of evolutionary genetics. The forces controlling their abundance within their host genomes have been the subject of a great deal of empirical and theoretical research (e.g., Langley, Brookfield, and Kaplan 1983; Capy 1997), although their nature and relative significance is still controversial. Such forces include selection against the deleterious effects of transposable elements in the host genome, self-regulation, and interaction with the host genome (Nuzhdin 1999). In previous studies on a set of mutation-accumulation lines, we found that the main source of variability affecting transposable elements was the generation of internal rearrangements. The rate of rearrangement was 8.5 3 10–6 (Domıńguez and Albornoz 1996; Albornoz and Domıńguez 1999; Domıńguez and Albornoz 1999). This applied to class I (297, 412, and copia), class II (P and hobo), and Foldback (FB) elements. The existence of this kind of mutation with a nonnegligible rate allows us to presume structural heterogeneity among transposable elements of the Drosophila genome. Structural heterogeneity in the elements pertaining to the same family is common for class II elements, retroposons, and the FB element of Drosophila (Potter et al. 1980; O’Hare and Rubin 1983; Vaury, Bucheton, and Pelisson 1989; Bucheton et al. 1992; Capy, David, and Hartl 1992). In contrast, the sequences of Drosophila retrotransposons (long terminal repeat [LTR] elements) have been shown to be closely conserved in the genome, present mainly as fullsized elements with only a few rearranged copies (Finnegan 1985). The existence of noncanonical elements Key words: Transposable elements, retrotransposons, rearrangements, heterochromatin, Drosophila melanogaster. E-mail: [email protected]. Mol. Biol. Evol. 20(3):401–409. 2003 DOI: 10.1093/molbev/msg047 Ó 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

in the heterochromatin of D. melanogaster has been reported for some retrotransposon families (Shevelyov, Balakireva, and Gvozdev 1989; Vaury, Bucheton, and Pellison 1989; Montchamp-Moreau et al. 1993). In plants, however, the structural heterogeneity of retrotransposon families is the rule (Marillonnet and Wessler 1998). Rearranged copies of transposable elements are often considered as old genomic components, usually embedded in heterochromatin (Bucheton et al. 1992; Vaury et al. 1994). There are different nonexclusive hypotheses to explain the accumulation of transposable elements in heterochromatin. This region may either contain insertional hotspots or may function like a trap in which transposable elements become immobilized by position-effect inactivation. Another possibility is that once inserted there, elements are not so easily eliminated by natural selection and recombination as are euchromatic insertions (reviewed in Charlesworth, Sniegowosky, and Stephan 1994). Recent evidence suggests that the relationship between transposable elements and heterochromatin will not be quite so straightforward. Constitutive heterochromatin contains genetically active domains, and there is evidence for elements that underwent fixation, probably under selective pressure for regulatory roles (reviewed in Dimitri 1997; Dimitri and Junakovic 1999). Recent studies have shown the existence of degenerate euchromatic elements pertaining to the 297 and 412 families (Cizeron and Bie´mont 1999; Domıńguez and Albornoz 1999) in D. melanogaster and D. simulans. Studies based on the released sequence of the D. melanogaster and Caenorhabditis elegans genomes have also reported structural heterogeneity of euchromatic LTRretrotransposons (Bowen and McDonald 2001; Frame, Cutfield, and Pulter 2001; Ganko, Fielman, and McDonald 2001). In this paper, we address the question of the structural heterogeneity and genomic distribution of five families of D. melanogaser LTR elements (297, 412, 1731, copia, and mdg1). Southern blot analysis of individuals allows us to reveal low-frequency bands and evaluate intrastock polymorphism for rearranged elements. The comparison of laboratory strains and a wild population show the existence of characteristic features of each 401

402 Alonso-Gonza´lez et al.

size 412 element (Shepherd and Finnegan 1984) and the 2.8-kb Hin dIII fragment from Dm 58 (Ilyin et al. 1980). These clones were digested with the appropriate combination of restriction enzymes, and, after electrophoresis, the fragment to be used as a probe was extracted from the agarose gel. Southern blots were performed as previously described (Albornoz and Domıńguez 1999). To check for the completeness of enzyme digestion, filters were retested with the 1.9-kb Eco RI-Bam HI fragment from Dm FB1, which is a piece of single-copy genomic DNA (Potter et al. 1980). The autoradiographs were scanned and analyzed with the GelReader software from the National Center for Supercomputing Applications (1991). In situ hybridization was carried out following the protocol of Gatti, Bonaccorsi and Pimpinelli (1994) and fluorescein-labeled probes. Deductions on the Proportion of Noncanonical Elements and Their Genomic Distribution

FIG. 1.—Sites for restriction enzymes that were used in Southern blots and probes. Size of the expected fragments for canonical copies of each retrotransposon family.

family both in the extent of heterogeneity and in the proportion of rearranged elements per individual. The genomic distribution of canonical and noncanonical elements was investigated in one fully homozygous line by comparing the results of Southern blot analysis and fluorescent in situ hybridization. Materials and Methods Fly Lines Three laboratory strains and a natural population were analyzed in this work. The laboratory lines were Oregon R, Canton S (both from the Bowling Green Stock Center) and the fully homozygous line C66 from Domıńguez and Albornoz (1996). Wild flies were collected from a natural population recently caught in Llanera (in the north of Spain). Laboratory Methods Genomic DNA was extracted from the salivary glands of third-instar larvae, single flies, and pools of flies as described in Di Franco et al. (1989), Domıńguez and Albornoz (1996), and Albornoz and Domıńguez (1999), respectively. Probes for 297, 1731, and copia were described in Di Franco, Galuppi, and Junakovic (1992). Probes for 412 and mdg 1 were obtained from clones pOR 708 and 5B257 bearing, respectively, a full-

To categorize elements, we used the sequences described in GenBank as representatives of each family as the canonical element (X03431 for 297, X59545 for mdg 1, X04132 for 412, X04456 for copia, and X07656 for 1731). Classification of elements as canonical or noncanonical was based on obtaining of the expected internal restriction fragments in Southern blots. Genomic DNA was digested with the restriction enzyme or the combination of two that rend the largest internal fragment for each family. Combinations of enzymes and the expected size of the fragment detected for the canonical element of each retrotransposon family are shown in figure 1. Noncanonical elements will generate fragments of a different size. A larger size than expected might be a consequence of the loss of a restriction site or insertion of a new sequence, and a smaller size might be due to either internal deletions or the presence of additional restriction sites. This approach is limited by the availability of restriction enzymes and adequate probes, it being possible that some noncanonical elements were not detected. The total number of elements of each family was scored from Southern blots. Digestions with Hin dIII were used for 297, copia, and mdg 1 and with Eco RI for 1731 and 412. The 1731 element has no Eco RI internal sites, thus, the number of bands will fit with the number of copies. Elements 412, copia, and mdg 1 have one or more internal sites for the restriction enzyme. Consequently, the digestion of genomic DNA produces two or more fragments, but only one of the ends of the element hybridizes with the probe, and hence the number of bands equals the number of copies. Element 297 has two internal Hin dIII sites, and so each element produces three fragments after digestion. The probe reveals one of the ends of the element and the central fragment. Consequently, the number of bands exceeds the number of copies by one, which corresponds to all the central fragments (see fig. 1). The genomic distribution of canonical and noncanonical elements was approached by comparing in situ hybridization on polytene chromosomes with Southern blots. In the salivary glands of Drosophila larvae,

Noncanonical Retrotransposons and Heterochromatin 403

euchromatin, and b-heterochromatin undergo polytenization (John 1988; Dimitri 1997). b-heterochromatin does not present the banding pattern characteristic of euchromatin and can only be seen as an amorphous region at the base of each chromosome arm. a-heterochromatin undergoes few, if any, rounds of replication during polytenization and is found as a dense region centrally located at the chromocenter. It corresponds to the bulk of constitutive heterochromatic segments so evident in the mitotic chromosomes. From these considerations, we have estimated the number of euchromatic elements in the fully homozygous line C66 as the number of signals detected in chromosome arms by in situ hybridization. The number of elements in b-heterochromatin was estimated as the difference between the number of bands in Southern blots from salivary glands and the number of in situ signals in chromosome arms. The comparison of the autoradiographs from adults and salivary glands was used to identify the elements located in underreplicated regions in a-heterochromatin. Finally, the number of elements on chromosome Y was deduced from the comparison of blots from adult males and females. We assume that hybridization signals are due to single elements. Hence the number of euchromatic elements may be underestimated if in some instances there is more than one element per hybridization signal. Results Southern blot pictures of digestions performed to reveal noncanonical elements in individuals from the four strains are shown in figure 2 and the data are summarized in table 1. The proportion of noncanonical elements was estimated from the densitometry analysis of autoradiographs as the relative intensity of bands other than that expected for the canonical element. The number of noncanonical elements per individual was computed as the number of restriction fragments other than that expected for the canonical elements (see fig. 2). This will be an estimation of the minimum number of structurally variant elements per individual, since the actual number depends on the heterozygosity of the lines and on the existence of multiple loci with noncanonical elements coincident in size. Bands present in all males from a strain, and only in the males, were classified as noncanonical elements located on the Y chromosome. The number of noncanonical size classes of each family of elements, as well as the percentage that they account for in the genome, were similar for the four strains. Families 297 and mdg 1 shared similar features: they have 17 to 20 classes of noncanonical elements per individual that account for about 80% of the hybridization signal. Rearranged elements were highly heterogeneous in size. Only two 297 (6.7 and 4.8 kb) and three mdg 1 (5.3, 4.3, and 3.5 kb) noncanonical classes were common to the four strains. In the wild population, noncanonical elements were also very heterogeneous, most of these segregating at low frequencies (fig. 3a and b). The total number of size classes in the sample from Llanera for element 297 was 64 with a mean frequency of 0.30, and for mdg1, the number of classes was 79 with a mean frequency of 0.24.

FIG. 2.—Banding patterns in Southern blots made to detect noncanonical elements in individuals from the four strains. Bands generated by potentially canonical elements (see fig. 1) are indicated by an asterisk.


Table 1 Number of Size Classes per Individual and Percentage Intensity of Noncanonical Elements in Southern Blots of the Four Strains Number of Individuals Element

Noncanonical Classes per Individual 6 SD Females

Males

Mean % of Noncanonical Copies per Individual

Strain

Females

Males

297

Llanera Canton S Oregon R C 66

9 2 2 2

9 2 2 2

20.33 18.50 17 20

6 6 6 6

0.37 1.50 0 0

16.89 17.00 17 20

6 6 6 6

0.42 1.00 0 0

79.91 80.35 76.25 80.30

6 6 6 6

1.87 4.03 2.87 1.92

mdg 1


7 3 3 3

7 2 3 2

17.29 16 18 18

6 6 6 6

0.68 0 0 0

18.71 21.5 20 20

6 6 6 6

1.23 (1) 1.50 (5) 0 (2) 0 (2)

81.10 78.30 76.26 81.46

6 6 6 6

0.76 1.37 0.66 1.14

412


10 3 5 2

10 5 4 2

6.20 5.33. 5 7

6 6 6 6

0.76 0.33 0 0

8.90 8.20 6.50 7

6 6 6 6

0.64 (2) 0.58 (1) 0.50 0

46.82 39.99 42.40 38.04

6 6 6 6

3.06 3.51 2.14 3.84

copia

Llanera Canton S Oregon R C66

9 3 3 2

8 5 3 2

3.33 6.20 3 3

6 6 6 6

0.47 0.49 0 0

4.62 9.80 4 3

6 6 6 6

0.56 0.77 0 (1) 0

26.73 55.37 53.07 44.62

6 6 6 6

3.50 3.45 8.34 2.26

1731


8 4 4 2

8 5 4 3

13.13 13.25 14 13

6 6 6 6

0.67 0.48 0 0

13.63 20.20 18 16

6 6 6 6

0.68 (2) 0.37 (6) 0 (4) 0 (3)

91.00 93.18 93.06 88.72

6 6 6 6

1.32 0.50 0,32 0.48

NOTE.—Numbers in parentheses indicates the number of size classes in chromosome Y.

Noncanonical classes of both families that appeared in the four strains were at frequency 1 in the Llanera population. For the 412 and copia families, the proportion of noncanonical elements per individual was lower, as was the number of size classes (table 1). Noncanonical elements were heterogeneous in size. As can be seen from figure 2, the autoradiographic bands corresponding to noncanonical elements are faint and quite close to the canonical element, particularly for copia. This observation contrasts with the results obtained for the other three elements included in the study, where prominent noncanonical bands can be observed. Only two 412 noncanonical classes (4.8 and 4.6 kb) were common to the four strains; these were also present in every individual from the wild population. In the sample from the Llanera population, the number of size classes of noncanonical elements for families 412 and copia were 32 and 23 with mean frequencies of 0.27 and 0.21, respectively (fig. 3c and d). The 1731 family was mainly composed of rearranged elements, the proportion of putative canonical copies being between 6% and 11%. Southern blots showed between 13 and 20 noncanonical size classes per individual in the different lines studied. The number of size classes in the sample of the wild population was 24 with a mean frequency of 0.55 (fig. 3e). Ten noncanonical classes were common to the four strains, but only four noncanonical classes were present in every individual; two of the latter were insertions on chromosome Y. The common noncanonical class of 3.6 kb was the most abundant in the four strains (fig. 2d) and accounted for 20% to 25% of the hybridization signal in every strain. The genomic distribution of structurally variant elements was analyzed in the line C66 (table 2). The complete homozygosity of this line allows us to test the

number of hybridization signals obtained by different methods and to compare them so as to deduce the distribution of elements in this genotype (see Materials and Methods). First, the number of elements per haploid genome was deduced from Southern blots on adult flies. The number of noncanonical size classes is the minimum number of noncanonical elements. With the exception of copia, these numbers agree quite well with those that can be estimated by the product of their relative intensity in autoradiographs by the number of elements: 26, 26, 8, 11, and 17 for families 297, mdg1, 412, copia, and 1731, respectively. This implies that most size classes of rearranged elements are present in one or a few loci per haploid genome. This estimation might contain a substantial error, since if some bands corresponding to noncanonical elements were close to those generated by the canonical elements, their absorption profiles would overlap and hence the proportion of noncanonical elements would be overestimated. If, on the other hand, the fullsize element is very abundant, saturation of the photographic sheet may be another cause of underestimation of the proportion of canonical elements. This source of error is of particular importance for copia (see fig. 2) and could explain the discrepancy between the two estimations of the number of noncanonical elements. The number of in situ hybridization signals is the minimum number of euchromatic elements, given that there might be more than one element per chromosome band. The 297 family had 32 elements per haploid genome. Five elements were inserted in a-heterochromatin and the remaining 27 were euchromatic. One of them is located in band 20A, at the border between euchromatin and bheterochromatin. At least 20 elements were noncanonical


FIG. 3.—Size distributions of autoradiographic bands in the samples from the Llanera population.

and only one of them was located in the a-heterochromatin. Consequently, there is a maximum of eight complete elements in the euchromatin and four in the a-heterochromatin. The mdg 1 family was represented by 32 elements per haploid genome. Seven elements were inserted in aheterochromatin (two on the Y chromosome) and 19 were euchromatic; the remaining six elements not detected by in situ hybridization were classified as b-heterochromatic.

Twenty elements were found to be noncanonical; of these, seven were a-heterochromatic (two on the Y chromosome). The remaining 13 noncanonical elements would be distributed between b-heterochromatin and euchromatin. Consequently, there is a maximum of 12 complete elements in euchromatin. The 412 family had 20 elements per haploid genome. Six were in a-heterochromatin (one on the Y chromosome) and 14 were euchromatic. Seven noncanonical


Table 2 Genomic Distribution of Elements in Line C 66 Distribution in Chromatin Number of Elements/Genome

a-Heterochromatin

Minimum Element Total Noncanonical Euchromatin 297 mdg 1 412 Copia 1731

32 32 20 27 19

20 20 7 3 16

27 19 14 19 2

Non-Y

Y

5 (1) 5 (5) 5 (3) 6 (0) 0

0 2 (2) 1 (0) 0 3 (3)

Noncanonical Elements in b-Heterochromatin b-Heterochromatin 1 Euchromatin 0 6 0 2 14

19 13 4 3 13

Maximum Canonical Elements in Euchromatin 8 12 10 18 2

NOTE.—The numbers were obtained by comparing results of in situ hybridization to polytenic chromosomes and Southern blots with different combinations of enzymes on DNA extracted from adult males and females and larval salivary glands (see text). Numbers in parentheses indicate the number of noncanonical elements in a-heterochromatin.

elements were observed; three of these being a-heterochromatic. Consequently, there is a maximum of 10 canonical elements in euchromatin and three in aheterochomatin. Copia was represented by 27 elements. Six elements were a-heterochromatic and 18 euchromatic. The other three elements not detected by in situ hybridization might be b-heterochromatic. Moreover, two elements were mapped to the polytene band 41F, at the border between b-heterochromatin and euchromatin. Only three noncanonical elements were detected, and these were not a-heterochromatic. Therefore, six potentially full-size elements were inserted in the a-heterochromatin. The element 1731 presented 19 copies per haploid genome, and the number of noncanonical size classes was 16. The noncanonical size class of 3.6 kb (see fig. 2) accounted for 25% of the Southern blot hybridization signal, whereas the canonical element accounted only for 11%. Therefore, the number of canonical elements would probably be one, whereas there would be two or three copies of the element giving the restriction fragment of 3.6 kb. Three noncanonical elements were a-heterochromatic and were located on chromosome Y. Only two signals were detected by in situ hybridization, one of which was in band 20A, at the b-heterochromatin–euchromatin boundary. Thus, the number of b-heterochromatic elements would be 14. Accordingly, an intense signal can be seen at the chromocenter region corresponding to b-heterochromatin.

Discussion Studies carried out by in situ hybridization to mitotic chromosomes have revealed prominent stable clusters of transposable elements located in heterochromatic regions (Carmena and Gonza´lez 1995; Pimpinelli et al. 1995). Studies of reassociation kinetics suggest that transposable elements represent 9% of the genome (Spradling and Rubin, 1981), that is, 16 Mb out of the 180 Mb that constitute the genome of D. melanogaster (Adams et al. 2000). The consideration that there are 50 families with an average size of 5.4 kb and an average copy number in the euchromatic arms of 32 (Maside et al. 2001) leads to the conclusion that approximately 8 Mb out of the 120 Mb that constitute

euchromatin corresponds to transposable elements. This implies that half of the sequences are heterochromatic, and hence the density of transposable elements in heterochromatin must be twofold higher than in euchromatin. With our technical approach, only 38% (49 out of a total of 130 elements for the five families considered) of the bands were heterochromatic in line C66. This value is somewhat lower than expected, bearing in mind the above considerations. One possible source of this discrepancy is that heterochromatic elements are linked to a fraction of DNA that remains a high-molecular-weight fraction in restriction digests and is not transferred to the membrane. Another possibility is that this fraction of DNA might contribute to a Southern blot picture as a smear of DNA fragments resulting from degradation during extraction (Terrinoni et al. 1997). Our study is hence restricted to the fraction of transposable elements that are detectable with the regular Southern blot method, a fraction that is biased towards euchromatic elements. It is worth noting that the comparison of DNA from adults and salivary glands (a method devised by Di Franco et al. 1989) allows aheterochromatic copies to be properly distinguished, as can be noticed by the fact that the elements on chromosome Y were independently classified as heterochromatic. On the other hand, Southern blot estimates of family copy numbers were always higher than in situ family copy numbers, as was expected, denoting a good level of resolution of this technique. Some specific characteristic of each family of transposable elements can be observed with regards to the intragenomic distribution of elements. Family 1731 was the only one preferentially inserted in heterochromatin, only two of the 19 copies detected in line C66 were viewable in situ and one of these was in band 20A, at the junction between euchromatin and bheterochromatin. The other four elements showed a small proportion of heterochromatic copies. On the other hand, families 297, 412, and copia had potentially active elements in a-heterochromatin. This is in line with some reports that showed that heterochromatic transposable elements may be active (Hochstenbach et al. 1996; Chalvet et al. 1998). The proportion of noncanonical elements is a particular characteristic of each family. Elements copia and 412


were mainly represented by putative full-size elements, mdg 1 and 297 had around 75% to 80% of rearranged elements that were not conserved between stocks, and most copies of 1731 were noncanonical elements with a remarkable conservation within and between stocks. These results can be compared with the study of LTRretrotransposons on the release of the complete euchromatic genome sequence of D. melanogaster by Bowen and McDonald (2001) that showed many of the characterized elements to contain sequence deletions. In previous studies (Domıńguez and Albornoz 1996), we estimated a rate of transposable element structural mutation of 8.5 3 10–6. A similar value was recently reported (6.8 3 10–6) by Maside et al. (2001). The existence of this kind of mutation is consistent with the structural heterogeneity observed and its rate is congruent with that reported by Petrov, Lozovskaya, and Hartl (1996) for small deletions in Drosophila, as dicussed in Albornoz and Domıńguez (1999) and Domıńguez and Albornoz (1999). However, several other processes, such as insertions, unequal crossing-over, ectopic recombination, and abortive gap repair may be responsible for the observed structural mutations (Brunet et al. 2002). It is rational to think that structurally variant elements are functionally different, and this must be taken into account in models to explain their population dynamics. Kaplan, Darden, and Langley (1983) proposed a model that allows mutation to functionally distinct mutant elements to describe the evolution of transposable elements. The model suggests that when an element enters a population, it can become extinct within the first few generations, and if not, then the average copy number per host genome of the wild type increases quickly in the early stages of the element’s evolution to then decrease slowly as the average copy number of the mutant starts to grow. The average copy number in the population of the wild type and mutant then appears to stabilize, but the wild type ultimately disappears from the population, leaving only the mutant. At this point, transposition stops and the mutant eventually becomes extinct due to the forces of deletion and drift. Following this model, the composition of the 1731 family is that expected for an ancient element that has become inactive. The abundance of noncanonical elements, in addition to the wild type that was observed for the 297 and mdg 1 families, will be associated with a reduction in transpositional activity. Finally, the composition of the copia and 412 families is that expected for young, actively transposing elements. The phylogenetic distribution of the 1731 family, which is widespread throughout the Drosophila genus as well as being present in the Scaptomyza and Zaprionus genera (MontchampMoreau et al. 1993), is consistent with this element being an old component of the Drosophila genome, leaving the defective elements that were inserted in the pericentromeric regions only as vestiges. In addition, 1731 was inactive in the three experiments on transposition rate that included this element (reviewed in Junakovic, Di Franco, and Terrinoni 1997). It has been proposed that during genome evolution, transposable elements that lose their transposition activity may conversely acquire new func-

tions (von Sternberg et al. 1992; McDonald 1993). In relation to this topic, Kalmykova, Maisonhaute, and Gvozdev (1999) proposed that the fused gag-pol polypeptide encoded by a fraction of 1731 element might serve for the normal development of host testes. In that case, the 1731 element would be subject to positive selection. This is congruent with the conservation observed within and between stocks of 1731 noncanonical elements and with the reported conservation between stocks and species of the Bam HI Sal I fragment (Vaury, Bucheton, and Pelisson 1989; Montchamp-Moreau et al. 1993) that includes the gag gene and most of the pol ORF (Champion et al. 1992). The age of the other four elements estimated on the basis of their phylogenetic distribution does not conform to the predictions of the model depicted above. The elements copia and 412 are ancient components of the Drosophila genome, as they have been found in representatives of all the major Drosophila radiations (Stacey et al. 1983; Cizeron et al. 1998; Bie´mont and Cizeron 1999). Data on mdg 1 is scarce. This family must have been acquired before the Sophophoran radiation because it is present in the Obscura group (de Frutos, Peterson, and Kidwell 1992). Element 297 is restricted to D. melanogaster, its sibling species D. simulans and D. mauritana, and the closely related D. yakuba; thus this family is probably of recent origin (Martin, Wiernasz, and Schedl 1983). The four elements have shown reduced transpositional activity in one or another stock (data reviewed in Junakovic, Di Franco, and Terrinoni 1997). The element copia has been shown to transpose actively, at a rate of 10–3 to 10–2, in some lines with permissive alleles (Pasyukova, Nuzhdin, and Filatov 1998), which is congruent with its structural conservation. It has been shown that the full-length elements from the D. melanogaster genome are very young (Bowen and McDonald 2001). These authors posed the question of the immediate source of the full-length LTR-retrotransposons and pointed to three possibilities. The full-length elements are descendants from older elements that have been actively eliminated from the D. melanogaster genome or from older elements sequestered within the heterochromatin. A third possibility is that the LTR-retrotransposons currently present in the D. melanogaster genome have been subject to vertical and horizontal transmission during their evolution, as has been suggested for copia (Jordan and McDonald 1998). This possibility could explain the lack of correspondence between the age of the elements as inferred on a phylogenetic basis and from the proportion of noncanonical elements in the genome. A final consideration concerns the close coincidence of the proportion and number of noncanonical elements in unrelated stocks within each of the different families studied. This observation suggests the possibility that noncanonical elements could play some role in the regulation of the transpositional activity of wild-type elements. Posttranscriptional inhibition at greater copy numbers has been demonstrated for I elements and is attributed to cosuppression (Jensen, Gassama, and Heidmann 1999). Cosuppression does not require any translatable sequence, and the severity of repression correlates with copy number. There is considerable evidence from a number of trans-


posable elements and hosts that this kind of interaction can be important to activity (Birchler, Pal-Bhadra, and Bhadra 1999). Acknowledgments We are grateful to D. J. Finnegan for providing clones pOR 708 and 5B257, to R. Pin˜eiro for providing samples of the Llanera population, to Hero S.A. for providing culture vials, to S. Pimpinelli for training L.A-G. in FISH and to an anonymous referee for their helpful comments. L.A-G. was supported by an FPI fellowship from the Ministerio de Ciencia y Tecnologıá. This work was supported by grant PB-97–1268 (to J.A.) from the Ministerio de Educacioń y Cultura. Literature Cited Adams, M. D., S. E. Celniker, R. A. Holt et al. (190 co-authors). 2000. The genome sequence of Drosophila melanogaster. Science 287:2185–2195. Albornoz, J., and A. Domıńguez. 1999. Spontaneous changes in Drosophila melanogaster transposable elements and their effects on fitness. Heredity 83:663–670. Bie´mont, C., and G. Cizeron. 1999. Distribution of transposable elements in Drosophila species. Genetica 105:43–62. Birchler, J. A., M. Pal-Bhadra, and U. Bhadra. 1999. Less from more: cosuppression of transposable elements. Nat. Genet. 21:148–149. Bowen, N. J., and J. F. McDonald. 2001. Drosophila euchromatic LTR retrotransposons are much younger than the host species in which they reside. Genome Res. 11:1527–1540. Brunet, F., T. Giraud, F. Godin, and P. Capy. 2002. Do deletions of Mos I-like elements occur randomly in the Drosophilidae family? J. Mol. Evol. 54:227–234. Bucheton, A., C. Vaury, M. C. Chaboissier, P. Abad, A. Pe´lisson, and M. Simonelig. 1992. I elements and the Drosophila genome. Genetica 86:175–190. Capy, P. ed. 1997. Evolution and impact of transposable elements. Kluwer, Dordretch, The Netherlands. Capy, P., J. R. David, and D. L. Hartl. 1992. Evolution of the transposable element mariner in the Drosophila melanogaster species group. Genetica 86:37–46. Carmena, M., and C. Gonza´lez. 1995. Transposable elements map in a conserved pattern of distribution extending from bheterochromatin to centromeres in Drosophila melanogaster. Chromosoma 103:676–684. Chalvet, F., C. Di Franco, A. Terrinoni, A. Pelisson, N. Junakovic, and A. Bucheton. 1998. Potentially active copies of the gypsy retroelement are confined to the Y chromosome of some strains of Drosophila melanogaster possibly as the result of the female-specific effect of the flamenco gene. J. Mol. Evol. 46:437–441. Champion, S., C. Maisonhaute, M. H. Kim, and M. BestBelpomme. 1992. Characterization of the reverse transcriptase of 1731, a Drosophila melanogaster retrotransposon. Eur. J. Biochem. 209:523–531. Charlesworth, B., P. D. Sniegowski, and W. Stephan. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215–220. Cizeron, G., and C. Bie´mont. 1999. Polymorphism in structure of the retrotransposable element 412 in Drosopila simulans and D. melanogaster populations. Gene 232:183–190. Cizeron, G., F. Lemeunier, C. Lœvenbruck, A. Brehm, and C. Bie´ mont. 1998. Distribution of the retrotransposable

element 412 in Drosophila species. Mol. Biol. Evol. 15:1589–1599. de Frutos, R., K. R. Peterson, and M. G. Kidwell. 1992. Distribution of Drosophila melanogaster transposable element sequences in species of the obscura group. Chromosoma 101:293–300. Di Franco C., D. Galuppi, and N. Junakovic. 1992. Genomic distribution of transposable elements among individuals of an inbred Drosophila line. Genetica 86:1–11. Di Franco, C., C. Pisano, P. Dimitri, S. Gigliotti, and N. Junakovic. 1989. Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster. Chromosoma 98:402–410. Dimitri, P. 1997. Constitutive hetrochromatin and transposable elements in Drosophila melanogaster. Genetica 100: 85–93. Dimitri, P., and N. Junakovic. 1999. Revising the selfish DNA hypothesis. New evidence on accumulation of transposable elements in heterochromatin. Trends Genet. 15:123–124. Domıńguez, A., and J. Albornoz. 1996. Rates of movement of transposable elements in Drosophila melanogaster. Mol. Gen. Genet. 251:130–138. ———. 1999. Structural instability of 297 element in Drosophila melanogaster. Genetica 105:239–248. Finnegan, D. J. 1985. Transposable elements in eukaryotes. Int. Rev. Cytol. 93:281–326. Frame, I. G., J. F. Cutfield, and R. T. M. Poulter. 2001. New BEL-like LTR-retrotransposons in Fugu rubipes, Caenorhabditis elegans, and Drosophila melanogaster. Gene 263:219– 230. Ganko, E. W., K. T. Fielman, and J. F. McDonald. 2001. Evolutionary history of Cer elements and their impact on the C. elegans genome. Genome Res. 11:2066–2074. Gatti, M., S. Bonaccorsi, and S. Pimpinelli. 1994. Looking at Drosophila mitotic chromosomes. Methods Cell. Biol. 44: 371–391. Hochstenbach, R., H. Harhangi, K. Schouren, P. Bindels, R. Suijkerbuijk, and W. Hennig. 1996. Transcription of gypsy elements in a Y-chromosome male fertility gene of Drosophila hydei. Genetics 142:437–466. Ilyin, Y. V., V. G. Chmeliauskaite, E. V. Ananiev, N. V. Lyubormirskaya, V. V. Kulguskin, A. A. Bayev, and G. P. Georgiev. 1980. Mobile dispersed genetic element mdg1 of Drosophila melanogaster: structural organization. Nucleic Acids Res. 22:5333–5346. Jensen, S., M. P. Gassama, and T. Heidmann. 1999. Taming of transposable elements by homology-dependent gene silencing. Nat. Genet. 21:209–212. John, B. 1988. The biology of heterochromatin. Pp. 1–128 in R.S. Verma, ed. Heterochromatin: Molecular and structural aspects. Cambridge University Press, Cambridge. Jordan, I. K., and J. F. McDonald. 1998. Evolution of the copia retrotransposon in the Drosophila melanogaster species subgroup. Mol. Biol. Evol. 15:1160–1171. Junakovic, N., C. Di Franco, and A. Terrinoni. 1997. Evidence for a host role in regulating the activity of transposable elements in Drosophila melanogaster: the case of the persistent instability of Bari 1 elements in Charolles stock. Genetica 100:149–154. Kalmykova, A., C. Maisonhaute, and V. Gvozdev. 1999. Retrotransposon 1731 in Drosophila melanogaster changes retrovirus-like expression strategy in host genome. Genetica 107:73–77. Kaplan, N., T. Darden, and C. H. Langley. 1985. Evolution and extinction of transposable elements in mendelian populations. Genetics 109:459–480. Langley, C. H., J. F. Y. Brookfield, and N. L. Kaplan. 1983.


Transposable elements in mendelian populations. I. A theory. Genetics 104:457–471. Marillonnet, S., and S. R. Wessler. 1998. Extreme structural heterogeneity among the members of a maize retrotransposon family. Genetics 150:1245–1256. Martin, G., D. Wiernasz, and P. Schedl. 1983. Evolution of repetitive-disersed DNA. J. Mol. Evol. 19:203–213. Maside, X., C. Bartolome´, S. Assimacopoulos, and B. Charlesworth. 2001. Rates of movement and distribution of transposable elements in Drosophila melanogaster: in situ hybridization vs. Southern blotting data. Genet. Res. 78: 121–136. McDonald, J. F. 1993. Evolution and consequences of transposable elements. Curr. Opin. Genet. Develop. 3:855–864. Montchamp-Moreau, C., S. Ronsseray, M. Jacques, M. Lehmann, and D. Anxolabe´here. 1993. Distribution and conservation of sequences homologous to the 1731 retrotransposon in Drosophila. Mol. Biol. Evol. 10:791–803. National Center for Supercomputing Applications, 1991. NCSA GelReader for the Macintosh. Version 2.0.5. University of Illinois at Urbana-Champaign, Urbana-Champaign. Nuzhdin, S. V. 1999. Sure facts, speculations, and open questions about the evolution of transposable element copy number. Genetica 107:129–137. O’Hare, K., and G. M. Rubin. 1983. Structures of P transposable elements and their sites of insertion and excision in the Drosophila melanogaster genome. Cell 34:25–35. Pasyukova, E. G., S. V. Nuzhdin, and D. A. Filatov. 1998. The relationship between rate of transposition and transposable element copy number for copia and Doc transposable elements of Drosophila melanogaster. Genet. Res. 72:1–11. Petrov, D. A., E. R. Lozovskaya, and D. L. Hartl. 1996. High intrinsic rate of DNA loss in Drosophila. Nature 384:346– 349. Pimpinelli, S., M. Berlocco, L. Fanti, P. Dimitri, S. Bonaccorsi, E. Marchetti, R. Caizzi, C. Caggese, and M. Gatti. 1995. Transposable elements are stable structural components of Drosophila melanogaster heterochromatin. Proc. Natl. Acad. Sci. USA 92:3804–3808. Potter, S., M. Truett, N. Phillips, and A. Maher. 1980. Eukaryotic

transposable genetic elements with inverted terminal repeats. Cell 20:639–647. Shepherd, B. M., and D. J. Finnegan. 1984. Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells and isolation of a free 412 long terminal repeat. J. Mol. Biol. 180:21–40. Shevelyov, Y. Y., M. D. Balakireva, and V. A. Gvozdev. 1989. Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1). Chromosoma 98:117–122. Spradling, A. C., and G. M. Rubin. 1981. Drosophila genome organization: conserved and dynamic aspects. Annu. Rev. Genet. 15:219–264. Stacey, S. N., R. A. Lansman, H. W. Brock, and T. A. Grigliatti. 1986. Distribution and conservation of mobile elements in the genus Drosophila. Mol. Biol. Evol. 3:522–534. Terrinoni, A., C. Di Franco, P. Dimitri, and N. Junakovic. 1997. Intragenomic distribution and stability of transposable elements in euchromatin and heterochromatin of Drosophila melanogaster: non-LTR retrotransposon. J. Mol. Evol. 45: 145–153. Vaury, C., A. Bucheton, and A. Pellison. 1989. The bheterochromatin sequences flanking the I elements are themselves defective transposable elements. Chromosoma 98:215–224. Vaury, C., M. C. Chaboissier, M. E. Drake, O. Lajoinie, B. Dastugue, and A. Pe´lisson. 1994. The doc transposable element in D. melanogaster and D. simulans: genomic distribution and transcription. Genetica 93:117–124. von Sternberg, R. M., G. E. Novick, G. P. Gao, and R. J. Herrera. 1992. Genome canalization: the coevolution of transposable and interspersed repetitive elements with single copy DNA. Genetica 86:215–246.

Diethard Tautz, Associate Editor Accepted November 6, 2002