* Dipartimento di Genetica e Biologia Molecolare Università "La Sapienza"
Centro di Genetica evoluzionistica del C. N. R., Roma, Italy
Centro di Studio per gli Acidi Nucleici, C. N. R., Dipartimento di Genetica e Biologia Molecolare, Università "La Sapienza," Roma, Italy
Dipartimento di Genetica, Biologia Generale e Molecolare, Università Federico II, Napoli, Italy
|| Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università "La Sapienza," Roma, Italy
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Abstract |
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Key Words: Heterochromatin transposable elements Drosophila
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Introduction |
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TEs can be highly deleterious to the hosts in that they can cause different types of mutations affecting gene expression and chromosome structure (Kidwell and Lish 1997; Labrador and Corces 1997). However, there is increasing evidence that TEs also contribute to the evolution of genomes and may even acquire a role beneficial to the host (McDonald 1993; Spradling 1994; Britten 1997; Kidwell and Lish 1997; Labrador and Corces 1997; Miller et al. 1999; Nekrutenko and Wen-Hsiung 2001). For example, the HeT-A and TART elements of D. melanogaster specifically move to telomeres and contribute to the maintenance of their integrity (Pardue et al. 1996). There is also evidence for a buildup of TEs in constitutive heterochromatin of evolutionarily distant organisms (Dimitri and Junakovic 1999). In particular, in D. melanogaster clusters of TEs map to the heterochromatin of sex chromosomes and autosomes at locations that are cytologically stable in unrelated strains (Pimpinelli et al. 1995). Some of these clusters cytologically map to regions that contain single-copy genes and are devoid of highly repetitive satellite DNAs (Lohe, Hilliker, and Roberts 1993; Pimpinelli et al. 1995; Berghella and Dimitri 1996). TEs have been found both in the flanking regions and in the introns of the heterochromatic gene light (Devlin, Bingham, and Wakimoto 1990). However, the molecular composition and the precise anatomy of those regions has not been fully clarified, and little is known about other single-copy heterochromatic genes on chromosome 2. In order to get further insights into the relationships between TEs and heterochromatin, we have carried out a Blast analysis of introns from five heterochromatic and 22 euchromatic genes, whose sequence was released by the Drosophila Genome Project (Adams et al. 2000). The results show that TE-related sequences appear to be enriched by 450-fold in heterochromatic introns compared with euchromatic introns. In addition, intraspecific and interspecific length polymorphisms of heterochromatic introns, which may be associated with changes in TE DNAs, were detected using PCR analysis. Together, these findings highlight a contribution of TEs to the evolution of heterochromatic genes in Drosophila and provide further insight on TEhost genome interactions.
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Materials and Methods |
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Nucleic Acid Manipulations, DNA Sequencing, PCR Amplification, and Cloning
Nucleic acids manipulations were performed as described in Ausubel et al. (1991) and in Sambrook, Fritsch, and Maniatis (1989). Introns were amplified using either the Expand Long Template PCR System (Roche) or the TaKaRa Ex Taq (Takara Shuzo Co.) with gene-specific oligonucleotide primers in the flanking exons. Typically, the initial denaturation (5 min at 94°C) was followed by 30 cycles of amplification (94°C, 30 s; 55°C, 1 min; and 72°C, 2 to 6 min, depending on the intron). The sequence of the oligonucleotide primers used for the amplification of intron I and II of rolled, intron V of light, and intron XVII of Nipped-B were, respectively, rlEx1 (5'-TGGGACATATTCAGTTGTAAAAGT-3'), rlEx2R (5'-TGTTAGCGTGTCATCCGC-3'), rlEx2F (5'-GCGGATGACACGCTAACA-3'), rlEx3 (5'-GGTCTATGCTATCAACTCG-3'), ltEx5 (5'-ATGGCAACTTTATTGCCTCG-3'), ltEx6 (5'-TGTCTGTGCAGATTTCCTCG-3'), NbEx17 (5'-ATCGTAAACAAACTTCGCCG-3'), and NbEx18 (5'-TTGTCAACAATTAGCCGACG-3').
Intron Sequences
Intron sequences of introns from rolled, Nipped-B, light, concertina, and chitinase-3 were retrieved from Release 1 and 2 (Adams et al. 2002; flybase.bio.indiana.edu/), because the preliminary version of Release 3 (http://www.fruitfly.org/annot/release3.html) does not encompass those genes. Sequences from 33 large introns (>1 kb) from the following euchromatic genes were analysed: yellow, armadillo, white, lozenge, cut, Multiple inositol polyphosphate phosphatase 2, vermillion, Beadex, rudimentary, aristaless, Star, glycerol-3-phosphate dehydrogenase, Segregation distorter, bicaudal D, vestigial, lola, CG12017, Antennapedia, Ultrabithorax, Delta, and Ca2+-channel protein alpha1 subunit D. The PCR amplified products of rolled intron II from Charolles and Farfield-2 strains and from Drosophila sibling species were cloned into the pCR2.1 plasmid vector (Invitrogen); sequences were obtained commercially (MWG-BIOTECH AG). BlastN searches for TE homology were conducted with FlyBlast using the recently updated transposons data set. BlastX searches were performed at NCBI (National Center for Biotechnology Information). Alignments were performed with the Bioedit Sequence Alignment Editor 4.8.10 program for windows 95/98/NT and were also manually controlled.
Microphotography
Chromosome preparations were analyzed using a computer-controlled Zeiss Axioplan epifluorescencemicroscope equipped with a cooled CCD camera (Photometrics). Fluorescent signals were recorded by IP Spectrum Lab Software and edited using the Adobe PhotoShop 5 software.
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Results |
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Nipped-B
Short regions with similarity to several TEs are scattered throughout all the big introns of Nipped-B. Larger TE-related portions are also present. For example, intron VIII carries an approximately 2-kb sequence similar to Burdock; intron X has Mercurio and Transib sequences of about 1.5 kb; and intron XVII carries a 1.1 kb 1360-like element with TIRs and a 7-bp target site duplication (TSD). In conclusion, about 50% of the Nipped-B intronic DNA is composed of TE-related sequences.
Light
The large introns V and VI of the light gene also contain several short regions related to narep1 and other TEs. As in Nipped-B, a 1360-like sequence carrying TIRs and 7-bp TSDs is found in intron V. At the 3' end of intron VI a 2.5-kb Doc sequence is present, which is interrupted by a 1360-like insertion (with TIRs and TSDs), followed by a GATE-like sequence. Together, these findings are consistent with the result obtained by Devlin, Bingham, and Wakimoto (1990) and show that the TE-derived portions represent about 59% of all light intronic regions.
Concertina
In the concertina gene, the very large intron I contains regions of similarity to the Tc1 transposase of C. elegans, HB, FB, and narep1, which together account for approximately 33% of its length.
Chitinase-3
Only intron II of the chitinase-3 gene is very large and includes regions related to narep1, I element, FB, Quasimodo, and 17.6. In particular, the highest identity (87%) is found in the long terminal repeat (LTR) of the Quasimodo-like element. Together, these sequences account for about 60% of the entire length of intron I.
In conclusion, as summarized in table 1, the analysis of 79 kb of intronic DNA from rolled, Nipped-B, light, concertina, and chitinase-3 revealed that approximately 40 kb of this DNA (about 50%) is composed by TE-related sequences with substantially higher values in the intron II (75%), of rolled, intron V of light (70%), introns VIII (76%) and X (68%) of Nipped-B, and intron II of chitinase-3 (60%). Moreover, we found regions not related to known TEs that shared similarity with multiple scaffolds and thus are likely to correspond to other repetitive sequences (fig. 2).
Sequences Analysis of Euchromatic Introns
We have also analyzed the sequence of 33 very large introns (1 to 70 kb) from 22 eucromatic genes of D. melanogaster (for a detailed descriptions of the genes see Materials and Methods). The heterochromatin of D. melanogaster has been found to contain roughly four to five times more TEs than euchromatin (Pimpinelli et al. 1995; Maside et al. 2001). Similar estimates can be obtained based on the amount of TEs found in euchromatin relative to the entire genome (J. S. Kaminker et al., in preparation). Then, to normalize this difference, Blast analysis was conducted with 4.5-fold excess of euchromatic intron sequences (355 kb versus 79 kb). Only one of the 33 introns tested was found to contain TE-related sequences. In particular, a 406-bp sequence with 95% identity to the 3' end of the Doc element was detected in the intron I of CG12017 gene. This corresponds to the 0.11% of the entire DNA tested. Although this analysis has been performed on a relative small sample of euchromatic genes, the results are consistent with previous findings showing that TE-related sequences are underrepresented in euchromatic introns of D. melanogaster (Moriyama, Petrov, and Hartl 1998; Comeron 2001).
Heterochromatic Introns May Vary in Length Among Strains of D. melanogaster
The finding that TEs undergo to high rate of DNA loss in Drosophila (Petrov, Lozovskaya, and Hartl 1996) together with our previous evidence for de novo insertions of I elements in rolled intron II (Dimitri et al. 1997) raised the question of how stable are heterochromatic introns. We then asked whether TE-containing introns may undergo size variation in unrelated strains of D. melanogaster. To this end, introns I and II of rolled, intron V of light, and intron XVII of Nipped-B were PCR-amplified from genomic DNA of 17 unrelated strains, including the y; cn bw sp strain employed for the genome sequencing project (Adams et al. 2000). Intron II was analyzed as an extension of our previous observations (see above), while the other three introns were chosen because they range from 2 to 4 kb in length in the y; cn bw sp strain, a size that can still be readily amplified by PCR. As shown in figure 3, length variation was observed for introns I (fig. 3a) and II of rolled (fig. 3b), and for intron XVII of Nipped-B (fig. 3c), whereas no variants were detected for intron V of light (data not shown). More specifically, the length of rolled intron I was reduced by a few hundred nucleotides in the California-1 line as compared with y; cn bw sp and the other strains (fig. 3a). The PCR product carrying the intron II of rolled was found to be about 4 kb in all strains except Farfield-2, which carries a variant of about 5 kb (fig. 3b). Finally, in both Altamura and HJ30, the amplified band with intron XVII of Nipped-B was about 1 kb shorter compared with the remaining strains (fig. 3c). Using a combination of cloning, sequencing, and PCR amplification, we found a 1078-bp duplication, which accounts for the size polymorphism of rolled intron II that the Farfield strain revealed. Such duplication extends from 549 to1626 of rolled intron II from the Charolles strain (GenBank accession number AJ441085) and includes a large part of Mercurio and a few hundred base pairs from 1360. It is likely that the intronic variants detected here represent only events compatible with the normal expression of heterochromatic genes, since mutations that affect the fitness should be selected against.
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Heterochromatic Introns May Vary in Length Among Drosophila Sibling Species
To get further insight into the evolutionary dynamics of heterochromatic introns, we have undertaken a comparative analysis of introns from orthologues of the same genes in closely related Drosophila species. To this end, PCR amplification of introns I and II of rolled and intron V of light was performed in D. mauritiana, D. simulans, D. sechellia, D. teissieri, and D. yakuba where both light and rolled are known to be located in heterochromatic regions (Yasuhara et al. 2002; P. Dimitri unpublished). PCR products were detected only in D. simulans, D. sechellia, and D. mauritiana (fig. 3df). The size of the amplified products differs from the y; cn bw sp reference strain of D. melanogaster by a few hundred bp (light intron V of D. simulans and D. sechellia [fig. 3f]) to about 4 kb (rolled intron I of D. simulans, [fig. 3d]). The molecular basis of these differences were further analyzed by cloning and sequencing the intron II of rolled. This intron is about 1.3 kb in both D. simulans (GenBank accession number AJ459415) and D. sechellia (GenBank accession number AJ459414), and approximately 1.5 kb in D. mauritiana (GenBank accession number AJ459416 [fig. 3e]). The sequence of the central region of intron II exhibits about 75% identity with the HB transposon in all three species. This region is nearly 750 bp in both D. simulans and D. sechellia, whereas it approachs 900 bp in D. mauritiana. Thus, size and sequence composition of rolled intron II are variable among D. melanogaster (fig. 2 and table 1) and closely related species due to changes in TE-related sequences.
TE Traces in Exons and Flanking Regions of Single-Copy Heterochromatic Genes
Extension of sequence analysis to exons and to regions flanking single-copy heterochromatic genes revealed TEs traces both in the Nipped-B and in the light genes. In particular, a 954-bp region with similarity to Rt1b (75% nucleotide identity, 30% amino acid identity in the RT domain) overlaps the last 300 bp of exon XXIV of Nipped-B and is followed by a 756-bp sequence related to Cr1a (85% identity). Another example is provided by a 120-bp sequence that is located about 100 bp upstream of the transcription initiation of light and shows high level of identity (86%) to the terminal inverted repeats (TIRs) of the S element. This sequence may represent the footprint left behind after the imprecise excision of a full-length S element. It is worth noting that potential transcription factor binding sites can be identified in this TIR homologous sequence by the AliBaba 2.1 software (http://www.gene-regulation.com/). Finally, a baggins-related sequence (84% identity, 1.5 kb) located about 2 kb upstream of the 5' end of rolled was also detected.
Determination of the Accuracy of TE Sequences
A warning has been issued about the uncertainty of transposable element sequences of Release 1 and 2, (www.fruitfly.org/sequence/faq.). In particular, the assembly of DNA sequences from genomic regions that contain many tandemly arranged repetitive elements may result in the omission of internal sequences (E. Myers, quoted in Bowen and McDonald 2001). However, four experimental observations indicate that the TE sequences examined here are likely to be accurate. First, the assembled sequence of rolled intron II in scaffold AE003090 is 99% identical to the one that we independently obtained from the wild-type Charolles strain (see Rolled above). Second, PCR amplification of intron I and II of rolled, intron V of light, and intron XVII of Nipped-B from the y; cn bw sp strain yielded products of the size expected on the basis of the scaffold sequences (fig. 3ac). Third, according to the strategy employed by Myers et al. (2000), we confirmed the accuracy of light intron V and Nipped-B intron XVII sequences by digesting with diagnostic restriction enzymes the corresponding amplification products from the y; cn bw sp strain. The light intron V was digested with KpnI and SacI, whereas Nipped-B intron XVII was cut with BglII. On the basis of the sequence reported in the respective scaffolds, these enzymes should cut once or more within the 1360-like sequence of the introns. In both cases, the expected restriction patterns were obtained (data not shown). Fourth, sequences corresponding to portions of some heterochromatic introns analyzed here were also retrieved from draft sequences of three bacterial artificial chromosomes (BACs) independently determined by the Berkeley Drosophila Genome Project (BDGP) (Hoskins et al. 2000): BACR07M22 (AC016129), which carries the last 3.5 kb of light intron VI and inludes three different TE-related sequences; BACR01O12 (AC016130), including almost the entire intron XII of Nipped-B; and BACR02C02 (AC008333), which contains the complete intron I of concertina. These independently assembled draft sequences did not substantially differ from those produced by whole genome shotgun sequence assembly (Adams et al. 2000). Together, these results converge to indicate that no substantial alteration occurred in the assembly of TE-homologous sequences of the heterochromatic introns analyzed in this work.
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Discussion |
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Sequences homologous to class I and class II elements represent 43% and 57%, respectively, of total intronic DNA. Class II elements have also been found to be more abundant on chromosome 4 of D. melanogaster, which exhibits heterochromatin-like features, whereas in euchromatin, they represent only about 8% of all TEs (J. S. Kaminker et al., in preparation). Sequences related to 1360 and narep1 (class II elements) appear to be scattered to several introns, presumably as result of multiple independent insertions. Some of the 1360-like sequences retain terminal inverted repeats (TIRs) flanked by target site duplications and are therefore likely to represent recent insertions in heterochromatin where this transposon is particularly abundant (Kholodilov et al. 1988).
These results, together with the finding that the presence of TEs in introns of the euchromatic genes tested is marginal (0.11% of all intronic DNA), show that TEs significantly contribute to the build up of large introns of genes located in the heterochromatin of D. melanogaster. Similarly, degenerate TEs are abundant in the huge introns of the Y-linked dynein gene of D. melanogaster (Kurek et al. 2000). TE enrichment in large introns might be a recurrent trait in the evolution of Drosophila heterochromatic genes, as is also suggested by the observation that in orthologues rolled genes from Drosophila sibling species, about 60% of intron II is made up of TE-related DNA.
Enrichment of TEs in Heterochromatic Introns Versus Enrichment in Heterochromatin As a Whole
The accumulation of TEs in Drosophila heterochromatin has been previously documented and discussed at length (Charlesworth, Sniegowski, and Stephan 1994; Pimpinelli et al. 1995; Dimitri and Junakovic 1999; Maside et al. 2001; Rizzon et al. 2002; Bartolomé, Maside, and Charlesworth 2002). Under the ectopic exchange model, selection against the deleterious effects of chromosomal rearrangements would be lower in heterochromatic regions where recombination is low or null. (Charlesworth and Langley, 1989). Consistent with this hypothesis is the finding that meiotic recombination is negatively correlated with TE density in D. melanogaster (Bartolomé, Maside, and Charlesworth 2002). Another possibility is that accumulation of TEs in heterochromatin reflects preferential targeting (Dimitri and Junakovic 1999). The results described in this report extend these observations in that TE density in heterochromatic introns appears to be 450-fold higher as compared with euchromatic introns. By contrast, the enrichment in the heterochromatin as a whole compared with euchromatin has been estimated to be only fourfold to fivefold (Pimpinelli et al. 1995; Maside et al. 2001). It is unclear at present whether preferential location of TEs in the intergenic regions of euchromatin is sufficient to account for this difference. Reduced selective pressure against intronic insertions in heterochromatin following the Hill-Robertson effects on insertional mutations may also be contributing (Bartolomé, Maside, and Charlesworth 2002). Finally, targeting of heterochromatic genes could also be relevant to the difference in density between euchromatic and heterochromatin introns (Dimitri et al. 1997).
TEs and the Evolution of Genes Located in Heterochromatin
In addition to enrichment of TE-derived sequences, we have shown that introns of heterochromatic genes can undergo intraspecific and interspecific length polymorphism (fig. 3) and that, at least in the case of the rolled intron II, such variation is due to TE-derived sequences. By contrast, the contribution of TE sequences to introns of euchromatic genes is marginal (Moriyama, Petrov, and Hartl 1998; Comeron 2001), and intron size of hortologues genes in euchromatin is quite stable between D. melanogaster and D. simulans (Akashi 1996). It appears then that TEs not only contribute to the build up of heterochromatic introns, but may also confer a dynamic state to the structure of genes located in heterochromatin. It has been suggested that TEs may become functional parts of such genes (Dimitri and Junakovic 1999), similar to the documented cases in euchromatin (McDonald 1993; Britten 1997; Kidwell and Lish 2001; Nekrutenko and Wen-Hsiung, 2001). Consistent with this speculation, evidence for the adaptive significance of TE-derived sequence in heterochromatic genes was recently reported for the LTR of Quasimodo located in intron I of chitinase-3 (McCollum et al. 2002). Additional candidates for comparable roles may be the LTR portion of 17.6 located in the intron IV of rolled, the TIR of the S element in the promoter region of light, or, most notably, the Rt1b-related region in exon XXIV of Nipped-B (see Results). Further genomic and molecular analyses will be required to assess a possible involvement of such sequences in the function and expression of heterochromatic genes.
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Acknowledgements |
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Footnotes |
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