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. 2001 Sep;11(9):1527–1540. doi: 10.1101/gr.164201

Drosophila Euchromatic LTR Retrotransposons are Much Younger Than the Host Species in Which They Reside

Nathan J Bowen 1,1, John F McDonald 1,2
PMCID: PMC311128  PMID: 11544196

Abstract

The recent release of the complete euchromatic genome sequence of Drosophila melanogaster offers a unique opportunity to explore the evolutionary history of transposable elements (TEs) within the genome of a higher eukaryote. In this report, we describe the annotation and phylogenetic comparison of 178 full-length long terminal repeat (LTR) retrotransposons from the sequenced component of the D. melanogaster genome. We report the characterization of 17 LTR retrotransposon families described previously and five newly discovered element families. Phylogenetically, these families can be divided into three distinct lineages that consist of members from the canonical Copia and Gypsy groups as well as a newly discovered third group containing BEL, mazi, and roo elements. Each family consists of members with average pairwise identities ≥99% at the nucleotide level, indicating they may be the products of recent transposition events. Consistent with the recent transposition hypothesis, we found that 70% (125/178) of the elements (across all families) have identical intra-element LTRs. Using the synonymous substitution rate that has been calculated previously for Drosophila (.016 substitutions per site per million years) and the intra-element LTR divergence calculated here, the average age of the remaining 30% (53/178) of the elements was found to be 137,000 ±89,000 yr. Collectively, these results indicate that many full-length LTR retrotransposons present in the D. melanogaster genome have transposed well after this species diverged from its closest relative Drosophila simulans, 2.3 ± .3 million years ago.

Retrotransposons are the most abundant and widespread class of eukaryotic transposable elements. For example, >50% of the maize genome (SanMiguel et al. 1996) and >40% of the human genome (Smit 1999) are comprised of retrotransposons. The biological importance of retrotransposons ranges from their contribution to mutation (Green 1988) and disease (Deininger and Batzer 1999) to their postulated role in evolution (McDonald 1990, 1993; Kidwell and Lisch 1997). The genome sequencing of humans and selected experimental and agriculturally important species is providing an unprecedented opportunity to view the patterns of variation existing among the entire complement of retrotransposons in complete genomes.

Retrotransposons are made up of short interspersed nuclear elements (SINES), long interspersed nuclear elements [LINES, also known as non-long terminal repeat (LTR) retrotransposons], LTR retrotransposons, and retroviruses. LTR retrotransposons are named for their long terminal repeats, which contain transcriptional regulatory sites and flank the internal coding regions of the elements (Boeke and Stoye 1997). LTR retrotransposons are classically divided into two groups, the Copia/Ty1 group and the Gypsy/Ty3 group. The distinguishing characteristic between these groups is the order of the three protein domains — protease (PR), reverse transcriptase (RT), and integrase (IN) — encoded within the polymerase (pol) gene of the elements. The pol region of Copia/Ty1 elements has the order (PR, IN, RT) whereas the Gypsy/Ty3 group has the more familiar arrangement (PR, RT, IN), which is also the order found in retroviruses. Recently, a third major group of LTR retrotransposons has been described containing the BEL element from Drosophila melanogaster as well as the Cer7-12 elements of Caenorhabditis elegans (Bowen and McDonald 1999; Malik et al. 2000). The IN domain is also found downstream of the RT domain in this third group of LTR retrotransposons.

LTR retrotransposons and retroviruses are nearly identical in structure and are clearly related phylogenetically (Xiong and Eickbush 1988). The main distinguishing characteristic is that some LTR retrotransposons, such as Ty1 in yeast, do not contain an envelope gene, which renders retroviruses infectious. Many LTR retrotransposons, such as gypsy from D. melanogaster, however, do encode Envelope proteins and are infectious (Song et al. 1994). Therefore, LTR retrotransposons also serve as excellent models for the study of the evolution of infectious retroviruses. Previous large-scale analyses of the LTR retrotransposons of Saccharomyces cerevisiae (Jordan and McDonald 1998, 1999a,b; Kim et al. 1998), C. elegans (Bowen and McDonald 1999), Zea mays, and Hordeum vulgare (Shirasu et al. 2000) have provided novel insights into the molecular evolution and phylogenetic distribution of these retrotransposons.

Because the long terminal repeats of LTR retrotransposons are synthesized from a single template during reverse transcription, they are identical at the DNA sequence level on integration. Therefore, if the nucleotide substitution rate for the host DNA polymerase is known, the relative integration time or age of the element can be estimated from the level of sequence divergence existing between an element's LTRs. Previously, LTR nucleotide identity has been used to estimate the time of insertion of LTR retrotransposons from S. cerevisiae, Zea mays, and humans. For example, the age of the Ty1 and Ty2 elements from S. cerevisiae has been estimated to be <100,000 years old (Jordan and McDonald 1999b; Promislow et al. 1999). In contrast, it has been reported that the LTR retrotransposons within the ADH-region of the maize genome are much older, having transposed in the past 2 to 6 million years (SanMiguel et al. 1998). In a similar study, it has been reported that most human endogenous retroviruses (HERVs) inserted into the human genome long before humans diverged from the Old World monkeys, more than 25 million years ago (Tristem 2000).

In an initial effort to characterize all of the LTR retrotransposons within the genome of D. melanogaster, we report the annotation, phylogenetic analysis, and estimated ages of 178 full-length elements (i.e., those containing two intact LTRs and intervening coding regions) from the nonredundant sequence found in GenBank (Benson et al. 2000). Our results indicate that there are three major groups of LTR retrotransposons found within the D. melanogaster genome. We find that these three groups consist of over 20 individual families of elements and that each family of elements is composed of a group of highly homologous individual elements (∼99% identity at the nucleotide level). We conclude that many LTR retrotransposons from each family have resulted from evolutionarily recent episodes of transpositional activity.

RESULTS

Isolation and Characterization of D. melanogaster LTR Retrotransposon Families from the Genome Sequence

The majority of the D. melanogaster genome sequence now available in GenBank is from the euchromatic regions of the genome (Adams et al. 2000). In contrast, only 2.5% of the genome sequence is derived from heterochromatic clones (Myers et al. 2000). Constitutive heterochromatin, which comprises roughly one-third of the D. melanogaster genome, is poorly represented in the genome sequence because these regions are not easily cloned into large inserts (Myers et al. 2000). Likewise, the assembly of DNA sequence from genomic regions that contain many tandemly arranged repetitive elements can result in the omission of internal sequences (E. Myers, pers. comm.). These issues are important to our study because D. melanogaster heterochromatin is thought to contain a substantial number of transposable elements (TEs) (Pimpinelli et al. 1995). Also LTR retrotransposons have been shown to exist in nested arrays in other species (SanMiguel et al. 1996a). Consequently, any LTR retrotransposons located in these regions of the genome are precluded from our analysis. Further sequencing and gap-filling efforts being conducted by Celera and the Berkeley Drosophila Genome Project (Myers et al. 2000) will likely identify additional elements within both the euchromatic and heterochromatic portions of the genome. Therefore, our results represent a large sampling of LTR retrotransposons from the euchromatin of D. melanogaster.

Following the initial characterization of each LTR retrotransposon (see Methods section), ClustalW (Thompson et al. 1997) was used to align the nucleotides of each element to known full-length LTR retrotransposons of D. melanogaster and other related organisms listed in Table 1. Information concerning all elements identified previously can be obtained through Flybase (http://flybase.harvard.edu). This initial alignment was done to group elements into known and unknown families. The phylogram generated from this preliminary alignment is shown in Figure 1. For clarity, each family is labeled once followed by the number of elements in each family. Because of the low level of interfamily nucleotide sequence identity, this initial phylogram may not accurately represent all interfamily relationships, but it does allow us to classify elements into distinguishable groups. The long interfamily branches and the large cluster of nearly identical elements at the termini of the family lineages apparent in this initial phylogram indicate that most families of D. melanogaster LTR retrotransposons consist of a group of highly homologous elements. Subsequently, computed pairwise nucleotide identities confirmed this finding in that each family was found to consist of elements with average pairwise nucleotide identities of ≥99% (Table 2).

Table 1.

LTR Retrotransposons Used to Categorize Elements from the Drosophila melanogaster Genome

Element name Accession no. Inserted element length (bp)
From D. melanogaster
17.6 X01472 7439
1731 X07656 4648
297 X03431 6995
412 X04132 6897
BEL U23420 6126
blastopia Z27119 5416
burdock U89994 6411
copia X04456 5146
gypsy AF033821 7469
HMS Beagle AH001020 ∼7300
Idefix AJ009736 7411
mdg1 X59545 7480
mdg3 X95908 5519
micropia X14037 5465
nomad AF039416 7592
tirant X93507 5184
transpac AF222049 5249
From other organisms
Anopheles gambiae Moose AF060859
Tribolium castaneum woot U09586
Drosophila buzzatii osvaldo AJ133521
Drosophila virilis Ulysses X56645
Ceratitis capitata yoyo U60529
Drosophila virilis gypsy M38438
Drosphila subobscura gypsy X72390
Trichoplusia ni TED M32662
Drosophila ananassae Tom Z24451
Drosophila virilis Tv1 AF056940
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Figure 1.

Figure 1

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Neighbor-joining phylogenetic tree of the ClustalW alignment of LTR retrotransposon nucleotide sequences. This tree indicates the high level of sequence homology within a family of elements. For clarity, the element families are listed once with the number of elements in each family in parentheses. Unclassified elements are identified by the accession number in which they are located.

Table 2.

Element Family Average Pairwise Nucleotide Identities

Element (sample size) Average pairwise identities Calculated divergence time (millions of years)
17.6 (6) 99.676525 ± 0.000862996 0.1078252 ± 0.028766517
297 (8) 99.846149 ± 0.000986654 0.051283679 ± 0.032888452
412 (19) 99.6790517 ± 0.001212138 0.106982767 ± 0.040404609
antonia (4) 98.8624153 ± 0.00499742 0.379194889 ± 0.166580662
blastopia (6) 99.7571929 ± 0.000753808 0.080935711 ± 0.025126928
blood (21) 99.8221958 ± 0.001247047 0.059268056 ± 0.041568245
copia (23) 99.7509078 ± 0.001175924 0.083030736 ± 0.039197471
wolfman (2) 99.889276 0.036908
hamilton (2) 99.973008 0.008997333
HMS Beagle (3)  #'s 4, 5 and 6 99.448482 ± 0.00441751 0.172349167 ± 0.138047175
HMS Beagle (4)  #'s 1, 2, 3, 7 and 8 99.852204 ± 0.0008726 0.04618625 ± 0.02726868
mazi (4) 99.8201147 ± 0.000824226 0.059961778 ± 0.027474189
mdg1 (5) 99.6931778 ± 0.000748225 0.102274067 ± 0.024940837
mdg3 (8) 99.6225424 ± 0.001529709 0.12581919 ± 0.050990305
nomad (4) 99.9075055 ± 0.000276762 0.0308315 ± 0.009225413
roo (40) 99.6887925 ± 0.001617852 0.103735823 ± 0.053928391
tirant (6) 99.9374788 ± 0.000253444 0.0208404 ± 0.008448124
transpac (4) 99.882341 ± 0.000408498 0.039219778 ± 0.013616585
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The nucleotide sequences of the LTR retrotransposons that did not group with known elements were translated and their RT motif was aligned to the RT of the known elements (Fig. 2A). Only those RT motifs that were uninterrupted by frame shifts or stop codons were used in the characterization of novel families. This alignment was then used to generate pairwise amino acid identities. Consistent with criteria established previously (Bowen and McDonald 1999), if an element had a pairwise identity of <90% to a known RT, it was classified as a new family. These novel elements are shown in boldface type in the first column of Table 3. Additional RT sequences from other Drosophila and invertebrate species were included in this analysis to ensure that novel elements from D. melanogaster did not represent previously characterized elements from other related species. Elements with RT pairwise identities >90% to a previously characterized element were given the name of that element followed by a number.

Figure 2.

Figure 2

Figure 2

Figure 2

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(A) Amino-acid alignment of the RT motif of Drosophila melanogaster LTR retrotransposons found in this study. The sequences were aligned using ClustalX as described in Methods. The seven conserved domains of RT (Xiong and Eickbush 1988) are indicated above the alignment. Residue coloring was performed by MacBoxshade v2.01 and is based on the similarity scheme (F, W, Y), (I, L, M, V), (P), (D, E), (G, A), (S, T, C), (N, H), (R, K), (Q). Members of a similar residue group are shaded identically. (B) Unrooted neighbor joining tree of sequences shown in A. Numbers found adjacent to branches indicate bootstrap support from 100 replicates. (C) Neighbor joining phylogenetic tree of the alignment shown in Arooted with the branch leading to the 1731 and copia lineages. Branches with bootstrap values <50 were collapsed to indicate only well-supported groups. Novel element families first characterized in this study are in boldface type and indicated by asterisks.

Table 3.

LTR Retrotransposons Characterized in This Study

Element Accession # Genomic location ITR DTR LTR length (bp) Inserted element length LTR % identity Element age in millions of years
17.6-1 AC005860 2R/47D1 AG/TT ATAT/ATAT 512 7202 100 0
17.6-2 AC005894 2R/46A1-46B2 AG/TT ATAT/ATAT 512 7200 100 0
17.6-3 AC004333 3L/62A1-62A4 AG/TT ATAT/ATAT 512 7245 100 0
17.6-4 AE003471 3L AG/TT ATAT/ATAT 514 6505 100 0
17.6-5 AE003082 2R AG/TT ATAT/ATAT 512 6329 99.419 0.181563
17.6-6 AE003545 3L AG/TT ATAT/ATAT 517 6509 99.189 0.253438
297-1 AC006303 2L/35E-36A AG/CT ATAT/ATAT 414 6808 100 0
297-2 AC007176 2L-2R/37C1-46B2 AG/CT ATAT/ATAT 414 6791 100 0
297-3 AC005558 2L/23A1-23B2 AG/CT ATAT/ATAT 414 6336 100 0
297-4 AC005720 3L/79F1-80A2 AG/CT ATAT/ATAT 414 6730 100 0
297-5 AC004306 2R/51E1-51E2 AG/CT ATAT/ATAT 414 6805 100 0
297-6 AC002441 2L/35F3-35F6 AG/CT ATAT/ 414/3′ LTR  DELETION 6809 100
297-7 AE003413 2L/34C4-36A7 AG/CT ATAT/ATAT 414 6808 100 0
297-8 AE003574 X AG/CT ATAT/ATAT 414 5708 100
297-9 AE003415 SL/34C4-36A7 AG/CT ATAT/ATAT 415 6809 100 0
297-10 AE003599 3L AG/CT ATAT/ATAT 414 5549 99.193 0.252188
412-1 AC005847 3L/61F3-62A2 TG/CT ATAT/ACAG 571 7592 99.789 0.065938
412-2 AC005639 2R/59E3-59F4 TG/CA AGGG/AGGG 481 7519 100 0
412-3 AC005267 3L/60A2-60B2 TG/CA ATAT/ATAT 514 7566 100 0
412-4 AL050231 X/1A-1B TG/TA CAAT/TATC 512 7572 100 0
412-6 AE003417 X TG/TA CCAAT/TCGAC 514 7578 99.415 0.182813
412-7 AE003429 X TG/CA TGAA/TGAA 515 99.610 0.121875
412-8 AE003553 3L TG/CA GGGAGA/TGGCGA 514 7582 99.805 0.060938
412-9 AE003709 3R TG/CA CTAC/CTAC 517 7558 99.368 0.197500
412-10 AE003461 2R TG/CA AGGG/AGGG 481 7452 100 0
412-11 AE003462 2R TG/CA ATAT/ATAT 518 7492 99.364 0.198750
412-12 AE003463 2R TG/CA ATAG/ATAG 482 7454 100 0
412-13 AE003463 2R TG/CA CGAT/CGAT 484 7461 99.583 0.130313
412-14 AE003464 2R TG/CA AGTATG/CTAGAG 472 7446 100 0
412-15 AE003470 3L TG/CA GTCCTT/CAAGTC 518 7529 100 0
412-16 AE003470 3L TG/CA GATATA/ACAGCA 572 7576 99.786 0.066875
412-17 AE003511 X TG/CA AAAC/AAAC 514 7520 100 0
412-18 AE003523 3L TG/CA GTGG/GTGG 514 7520 99.790 0.065625
412-19 AE003718 3R TG/CA ACAG/ACAG 483 7550 100 0
antonia-1 AC005891 2L/33F3-34A2 AG/TT ATAT/ATAT 659 6953 100 0
antonia-2 AC004362 2L/35E3-35E6 AG/TT ATAT/ATAT 659 6943 100 0
antonia-3 AE003640 2L AG/TT ATAT/ATAT 661 6170 99.697 0.094688
antonia-4 AE003786 2R AG/TT ATAT/ATAT 661 6116 98.573 0.445938
blastopia-1 AC007177 2R/59C1-59C5 TG/CA TATA/TATA 276 5029 100 0
blastopia-2 AC006402 2L/38C-38D TG/CA TATA/TATA 276 5029 100 0
blastopia-3 AC005638 2R/47E1-47E3 TG/CA TTTA/TTTA 276 4264 100 0
blastopia-4 AL033125 DISTAL X TG/CA TATA/TATA 276 4645 100 0
blastopia-5 AE003417 X TG/CA TACA/TACA 276 4272 100 0
blastopia-6 AE003489 X TG/CA TGTA/TGTA 275 3851 100 0
blood-1 AC002474 2L/38D1-38D2 TG/CA GTAC/GTAC 399 7411 100 0
blood-2 AC005734 2L/31A1-31B1 TG/CA AAGG/AAGG 401 7413 100 0
blood-3 AC004442 2L/22A3-22A7 TG/CA GTGG/GTGG 399 7408 100 0
blood-4 AC005130 2L/39C1-39D1 TG/CA AGCA/AGCA 399 7415 100 0
blood-5 AC007082 2L/37D TG/CA CCTG/CCTG 401 7417 100 0
blood-6 L49394 2L/35E1-35E2 TG/CA AGGG/AGGG 399 7411 100 0
blood-7 AC004115 2L/27B7-21C2 TG/CA GAAT/GAAT 415 7443 99.758 0.07563
blood-8 AC003727 3R TG/CA ATAT/AGTC 395 7425 99.500 0.15625
blood-9 AE003413 2L/34C4-36A7 TG/CA AGGG/AGGG 399 7411 100 0
blood-10 AE003647 2L TG/CA AGGG/AGGG 399 7421 100 0
blood-11 AE003718 3R TG/CA ATAC/ATAC 399 7421 100 0
blood-12 AE003775 3R TG/CA GTAC/GTAC 399 7421 100 0
blood-13 AE003555 3L TG/CA ACAT/ACAT 400 7422 99.750 0.078125
blood-14 AE003667 2L TG/CA GTAC/GTAC 400 7420 99.748 0.078750
blood-15 AE003670 2L TG/CA AGCA/AGCA 400 7420 99.751 0.077813
blood-16 AE003633 2L TG/CA GCAG/GCAG 400 7422 100 0
blood-17 AE003709 3R TG/CA ACAG/ACAG 401 7425 100 0
blood-18 AE003598 3L TG/CA CTGC/CTGC 401 7423 100 0
blood-19 AE003780 3R TG/CA CTAC/CTAC 402 7427 100 0
blood-20 AE003589 2L TG/CA GAAT/GAAT 415 7452 99.758 0.075625
blood-21 AE003638 2L TG/CA GTAC/GTAC 377 7403 99.24 0.237500
burdock-1 AE002976 2R/41C-41D AG/TT TATA/TATA 276 6425 100 0
copia-1 AC007146 2L/33A TG/CA GATCC/GATCC 276 5146 100 0
copia-2 AC001657 2L/35B7-35C1 TG/CA GTTTC/GTTTC 276 5145 100 0
copia-3 AC004445 2L/22A3-22A5 TG/CA GTTGG/GTTGG 276 5145 100 0
copia-4 AC005554 2L/23C4-23D4 TG/CA TTATC/TTATC 276 5145 100 0
copia-5 AC004276 2L/22E1-22E2 TG/CA GTATC/GTATC 276 5147 100 0
copia-6 AC005647 2R/53C7-53C14 TG/CA TATGT/TATGT 276 5151 100 0
copia-7 AC002501 2L/35F1-35F2 TG/CA AAAAT/AAAAT 276 5149 100 0
copia-8 AC004735 2L/38A7-39C9 TG/CA GAGCA/GAGCA 276 5144 100 0
copia-9 AC003053 2L/24A3-24C2 TG/CA ATCAC/ATCAC 276 5145 100 0
copia-10 AC011662 2L/36D TG/CA ATAAC/ATAAC 275 5145 100 0
copia-11 AC006215 2L/38C1-38C2 TG/CA ACTGC/ACTGC 275 5252 100 0
copia-12 AE003494 X TG/CA TTGAC/TTGAC 276 4835 99.638 0.113125
copia-14 AE003794 2R TG/CA GGTTC/GGTTC 276 4837 100 0
copia-15 AE003804 2R TG/CA ACGCC/ACGCC 276 4742 100 0
copia-16 AE003777 3R TG/CA GATGG/GATGG 277 4836 100 0
copia-17 AE003806 2R TG/CA TATGT/TATGT 277 4511 99.628 0.116250
copia-18 AE003579 2L TG/CA ATCAC/ATCAC 277 4747 100 0
copia-19 AE003729 3R TG/CA AAAAC/AAAAC 277 4506 100 0
copia-21 AE003466 2R TG/CA CTTTG/CTCAG 278 5176 100 0
copia-22 AE003783 2L TG/CA AACAC/AACAC 278 4509 100 0
copia-23 AE003526 3L TG/CA AATAT/AATAT 288 4564 100 0
copia-24 AE003778 3R TG/CA ATTAT/ATTAT 307 4741 98.536 0.457500
copia-25 AE003473 3L TG/CA AGAAC/AGAAC 276 4120 100 0
hamilton-1 AC006215 2L/38C1-38C2 AG/TT TGTA/TGTA 495 6973 100 0
hamilton-2 AC006215 2L/38C1-38C2 495 6973 100 0
HMS Beagle-1 AC004563 2R/56D11-56E6 AG/CT TATA/TATA 266 6883 100 0
HMS Beagle-2 AC007977 2L/27C AG/CT TATA/TATA 266 6885 100 0
HMS Beagle-3 AL121804 DISTAL X AG/CT TATA/TATT 266 6887 100 0
HMS Beagle-4 AE003116 3L AG/CT TATA/TATA 332 7180 99.034 0.301875
HMS Beagle-5 AE003574 X AG/CT TGTA/TGTA 332 7008 99.698 0.094375
HMS Beagle-6 AE003600 3R AG/CT TATA/TATA 332 7217 100 0
HMS Beagle-7 AE003679 AG/CT TGCA/TGCA 266 4441 100 0
HMS Beagle-8 AE003651 2L AG/CT TGTA/TGTA 266 6906 100 0
mazi-1 AC004377 2R/58E8-58F4 TG/CA ATGCTT/ATGGAA 224 6112 100 0
mazi-2 AE003482 3L TG/CA AAGGG/AAGGG 245 6150 100 0
mazi-3 AE003615 2L TG/CA GCTGG/GCTGG 245 6148 100 0
mazi-4 AE003777 3R TG/CA AGTGT/AGTGT 245 6149 100 0
mdg1-1 AL138971 DISTAL X TG/CA CTAG/CTAG 442 7355 100 0
mdg1-2 AE003736 3R TG/CA CTCC/CTCC 442 8170 99.582 0.130625
mdg1-3 AE003828 2R TG/CA TCAC/TCAC 444 7420 99.320 0.212500
mdg1-4 AE002943 TG/CA CTAT/CTAT 425 8213 100 0
mdg3-1 AC004574 2L/22B1-22B2 TG/AA GTAG/GTAG 267 5519 100 0
mdg3-2 AC004735 2L/38A7-38C9 TG/AA GCAC/GCAC 267 5504 100 0
mdg3-3 AE003607 3R TG/AA ATTG/ATTG 265 5526 100 0
mdg3-4 AE003492 X TG/AA ATGT/ATGT 267 5399 100 0
mdg3-5 AE003514 3L TG/AA GTAT/GTAT 267 5331 100 0
mdg3-6 AE003604 3R TG/AA CCAT/CCAT 267 5330 100 0
mdg3-7 AE003686 3R TG/AA GTTG/GTTG 267 5526 100 0
mdg3-8 AE003502 X TG/AA TACT/TACT 268 5529 99.247 0.235313
micropia-1 AE003672 3R TG/CA TATA/TATA 514 5124 99.405 0.185938
nik AE003485 X AG/TT CCAG/CCAG 430 6938 100 0
nomad-1 AE003411 2L/35B4-35C1 AG/CT TTATA/TATA 518 7389 100 0
nomad-2 AC004716 2L/22C1-22C2 AG/CT TTTA/TTTA 518 7473 100 0
nomad-3 AC005111 2L/31F5-32A1 AG/CT TTCA/TTCA 518 7473 100 0
nomad-4 AL121805 DISTAL X AG/CT TATA/TATA 517 7475 100 0
roo-1 AC005333 2L/26A1-26A3 TG/CA GGTAC/GGTAC 428 8067 100 0
roo-2 AC004728 2L/29D1 TG/CA AGCGC/AGCGC 429 8218 100 0
roo-3 AC001647 2R/35A1 TG/CA GTTAT/GTTAT 428 8211 100 0
roo-4 AC001659 2L/34D1-34D4 TG/CA CTAAC/CTAAC 429 8209 99.528 0.147500
roo-5 AC007453 2R/49D1-49D3 TG/CA TGGCC/TGGCC 428 7411 100 0
roo-6 AC004438 3L/68C2-68C3 TG/CA CATTT/CATTT 428 8251 99.766 0.073125
roo-7 AC004767 3L/65A6-65A10 TG/CA AACGG/AACGG 428 8211 99.766 0.073125
roo-8 AC004245 2L/21D1-21D2 TG/CA GCCCC/GCCCC 428 8087 100 0
roo-9 AC005974 2R/46E1-46F6 TG/CA TGTAT/TGTAT 428 8215 99.766 0.073125
roo-10 AC011662 2L/36D TG/CA CCCCC/CCCCC 428 8209 100 0
roo-11 AC006415 2L/40B-40C TG/CA GCTTCC/GCTTCC 428 8212 100 0
roo-12 AL035311 DISTAL TIP OF X TG/CA GCATC/GCATC 427 7788 100 0
roo-13 AL121805 DISTAL TIP OF X TG/CA TTAAT/TTAAT 428 8197 100 0
roo-14 AC004349 3L/65B1-65B2 TG/CA TAATA/ 428/3′ END   DELETION 5895 100 0
roo-16 AL031366 DISTAL TIP OF X TG/CA ATTA/ATTA 428 7758 100 0
roo-17 AL138971 DISTAL X TG/CA GGGAC/GGGAC 428 7593 100 0
roo-19 AE003606 3R TG/CA ATAAG/ATAAG 429 8211 100 0
roo-21 AE003428 X TG/CA CATAC/CCAGA 429 8086 99.532 0.146250
roo-22 AE003703 3R TG/CA AGAC/AGAC 429 8317 99.766 0.073125
roo-23 AE003497 X TG/CA GTCAT/GTCAT 429 8087 100 0
roo-24 AE003552 3L TG/CA TGGAG/TGGAG 429 8086 100 0
roo-25 AE003421 X TG/CA CAGCT/CAGCT 430 8251 100 0
roo-26 AE003773 3R TG/CA CAAAT/CAAAT 430 7332 99.767 0.072813
roo-27 AE003753 3R TG/CA AGGGC/AGGGC 430 8229 99.765 0.073438
roo-28 AE003756 3R TG/CA ATAGT/ATAGT 430 8229 99.766 0.073125
roo-29 AE003455 2R TG/CA ACTAC/ACTAC 431 8089 99.523 0.149063
roo-30 AE003544 3L TG/CA CATTT/CATTT 431 8086 99.769 0.072188
roo-31 AE003679 3R TG/CA GCCGG/GCCGG 431 8240 99.768 0.072500
roo-32 AE003738 3R TG/CA AGACC/AGACC 431 8240 99.766 0.073125
roo-33 AE003529 3L TG/CA ATAGG/ATAGG 431 8087 100 0
roo-34 AE003470 3L TG/CA ATAA/ATAA 432 8144 99.295 0.220313
roo-35 AE003455 2R TG/CA GCCAG/GCCAG 432 8086 99.766 0.073125
roo-36 AE003519 2R TG/CA GAAGG/GAAGG 432 8090 99.768 0.072500
roo-37 AE003520 3L TG/CA AATAG/AATAG 432 8089 99.766 0.073125
roo-38 AE003467 3L TG/CA TAAAC/TAAAC 433 8241 99.769 0.072188
roo-39 AE003515 3L TG/CA CTTGC/CTTGC 433 8091 99.761 0.074688
roo-40 AE003419 X TG/CA TTAC/TTAC 434 8090 99.529 0.147188
roo-41 AE003781 2L TG/CA ATAAC/ATAAC 431 8251 100 0
roo-42 AE003463 2R TG/CA CTTTA/CTTTA 430 8088 100 0
roo-43 AE003410 2L, REGION 34C4-36A7 TG/CA ATAAC/ATAAC 428 8142 100 0
tirant-1 AC005444 2L/36D3 AG/CT CGCG/CGCG 417 8109 100 0
tirant-2 AC004759 2L/38E1-38E9 AG/CT CCCG/CCCG 417 7399 100 0
tirant-3 AE003829 2R AG/CT CGTG/CGTG 418 8016 100 0
tirant-4 AE003601 3R AG/CT CCCG/CCCG 418 8016 100 0
tirant-5 AE003593 3L AG/CT CGCG/CGCG 418 8016 100 0
tirant-6 AE003843 4 AG/CT CGCT/CGAT 419 8017 100 0
transpac-1 AC006302 2L/34C4-34D2 AG/CT ATAT/ATAT 330 5249 100 0
transpac-2 AE003426 X AG/CT ATAT/ATAT 330 5251 100 0
transpac-3 AE003762 3R AG/CT ATAT/ATAT 330 5247 100 0
transpac-4 AE003508 X AG/CT ATAT/ATAT 331 5253 100 0
wolfman-1 AC007146 2L/33A-33A TG/CA AAGC/AAGC 506 7345 100 0
wolfman-2 AE003439 X TG/CA CCTG/CCTG 506 7231 100 0
unclassified AE002665 3L TG/CA AATG/AATG 266 7436 100 0
unclassified AE002987 X AG/TT TATA/TATA 414 8437 99.510 0.153125
unclassified AE003268 AG/CT TATA/TATA 416 7185 99.760 0.075120
unclassified AE003450 X AG/TT TATA/TATA 405 6538 100 0
unclassified AL035631 DISTAL X AG/TT TCTA/TCTA 353 5023 99.433 0.177054
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All elements characterized in this study are listed individually in Table 3. Included in this table are other distinguishing characteristics of the LTR retrotransposons, including their accession numbers, chromosomal locations, inverted terminal repeats (ITRs), direct terminal repeats (DTRs), LTR length, complete element length, and estimated age of each element (see below). The DTRs result from a duplication of the unoccupied insertion site following proviral or element insertion (Coffin et al. 1997). In our study, the DTRs served as internal controls for the assembly process following the whole genome shotgun sequencing of D. melanogaster (Myers et al. 2000). If proviral elements located at different loci were incorrectly assembled, they would contain a mixed set of DTRs. For the elements that contained unique DTR sequences, 93% were identical. The other 7% are either incorrectly assembled or are possibly the result of ectopic recombination between proviral elements at different loci. This hypothesis is currently under further investigation. In summary we identified 23 copia, six 17.6, 10 297, 18 412, four antonia, six blastopia, 21 blood, one burdock, two hamilton, eight HMS Beagle, four mazi, five mdg1, eight mdg3, one micropia, one nik, four nomad, 40 roo, six tirant, four transpac, two wolfman, and five unclassified elements (Table 4). The elements' combined lengths totaled 1, 279, 046 nucleotides or nearly 1% of the sequenced component of the genome.

Table 4.

The Content of LTR-Retrotransposons Analyzed from the Drosophila melanogaster Genome in This Study

LTR retrotransposon family Total number of elements characterized in this study Elements estimated by in situ hybridization in 10 natural populationsa
copia 23 24 ± 4
17.6 6 3 ± 3
297 10 23 ± 6
412 18 28 ± 5
antonia 4 n.d.
blastopia 6 n.d.
blood 21 17 ± 3
burdock 1 10 ± 2
hamilton 2 n.d.
HMS Beagle 8 9 ± 3
mazi 4 n.d.
mdg1 4 21 ± 5
mdg3 8 14 ± 5
micropia 1 n.d.
nik 1 n.d.
nomad 4 n.d.
roo 40 68 ± 14
tirant 6 11 ± 3
transpac 4 n.d.
wolfman 2 n.d.
Unclassified 5
Total 178
Total nucleotide  sequence length of  LTR retrotransposons 1,269,435
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a

Vieira et al. 1999 

In general, the number of individual elements that we have characterized for each LTR retrotransposon family is consistent with average copy numbers estimated previously by in situ hybridization (Table 4). In situ hybridization also detects only those elements located in the polytenized, euchromatic component of the genome. For example, the most abundant element found is the roo element, which occupies, on average, 68 ± 14 sites within the polytene chromosomes of natural D. melanogaster populations (Vieira et al. 1999). We also found that roo was the most abundant element with at least 40 full-length copies in the genome of the sequenced D. melanogaster lab strain. In contrast, the least abundant elements in natural populations are 1731, gypsy, and zam. Each of these elements has, on average, less than two copies in natural populations (Vieira et al. 1999). We did not find any copies of these elements in our analysis of the D. melanogaster genome sequence. This is not surprising in that both gypsy and zam are known to be most abundant in constitutive heterochromatin and are in low abundance or absent from the euchromatic regions of some D. melanogaster strains (Pimpinelli et al. 1995; Baldrich et al. 1997).

Phylogenetic Characterization of D. melanogaster LTR Retrotransposons

The aligned RTs of all D. melanogaster LTR retrotransposons shown in Figure 2A were used to generate the phylogenetic trees presented in Figure 2, B and C. Additional RT sequences from other Drosophila and invertebrate species as well as D. melanogaster elements that were not identified in our analysis are also included in this phylogeny. The RT phylogeny indicates that there are three major groups of LTR retrotransposons within the D. melanogaster genome.

Copia Group

To date, members of the Copia group found in D. melanogaster include only copia and 1731. In our study, we did not find any representatives of the 1731 family. In one instance we found a copia element, copia-8, inserted into another element (mdg3-2). Similar composite insertions have been observed previously in maize (SanMiguel et al. 1996) and barley (Shirasu et al. 2000).

Gypsy Group

Previously characterized Gypsy group members that we identified in this study include 17.6, 297, 412, blastopia, blood, burdock, HMS Beagle, mdg1, mdg3, micropia, nomad, tirant, and transpac. Novel Gypsy group members first identified and named here are antonia, hamilton, nik, and wolfman. Five additional elements were identified that are closely related to Gypsy group elements and not characterized previously at the level of RT amino-acid identity. These elements are listed by accession number only in Figure 1. The RTs of these elements contain frame shifts or stop codons and are difficult to characterize (see above discussion). These five elements will require further analysis before they can be confidently placed phylogenetically with respect to their RT identity.

The Gypsy group found in D. melanogaster is composed of at least 20 different families that form three divergent clades seen in Figure 2, B and C. These clades all emerge from a central unsupported region deep within the Gypsy group. This is best illustrated in the unrooted phylogram shown in Figure 2B. One clade is composed of the elements 412, blood, mdg1 , and the novel element we named wolfman. This clade is well supported with a bootstrap value of 100. These four element families are closely related and form a very tight cluster at the end of a long branch that separates them from the rest of the Gypsy group.

A second clade that is less well supported (bootstrap value= 63) is composed of the elements micropia, mdg3, and blastopia. In contrast to the previously described group, these three elements are very distantly related to each other as indicated by very long branch lengths leading to each element.

The third clade that is found within the Gypsy group is better supported with a bootstrap value of 71. This is the most abundant clade within the D. melanogaster genome and contains, to date, 13 different families of elements. This clade can be divided further into two well-supported lineages containing five and eight families each. In addition to gypsy, burdock, HMS Beagle, and nomad, the group of five contains one novel element we have named hamilton. Previously, only LTR nucleotide sequences were available for the HMS Beagle element. Here we describe the first full-length copies of this element family. HMS Beagle is most closely related to the yoyo element first characterized in the Mediterranean fruit fly, Ceratitis capitata.

As mentioned earlier, each LTR retrotransposon family that we have characterized consists of a group of nearly identical elements (≥99% identity at the nucleotide level). One exception to this is the HMS Beagle family, which contains elements that are highly related yet show some level of phylogenetic structure (Figure 1). HMS Beagle elements consist of two well supported phylogenetic groups that share 97% RT identity at the amino acid level (Figure 2C). An additional phylogenetic comparison based on the entire DNA sequence of the HMS Beagle elements supports the conclusion that HMS Beagle elements consist of two well-defined subgroups (Fig. 3).

Figure 3.

Figure 3

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Unrooted neighbor joining phylogram of the nucleotide alignments of the HMS Beagle elements. Bootstrap values are shown on branches. The age of the individual elements as calculated by LTR sequence divergence is shown in parentheses following the name of the individual elements.

The novel element found within the aforementioned group of five, hamilton, is most closely related to the D. melanogaster endogenous retrovirus gypsy. Interestingly, we found that the two members of the hamilton family of elements are tandemly duplicated and separated by a single LTR. LTR retrotransposons having this same duplicate structure have been identified previously in yeast (Roeder and Fink 1983) and flies (Csink and McDonald 1995) and are postulated to be the products of homologous recombination between two full-length elements within the LTR region.

The group of eight families within the third Gypsy group clade contains tirant, zam, idefix, 17.6, 297, transpac, and two novel elements we have named antonia and nik. antonia is most closely related to idefix and Tv1 from D. virilis. nik is most closely related to tirant, zam and TED. TED was first found integrated into the genome of a baculovirus in the cells of the cabbage looper, Trichoplusia ni (Friesen et al. 1986).

BEL Group

In addition to the Gypsy and Copia clades, there is a third well-supported clade (bootstrap value  = 100) that contains the BEL, mazi, and roo families.

The complete sequence of BEL has been published previously (Bell et al. 1985) and partial characterization of roo (first described as B104) has been reported (Scherer et al. 1982; Lerat and Capy 1999). mazi, however, is a novel element. This is the first phylogenetic characterization of both roo and mazi. Previously, BEL was the only described member from D. melanogaster belonging to this third group of LTR retrotransposons now referred to as the BEL group (Malik et al. 2000). Interestingly, the roo element seems to encode a single, long open reading frame (ORF) of 2360 amino acids that contains homology to all of the previously described motifs found in LTR retrotransposons and retroviruses (McClure 1991) (Fig. 4). In most instances a frame shift is present after the gag (group specific antigen ) gene to regulate differential expression of the gag and pol regions of LTR retrotransposon and retrovirus genomes.

Figure 4.

Figure 4

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Translation of roo reveals one long ORF. The characteristic amino acid motifs of Gag, Protease, RT, Ribonuclease H (Rnase H), and Integrase of LTR retrotransposons and retroviruses (McClure 1991) are shaded and labeled in the margin. The region containing motifs similar to other Envelope proteins (Lerat and Capy 1999) is indicated near the end of the ORF.

Aging the LTR-Retrotransposons of D. melanogaster

As described previously, LTR nucleotide identity can be used to estimate the time of integration (SanMiguel et al. 1998) of LTR retrotransposons and retroviruses. We have found that 125 of the LTR retrotransposons described here have identical LTRs, whereas the remaining 53 have low levels of nucleotide divergence. Identical LTRs indicate that the elements have inserted recently and have not had time to accumulate mutations between the LTRs. Using the synonymous substitution rate for Drosophila (Li 1997) of .016 substitutions per site per million years and the intra-element LTR divergence calculated here, we have calculated the integration time of the 53 elements with LTR nucleotide divergence. The average age of the remaining 30% (53/178) of the elements was found to be 137,000 ± 89,000 yr. These results are shown in Table 3 and Figure 5A. Our data indicate that all of the D. melanogaster LTR retrotransposons analyzed in this study have integrated within the last 500,000 years. Moreover, the level of divergence for most elements indicates integration times of <200,000 years.

Figure 5.

Figure 5

Figure 5

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(A) Graph of LTR retrotransposon element ages of those elements that contain LTR nucleotide divergence values other than zero. (B) Graph of LTR retrotransposon family ages based on average pairwise identities of elements contained within a single family. The number of elements is shown in parentheses following the family name. Error bars indicate standard deviation of ages.

A second method for dating TEs is to calculate the average pairwise nucleotide identity across the complete sequences of the elements that are very closely related at the phylogenetic level (Kapitonov and Jurka 1996; Costas and Naveira 2000). The assumption underlying this method is that phylogenetically related elements are identical at the time of integration and have subsequently accumulated differences attributable to host DNA polymerase substitutions. This method also assumes that no homogenization of the element sequences by molecular mechanisms related to gene conversion has occurred subsequent to their integration. Most elements we characterized were found to contain unique flanking sequences in the DTRs (see above). This indicates that gene conversion has not affected any of the sequence directly adjacent to the elements since their insertion. Although it is a formal possibility that gene conversion may have some role in homogenizing repetitive sequences, available data indicate that the magnitude of its influence is not sufficient to account for the degree of similarity we observe (Nevo-Caspi and Kupiec 1996). We analyzed each independent family of elements using this second method. The results of this independent method of aging elements also indicate that the full-length D. melanogaster LTR retrotransposons have integrated within the last 500,000 years (Table 2; Fig. 5B).

Therefore, both available methods of computing the age of LTR retrotransposon integration are consistent and indicate that many full-length LTR retrotransposons in D. melanogaster are much younger than the age of the genome in which they reside. The estimated divergence time of D. melanogaster from its closest relative D. simulans is 2.3 ± .3 million years ago (Li et al. 1999).

DISCUSSION

We have identified 178 full-length LTR retrotransposons from the sequenced, euchromatic component of the D. melanogaster genome. We have characterized the D. melanogaster LTR retrotransposons phylogenetically with respect to other known LTR retrotransposon families. In doing so, we have identified five novel families of LTR retrotransposons within the genome of D. melanogaster that we have named antonia, hamilton, mazi, nik, and wolfman. Four of these elements fall into the canonical Gypsy group of LTR retrotransposons. mazi groups with a third well-defined group of LTR retrotransposons present within the genome of D. melanogaster. Also found within this third group is the abundant element roo, which we found encodes a single polyprotein that contains all of the enzymes necessary for LTR retrotransposon replication. We have previously characterized six families of elements from C. elegans (Cer7-12) belonging to this newly defined third clade (Bowen and McDonald 1999), which also contains Pao from Bombyx mori and Tas from Ascaris lumbricoides (Xiong et al. 1993). This group is most closely related in structure to the Gypsy group of elements in that its integrase gene is found downstream or 3′ of reverse transcriptase. In the Copia group, integrase is found upstream or 5′ of reverse transcriptase. Judging from its almost equal phylogenetic distance from both Copia and Gypsy groups, however, this third clade likely diverged at or near the time of divergence of the Copia and Gypsy groups and represents an ancient group of LTR retrotransposons. Additional elements belonging to this third clade have since been characterized from the genomes of Anopheles mosquitoes (Cook et al. 2000). Even more recently, it has been claimed that elements belonging to this clade have been identified in the pufferfish Fugu rubripes, the ascidian urochordate Ciona intestinalis, and the blood fluke Schistosoma mansoni (Malik et al. 2000). Therefore, this third major group of LTR retrotransposons is likely to be widespread within the metazoan lineage.

Most LTR retrotransposons and retroviruses contain at least one translational frame shift following the gag gene to regulate the necessary overproduction of Gag relative to the other element proteins (Coffin et al. 1997). In addition to roo, other elements with single ORFs include copia from D. melanogaster as well as the Gypsy group members Cer1 from C. elegans (Britten 1995) and Tf1 from Schizosaccharomyces pombe (Levin et al. 1990). In the case of Tf1, a differential protein degradation process regulates the overproduction of Gag (Atwood et al. 1996). The presence of a long, single ORF in the roo element (Fig. 4) indicates that this characteristic is present within all three major groups of LTR retrotransposons.

Perhaps the most intriguing result to appear from our study is the fact that the D. melanogaster genome contains many families of full-length LTR retrotransposons, all of which have been transpositionally active in the very recent evolutionary past. Interestingly, this finding is similar to what has been observed previously for the LTR retrotransposons in S. cerevisiae (Jordan and McDonald 1998, 1999b) and C. elegans (Bowen and McDonald 1999). As shown in our results, the age of the full-length LTR retrotransposons in the D. melanogaster genome is substantially younger than the melanogaster species itself. Interestingly, the average ages of all full-length LTR retrotransposons in yeast (<100,000 yr) (Promislow et al. 1999) and nematode (<500,000 yr) (N. Bowen, unpubl.) are also much younger than the age of the species in which they are contained. In contrast to these findings, it has been reported using the same criteria we have used here that several full-length LTR retrotransposons within the ADH-region of the maize genome are much older, having transposed in the past two to six million years (SanMiguel et al. 1998). Likewise, the average age of full-length HERVs (>25 million years) (Tristem 2000) is significantly older than the age of the human species (4–6 million years) (Yang 1996; Goodman et al. 1998).

One possible explanation for these contrasting comparisons may be differential genome size constraints placed on these species. In this regard, Adrian Bird (Bird 1995) has postulated that large increases in genome size are necessarily associated with increases in informational noise. Bird believes that the evolution of global epigenetic control mechanisms, such as methylation, were prerequisite to the significant expansions in genome size observed over the evolutionary history of higher eukaryotes. Although methylation is known to have a key role in the silencing of LTR retrotransposons in plants and vertebrates (Yoder et al. 1997), it appears to be lacking this function in many invertebrate species, including yeast, nematodes, and Drosophila (Russo et al. 1996). We believe that it may be for reasons such as this that full-length LTR retrotransposons have not accumulated over evolutionary time within these invertebrate genomes. As a consequence of the lack of methylation-mediated silencing in invertebrates, there would be strong selective pressure to eliminate LTR retrotransposons from these genomes.

Evidence has been presented that supports the existence of an active mechanism for the deletion of TEs in S. cerevisiae (Jordan and McDonald 1999b) and Drosophila (Petrov et al. 1996). Numerous solo LTRs exist in the S. cerevisiae genome as the result of intra-element LTR recombination, which serves to eliminate Ty elements from the host's genome (Jordan and McDonald 1999b). In D. melanogaster, as well as in other Drosophila species, DNA deletions of <400 bp are thought to occur at an astonishingly high rate within the genome, leading to a very high incidence of DNA loss (Petrov and Hartl 1997). The level of DNA loss attributable to deletions in Drosophila is estimated to be 75 times higher than that produced by deletions in mammals (Petrov and Hartl 1997). Consistent with this hypothesis, many of the elements that we have characterized contain sequence deletions when compared to the length of the canonical elements found in the public database (Tables 1 and 3). For example, every 17.6 element that we characterized from the D. melanogaster genome is shorter than the 7439 bp reported for the canonical 17.6 element (Saigo et al. 1984). These active processes that eliminate elements from genomes supply selective pressure for these elements to continually replicate or risk elimination (Jordan and McDonald 1999b). In turn, this results in only young, full-length elements within these genomes. Our results indicate that the full-length elements from the melanogaster genome are very young. Further support that genome size constraints can limit the accumulation of older retrotransposons comes from the recent characterization of BARE-1 insertion patterns in Hordeum spontaneum (barley) (Kalendar et al. 2000). These authors have shown that there is a positive correlation between full-length BARE-1 elements and increased genome size in barley. They further suggest that, if needed, selection for increased genome size can be regulated by limiting the amount of intra-element LTR recombination as described above for S. cerevisiae.

A final question concerns the immediate source of the full-length LTR retrotransposons present within the D. melanogaster genome. One possibility is that the full-length LTR retrotransposons are descendants from older elements that have been actively eliminated from the D. melanogaster genome or from older elements sequestered within the yet to be sequenced heterochromatin (see above discussions). An additional possibility is that the LTR retrotransposons currently present in the melanogaster genome have derived from elements recently introduced from other species via horizontal transfer. Recent analyses of specific families of Drosophila LTR retrotransposons indicate that horizontal transfer of LTR retrotransposons can occur (Jordan et al. 1999; Terzian et al. 2000). The extent of horizontal transfer and the degree to which it may have contributed to the overall composition of LTR retrotransposons that are present within the D. melanogaster genome remains to be determined.

Subsequent to the submission of this manuscript for publication, others (Frame et al. 2001) have reported a similar phylogenetic characterization for the members of the BEL clade. In their report, the element Tinker is identical to the element family that we call mazi. Similarly, a database of repetitive elements including a section for Drosophila has been made available by Genetic Information Research Institute (Jurka 2000) in which individual members of the families that we identify as antonia, hamilton, mazi, nik, and wolfman have been given the names Quasimodo, Gtwin, Diver, Gypsy5, and Tabor, respectively.

METHODS

Genome Query

Searches of the entire sequenced component of the D. melanogaster genome (using Advanced BLAST, http://www.ncbi.nlm.nih.gov/blast/blast.cgi) were initiated by performing TBLASTN (Altschul et al. 1997) searches using the RT amino-acid sequence of the Drosophila LTR retrotransposons BEL (U23420), copia (M11240), and mdg3 (X95908). Based on preliminary phylogenies we have constructed using the RT amino acid sequences of D. melanogaster LTR retrotransposons characterized previously, these three elements were chosen to represent the most divergent lineages. Nucleotide sequences with homology to the RTs were then subjected to a dot matrix (see below) analysis to reveal the presence of LTR sequences. Accession numbers that did not contain LTRs (as revealed by dot matrix analysis) were not included for further characterization. The characteristic ITRs as well as the DTRs that flank the LTRs were identified (Coffin et al. 1997). The region between LTRs was then translated to reveal coding sequences. Subsequently, the RT of each identified element was used to query the genome until all queries produced TBLASTN hits that overlapped into other element families.

Element Characterization

Each accession number containing a match to RT was retrieved from NCBI and ∼10,000 bp on each side of the TBLASTN hit were subjected to further analysis. Sequences were characterized using SeqLab: The Graphical User Interface to the Wisconsin Package (GCG 1999), maintained, and made accessible by the Research Computing Resource (RCR) at the University of Georgia (UGA) (http://www.rcr.uga.edu/biosci/home.html). The dot matrix program COMPARE was used to identify regions of identity within each sequence. DOTPLOT was used to visualize the dot matrixes generated with COMPARE. LTRs appeared as a line offset from and parallel to the identity diagonal. The terminal direct repeats were characterized from the flanking sequences of the LTRs. The terminal dinucleotides of each element LTR were also identified. RT motif amino-acid sequences of each element and the polyprotein of roo were predicted using TRANSLATE.

Multiple Sequence Alignments and Phylogenetic Analyses

Se-Al (courtesy of Andrew Rambaut, andrew.rambaut@zoo.ox.ac.uk) was used for multiple sequence file format manipulation and labeling. The ClustalW (Thompson et al. 1997) extension to SeqLab (GCG 1999) and ClustalX (Thompson et al. 1997) were used to generate nucleotide and amino acid alignments as described previously (Bowen and McDonald 1999). The seven conserved domains of the RT motif (Xiong and Eickbush 1988), also known as the RT ordered series of motifs (OSM) (Hudak and McClure 1999), are shown boxed in Figure 1A. Amino-acid and nucleotide alignment files may be obtained from the authors by request. PHYLIP (Felsenstein 1993) was used for distance calculation, tree production and bootstrap analysis. Phylogenetic analyses were performed on the multiple sequence alignments using distance methods employed by PHYLIP (Felsenstein 1993). The PRODIST program of PHYLIP, employing the Categories model, was used to generate distance matrices that were analyzed with the NEIGHBOR program to generate neighbor-joining tree files. SEQBOOT was also used to generate 100 data replicates that were subsequently analyzed with PRODIST (Categories model), followed by NEIGHBOR, and finally with CONSENSE to generate an unrooted bootstrapped tree as presented in Figure 2B. The phylogram presented in Figure 2C was rooted with the 1731 and copia elements. All trees generated were visualized with TreeViewPPC version 1.5.3 (Page 1996).

LTR Retrotransposon Age Calculation

PAUP (Swofford 1999) was used to calculate intra-element LTR identities and entire element family pairwise identities using the Kimura-2 parameter method. Ages were calculated using the formula T = K/2‘r’, where T = time of divergence, K =divergence, and r= substitution rate (Li 1997). The average synonymous or silent site substitution rate used was .016 substitutions per site per million years as calculated by E.N. Moriyama from 39 genes between the melanogaster and obscura groups where the time of divergence was set at 30 million years ago (Li 1997).

Acknowledgments

We are grateful to Drs. John Avise, Susan Wessler, Kelly Dawe, Michael Bender, and members of our laboratory for comments on earlier drafts of this manuscript. We thank Maney Mazloom for assistance in searching Genbank for Drosophila elements. This work was supported by a National Institutes of Health grant to J.F.M.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Footnotes

E-MAIL mcgene@arches.uga.edu; FAX (706) 542-3910.

Article published on-line before print: Genome Res., 10.1101/gr.164201.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.164201.

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