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Genetics, Vol. 173, 1433-1445, July 2006, Copyright © 2006
doi:10.1534/genetics.106.056069
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* Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada, Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242 and Istituto Pasteur, Fondazione Cenci Bolognetti and Dipartimento di Genetica e Biologia Molecolare, Università "La Sapienza," 00185 Roma, Italy
2 Corresponding author: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada.
Manuscript received January 20, 2006. Accepted for publication April 29, 2006. |
ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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30% of the Drosophila melanogaster genome, forming a transcriptionally repressive environment that silences euchromatic genes juxtaposed nearby. Surprisingly, there are genes naturally resident in heterochromatin, which appear to require this environment for optimal activity. Here we report an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D. melanogaster. DmDbp80 is typical of previously described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast, DmRpL15 is uncharacteristically small. The orthologs of these genes were examined in D. pseudoobscura and D. virilis. In situ hybridization and whole-genome assembly analysis show that these genes are adjacent, but not centromeric in the genome of D. pseudoobscura, while they are located on different chromosomal elements in D. virilis. Dbp80 gene organization differs dramatically among these species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species demonstrates active repositioning of these genes both within and between chromosomal elements. This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these genes also provides unique insight into the dynamic nature of genome evolution.
A small number of essential genes have been identified in autosomal heterochromatin of D. melanogaster (HILLIKER and HOLM 1975; HILLIKER 1976; MARCHANT and HOLM 1988a,b; SCHULZE et al. 2001; reviewed by COULTHARD et al. 2003; DIMITRI et al. 2005; FITZPATRICK et al. 2005). Their ability to function in this inactivating environment is puzzling, as is their repression when moved to distal sites within euchromatin (WAKIMOTO and HEARN 1990; EBERL et al. 1993). There is also genetic and molecular evidence indicating that heterochromatic gene expression is dependent on constituents of heterochromatin even in the absence of chromosomal rearrangements. For example, genes in heterochromatin exhibit compromised transcription in a genetic background deficient for heterochromatin-associated protein (CLEGG et al. 1998; LU et al. 2000; SINCLAIR et al. 2000; SCHULZE et al. 2005). Thus, the regulation of heterochromatic loci and the dependence of gene expression on the surrounding chromatin environment are intensely active areas of research.
A number of issues have impeded progress in the genetic and molecular characterization of centric heterochromatin using standard methods. These problems include the absence of good polytene banding, the lack of standard meiotic recombination, and the presence of numerous repetitive DNA sequences. Release 3 of the Drosophila genome sequence contains 21 Mb of putative heterochromatic DNA (HOSKINS et al. 2002). These data are extremely valuable in providing both an overview and a preliminary model for the molecular organization of heterochromatic genes, although there are questions concerning the discrepancy between the numbers of predicted (at least 300) gene models vs. the few dozen essential loci defined by genetic methods. An additional difficulty concerning the molecular analysis of heterochromatic genes is presented by their size: most that have been characterized to date tend to be very large, due to the presence of repetitive sequences in their introns (DEVLIN et al. 1990a,b; RISINGER et al. 1997; WARREN et al. 2000; TULIN et al. 2002; DIMITRI et al. 2003; SCHULZE et al. 2005).
Another puzzle to be addressed is how genes come to reside and function in such a transcriptionally repressive environment. One way of elucidating the evolutionary history of heterochromatic genes is to examine the structure and position of these genes in a set of related species. The genus Drosophila is ideal for a comparative study of heterochromatic genes: it consists of a large number of well-studied species and possesses a relatively stable karyotype, while nevertheless exhibiting exceptionally malleable evolutionary dynamics at the intrachromosomal level. Six chromosomal elements, identified with letter designations to indicate homology ("Muller elements"), are the basis for chromosomal organization in the genus (MULLER 1940). Gene content of the chromosomal elements is largely shared among species, and movement between elements is considered unusual, but genes are extensively rearranged within elements (VIEIRA et al. 1997; RANZ et al. 2003). Chromosome number differs among Drosophila species due to large-scale rearrangements involving fusion events between acrocentric chromosomes that produce metacentric arrangements. This is exemplified by comparing the chromosomal configurations of representative species in Figure 1. For example, the haploid karyotype of D. virilis possesses the inferred ancestral condition with six acrocentric chromosomes. D. melanogaster has only four chromosomes in the haploid set due to two fusion events that formed the large metacentric autosomes, whereas D. pseudoobscura has five chromosomes with one of these resulting from a fusion event between elements A (the X chromosome) and D.
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10 kb downstream of a highly active gene, DmRpL15, which encodes an essential component of the ribosome (SCHULZE et al. 2005). DmRpL15 is surprisingly small for a heterochromatic gene, although it is likewise characterized by the presence of repetitive DNA upstream, downstream, and within its introns. General evolutionary constraints on these genes, and the resultant diversity in gene structure and location, were determined through a comparative genomic framework. The genes are not adjacent to each other in the genome of D. virilis and appear to reside on separate chromosomal elements in this species. DvDbp80 is a small gene located on element D in euchromatin, while DvRpL15 is similar to D. melanogaster in both structure and heterochromatic position, but is on element E in D. virilis. In D. pseudoobscura, the genes are adjacent to each other as they are in D. melanogaster and have retained their fundamental structures. Surprisingly, however, in D. pseudoobscura, they are located in the middle of the euchromatic portion of element E. These genes have therefore undergone a rare interelement relocation at least twice during the course of Drosophila evolution, and these movements have been confined to the chromosomal elements (D and E) that form the metacentric chromosome 3 in D. melanogaster. In addition, both genes present a study in contrasts with respect to gene structure. Collectively, these findings provide novel insight into the dynamic nature of Drosophila heterochromatin and into how the local environment influences gene structure in relation to functional constraints.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Isolation of D. virilis cDNAs:
The D. virilis cDNAs were isolated from a mixed embryonic plasmid library in pOT7B constructed for the Berkeley Drosophila Genome Project (http://www.fruitfly.org/) and kindly provided by Ling Hong. Plasmid libraries were screened in three stages, with the primary screen plated with liquid culture to near confluence, the secondary plates patched, and the tertiary plates streaked to ensure unique isolates. Colonies were grown overnight on 2YT agar plates with 25 µg/µl chloramphenicol and then transferred to 4° for at least 2 hr before treatment with nylon filters (lifts). In all cases, Hybond-N+ (Amersham, Buckinghamshire, UK) circular filters were used. The filters with colonies were transferred to fresh plates, which were placed at 37° for a minimum of 4 hr, after which the filters were twice soaked (colony side up) for 3 min in denaturing solution (1.5 N NaCl, 0.5 N NaOH) and then once in neutralizing solution (0.5 M Tris-HCl pH 7.5, 1.5 N NaCl) for 7 min. They were then rinsed in 2x SSC, and the colony debris actively scrubbed off. After the filters were drained briefly on Whatman filter paper, they were irradiated with UV light using a Stratagene (La Jolla, CA) stratalinker and subjected to hybridization under the conditions described above for genomic Southern analysis, except that the stringency was lower (6x SSC, 2x Denhardt's, 0.5% SDS, 59°). Putative clones were purified after the tertiary screen by alkaline lysis (SAMBROOK et al. 1989) and inserts were sequenced by the University of Calgary Core DNA and Protein services (accession no. DQ426903 for DvRpL15 and DQ426902 for DvDbp80).
In situ chromosome analysis:
Preparation of chromosome squashes from the salivary glands of third instar larvae of D. virilis was as described by KRESS (1993). Probes were labeled by nick translation with biotin-14-dATP (Invitrogen Life Technologies, San Diego) or digoxigenin-11-dUTP (Roche Diagnostics) according to the manufacturer's instructions. After hybridization at 37° overnight, and three washes in 2x SSC, signal was detected with antidigoxygenin–rhodamine (Roche Diagnostics) at a 1:550 dilution, or with antibiotin-FITC (Vector Laboratories) diluted 1:45 for 1 hr. DNA was counterstained with DAPI before image capture, using a cooled CCD camera (MicromaxYHS 1300, Roper Scientific) mounted on a DMRXA Leica microscope. Polytene in situ analysis for D. pseudoobscura and mitotic (from neuroblasts) in situ analysis for D. virilis were carried out according to PIMPINELLI et al. (2000). Probes were differentially labeled by nick translation with digoxigenin- or biotin-coupled dUTP and detected with fluorescein avidin or antidigoxigenin–rhodamine antibody. For D. virilis, either cDNA (for polytene chromosomes) or genomic DNA (for mitotic chromosomes) probes were used. For the latter, a DvRpL15 probe was prepared by labeling a 3.5-kb EcoRI subclone (also having an internal EcoRI site), which contains the DvRpL15 gene and just under 1 kb of flanking sequence on either side. The DvDbp80 probe was prepared by labeling a 4.6-kb EcoRI fragment containing the DvDbp80 gene. For D. pseudoobscura polytene analysis, probes were prepared by PCR amplification from genomic DNA of unique sequences containing the genes in question, and primer selection was based upon the published genome sequence (at http://www.genome.gov/11008080, the portal for the comparative Drosophila genome-sequencing project). Therefore, for DpRpL15, two primers, 5'-GGGCCTATCGTTATATGCAAG-3' and 5'-ACGGTTCTTGCGCTTCCAGGC-3', were chosen to amplify a 760-bp probe, while 5'-CTGAGTGGTAATCTGGTCA-3' and 5'-TAATGCTATCCAGTGTTCGA-3' amplified an 870-bp probe for DpDbp80.
Phage library screening:
The genomic regions for DvRpL15 and DvDbp80 were subcloned from a phage 
EMBL3 D. virilis genomic library (THUMMEL 1993), generously provided by Ron Blackman. Phage libraries were screened using the D. virilis cDNAs as probes in three stages: for the primary screen, plates were almost confluent, and the secondary and tertiary screens were plated at low titer to ensure unique isolates. Phage were plated in NZYM Top agarose (SAMBROOK et al. 1989), grown overnight, and then transferred to 4° for at least 2 hr before transfer to filters (lifts). Hybond-N+ (Amersham) circular filters were used for plate lifts and treated with the same denaturing and neutralizing solutions described above for plasmid library screening. The filters were rinsed briefly in 2x SSC, drained on Whatman filter paper, irradiated with UV light using a Stratagene stratalinker, and subjected to hybridization under the conditions described above. Genomic inserts from positive phage were cut with SalI to liberate the inserts. The restriction digest products were then diluted, run on an agarose gel, transferred to nylon membrane by Southern blotting, and hybridized with relevant cDNA probes to establish which bands contained coding regions. These bands were then extracted, using a GFX gel extraction kit (Amersham) according to the manufacturer's instructions, and subcloned into pBluescript. Small SalI fragments were also gel extracted and cloned into SalI-cut pBluescript.
Sequence analysis:
Sequence assembly (D. virilis genomic subclones) was carried out using the BLAST algorithm for two sequences (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml). Identifying D. virilis coding and noncoding regions was carried out using BLASTN and TBLASTN on genomes of those model organisms that have already been sequenced. All the default BLAST settings were used, except that low-complexity sequence was not masked. Multiple protein or nucleic acid alignments were made first by using CLUSTAL W (THOMPSON et al. 1994) to generate the alignments and then BOXSHADE (Boxshade version 3.3.1 by Kay Hofmann and Michael D. Baron) for coloring conserved regions. CLUSTAL W and BOXSHADE are available at http://workbench.sdsc.edu/. Sequencing was carried out by the University of Calgary Core DNA and Protein Services and assembled (see below). Accession numbers are DQ426901 (DvDbp80 genomic) and DQ426900 (DvRpL15 genomic).
Bioinformatic analysis:
Sequence of the DmDbp80 and DmRpL15 region in the D. melanogaster genome was obtained from GenBank (accession no. AABU01002497) along with the cDNA sequences of these genes (accession nos. AF005239 and AY094841, respectively). Sequences of cDNAs for both Dbp80 and RpL15 were constructed from 5' and 3' EST reads of D. pseudoobscura and D. ananassae available from the NCBI Trace Archive (deposited by the Human Genome Sequencing Center at Baylor and Agencourt Bioscience). The cDNA sequence of RpL15 was also available for D. yakuba (AY231804; DOMAZET-LOSO and TAUTZ 2003). Genomic sequences were obtained from the following sources: D. pseudobscura (Human Genome Sequencing Center at Baylor, Release 1.0; RICHARDS et al. 2005), D. yakuba (Genome Sequencing Center at Washington University, release 1), D. willistoni (J. Craig Venter Institute, NCBI Trace Archive), and D. ananassae, D. virilis, and D. mojavensis (Agencourt Bioscience, freeze 1 assemblies). Sequences of Dbp80 and RpL15 were identified in each genome sequence using blastn (ALTSCHUL et al. 1997). A CLUSTAL W alignment was obtained for the D. melanogaster, D. pseudoobscura, and D. virilis cDNA sequences of Dbp80 with genomic sequences from D. virilis and D. mojavensis. Positions of exon/intron boundaries were inferred by the position of gaps and conserved splice sites. Positions of exon/intron boundaries in the remaining species were inferred from BLAST alignments of cDNA sequences with genomic sequences. The most similar cDNA sequence was used as the query for each of the alignments. A CLUSTAL W alignment of RpL15 was obtained for the cDNA and genomic sequences of all species, and the positions of the two conserved introns were inferred from gaps and splice sites in the alignment. Genomic context of the Dbp80 and RpL15 genes in each of the genomes was assessed on the basis of the initial assemblies. Contigs containing these genes were screened for the presence of repetitive sequences using Repeat Masker to mask repeats in a sequence (http://www.repeatmasker.org). Furthermore, the presence of genes in the masked contig was assessed using the NCBI BLAST server to screen the D. melanogaster genome. Inferences of gene content were also evaluated in the annotations represented in the UCSC Genome Browser (http://genome.ucsc.edu).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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To initiate a study of the evolutionary history of these genes, cDNAs for both were isolated in a low-stringency screen of a mixed embryonic D. virilis plasmid cDNA library using D. melanogaster probes. For DvRpL15, a single transcript was obtained; for DvDbp80, two transcripts resulted, differing only slightly in length at the 5'-end. Both genes encode products that are comparable in size to their D. melanogaster orthologs. cDNA sequences are available for D. pseudoobscura and Anopheles gambiae, two additional dipteran species that have assembled genome sequences (HOLT et al. 2002; RICHARDS et al. 2005). Alignments of the conceptually translated sequences of both genes from the four available dipteran species with sequences of the human and yeast orthologs are shown in Figure 2, A and B. All of the Dbp80 sequences possess a unique six-amino-acid residue motif indicative of DEAD box helicases, which in yeast have been shown to be involved in mRNA export (SNAY-HODGE et al. 1998; ROLLENHAGEN et al. 2004). Southern and Northern analyses of both genes in D. virilis demonstrate that they are likely to be single copy and expressed in a manner similar to that of their D. melanogaster orthologs (data not shown). A BLAST search of D. virilis sequence traces in the NCBI Trace Archive also revealed single haplotypes, indicating that both genes are present in single copy. Since both genes appear to be single copy and functional in this distantly related species, they are good candidates for a study of heterochromatic gene evolution.
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The in situ data are shown in Figure 3. For both species, a single signal resulted in each case, supporting the molecular and bioinformatic evidence that Dbp80 and RpL15 exist as intact single copies in these genomes. Both genes reside in the heterochromatin of element D (3L) in D. melanogaster, but this arrangement is not conserved in D. virilis or D. pseudoobscura. In D. virilis, Dbp80 appears to be euchromatic (Figure 3A), mapping approximately to position 35F (KRESS 1993) on chromosome 3, which is element D and therefore homologous to 3L in D. melanogaster (see Figure 1). DvRpL15, on the other hand, consistently gave a chromocenter signal (Figure 3B), indicating that this gene is heterochromatic in this species. To establish on which chromosome arm DvRpL15 resides, it was necessary to perform an in situ analysis on fully condensed mitotic chromosomes, which showed that this gene is located on a different chromosomal element than DvDbp80 (Figure 3C). The identity of this element can be inferred from two observations. First, the DvRpL15 probe hybridized to a homologous chromosome pair with a centromeric region that stains brightly with DAPI. Previous studies (HOLMQUIST 1975) demonstrate bright staining of the centromeres of chromosome 2 and 4 with Hoechst, and although DAPI was used as the counterstain in this analysis, the two fluorochromes exhibit identical staining patterns (PIMPINELLI et al. 2000), probably due to their high affinity for AT-rich regions. Second, rare polytene spreads were observed in which the heterochromatic region bearing the DvRpL15 signal was pulled out from the chromocenter and clearly linked to chromosome 2, which has the longest polytenized euchromatic arm in this species (data not shown). Collectively, the cytological evidence indicates that in D. virilis, RpL15 is located in heterochromatin on element E (chromosome 2), while Dbp80 is located in euchromatin on element D (chromosome 3).
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Dbp80 gene structure is evolutionarily dynamic while RpL15 is highly conserved:
The in situ data provide clear evidence of at least two interelement movements involving Dbp80 and RpL15 during the evolutionary history of these species. It is also clear, that while Dbp80 and RpL15 are different in terms of gene structure and biological function (SCHULZE et al. 2005), their activity is not dependent on chromatin environment, since it appears that both can exist in heterochromatin or euchromatin. To study the connection between gene structure and genomic context, we employed a combination of molecular and bioinformatic approaches to determine the structure for each gene in seven Drosophila species (see Figure 1). As a starting point, the gene structures in D. virilis were determined by cloning and characterizing genomic sequence: D.virilis cDNAs were used in high-stringency screens of a D. virilis phage genomic library and the genomic structures for both genes were resolved by alignment. Sequences derived from these clones were compared with the sequence obtained from the Agencourt whole-genome assembly for D. virilis (freeze 1 assembly). In addition, cDNAs for Dbp80 and RpL15 from D. virilis, D. pseudoobscura, and D. melanogaster were used to determine the genomic organization for both genes in five additional species (D. yakuba, D. ananassae, D. pseudoobscura, D. willistoni, and D. mojavensis) that form a part of the Drosophila comparative genome-sequencing project (http://www.genome.gov/11008080). As a potential outgroup for comparison, genomic structures were also derived by analysis of cDNA and genomic sequence obtained from the Anopheles genome project (http://www.ensembl.org/Anopheles_gambiae/).
As can be seen in Figure 4A, Dbp80 exhibits an evolutionarily labile structure: it is extended in genomic size with many introns in species of the subgenus Sophophora (D. willistoni, D. pseudoobscura, D. ananassae, D. yakuba, and D. melanogaster) and condensed with only a single intron in species of the subgenus Drosophila (D. mojavensis and D. virilis). This single intron is conserved both in position and approximate size in species from both subgenera. Where contiguous sequence is available, the extended Dbp80 is interrupted by introns in precisely the same positions in all species of the subgenus Sophorphora; however, intron sizes vary remarkably among these species. The structure of Dbp80 in An. gambiae is intermediate, with five introns, but these appear to be completely conserved in position with respect to species in the Sophophoran lineage. The same comparative study for RpL15 reveals a contrasting picture: this gene retains its structure throughout all species examined (Figure 4B), including the polypyrimidine tract that has been shown to play an important role in both the transcriptional and translational regulation of ribosomal protein genes across taxa (HARIHARAN and PERRY 1990; LEVY et al. 1991; BARAKAT et al. 2001). Thus Dbp80 appears to tolerate great flexibility with respect to genomic organization, while the structure of RpL15 has remained highly conserved during the divergence of these lineages.
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Genomic context and arrangement of Dbp80 and RpL15 in other Drosophila species:
Analysis of the flanking sequence of both Dbp80 and RpL15 throughout the genus Drosophila is possible using the data available from the comparative genome-sequencing project (http://www.genome.gov/11008080). From genome sequence data, the position of these genes relative to each other, and the identity of genes in flanking regions can be determined. The in situ data indicate that these genes are very close to each other in D. pseudoobscura (Figure 3, D and E), and the location of these genes in the assembled sequence also demonstrates that they are adjacent, but they are not transcribed in the same orientation as in D. melanogaster (Figure 5). Analyses of the available genome sequence data from D. yakuba and D. willistoni indicate that Dbp80 and RpL15 are located close to each other in a scaffold and a contig, respectively, and thus are likely adjacent in these species as well. These genes are located on the same scaffold in D. ananassae, but are separated by 2 Mb of intervening sequence. On the basis of in situ hybridization in D. virilis, the genes are present on different chromosomal elements (Figure 3C), supported by the observation that they are located in different large scaffolds of assembled D. virilis genomic sequence. Similar results were also obtained for D. mojavensis. Therefore, Dbp80 and RpL15 are adjacent in all of the species in the subgenus Sophophora, except D. ananassae, and they are separate in the two representatives of the subgenus Drosophila (Table 1).
Repeat content of the sequence in the immediate vicinity of Dbp80 and RpL15 and the position of additional identifiable orthologs of D. melanogaster provide further indication of the surrounding genomic environment. The region surrounding Dbp80 in D. virilis contains few interspersed repeats (Table 1), consistent with the euchromatic localization of this gene determined by in situ hybridization (Figure 3A). The genomic context of Dbp80 in D. virilis appears to be conserved with D. mojavensis, including the relative positions of the flanking genes CG6513, CG14561, and CG7139 (Figure 5), which indicates microsynteny for this region in these two representative species of the subgenus Drosophila. The genes in this conserved region are all located on element D of D. melanogaster.
The region surrounding RpL15 contains an abundance of interspersed repeats in all the species examined, with the exception of D. willistoni and D. mojavensis (Table 1). Although repeat content differs in the region around RpL15 between D. virilis and D. mojavensis (Table 1), two single-copy genes [Crc (CG9429) and CG40228] flanking RpL15 are conserved between these species (Figure 5). Conservation of genes around RpL15 (and including Dbp80 when adjacent) is also evident in comparison with species in the subgenus Sophophora (Figure 5). The most striking of these conserved flanking genes is CG40228, which is present in single copy and near RpL15 in D. pseudoobscura, D. willistoni, D. virilis, and D. mojavensis, and thus reflects a conserved ancestral arrangement. The proximity of RpL15 and CG40228 in the genome of D. melanogaster remains difficult to assess on the basis of the current heterochromatic assembly. In D. virilis, D. mojavensis, and D. willistoni, genes flanking RpL15 are present on either element D or element E in the genome of D. melanogaster, which corresponds to the left and right arms of chromosome 3 (Figure 1). Thus, whereas Dbp80 is associated with two different sets of flanking genes in the subgenera Drosophila and Sophophora, RpL15 is embedded among a number of conserved genes in both lineages.
The high density of interspersed repeats surrounding RpL15 is consistent with the heterochromatic location in D. virilis and D. melanogaster, but the relatively high density of repeats around RpL15 is not consistent with its apparent euchromatic position in D. pseudoobscura. Additionally, the region directly flanking RpL15 and Dbp80 in the assembled genome of D. pseudoobscura contains several other genes present on element D of D. melanogaster (Figure 5), which is inconsistent with the in situ localization of probes of both Dbp80 and RpL15 to element E of D. pseudoobscura (Figure 3, D and E). However, this discrepancy may be misleading, because the original scaffold (Contig3286_Contig7811B) containing Dbp80 and RpL15 is a manual subdivision of a larger scaffold (Contig815_Contig5737) obtained from the automated assembly (RICHARDS et al. 2005). Interestingly, the adjacent subdivided scaffold (Contig4971_Contig7717A) contains both CG8327 and Crc, which are near RpL15 in several other species (Figure 5). Therefore, the Dbp80 and RpL15 genes in D. pseudoobscura reside in a moderately repetitive environment in the middle of element E, and this has been confirmed in the current reassembly of the genome (S. SCHAEFFER, personal communication). Furthermore, this region contains a mosaic of genes from elements D and E of D. melanogaster.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Dbp80 and RpL15 show contrasting evolutionary dynamics with respect to gene structure:
This study suggests that the surrounding genomic environment does affect gene structure, although the effect of gene expansion in heterochromatin is subject to constraints on gene function. Dbp80 is a small gene with a single intron in the subgenus Drosophila, but is expanded with many introns in the subgenus Sophophora, while RpL15 maintains a conserved gene structure among all the examined species (Figure 4). There is some evidence suggesting that the expanded version of Dbp80 is ancestral, on the basis of the structure in An. gambiae, which, although separated from Drosophila by a quarter of a billion years, shares the position of all five of its introns with species from the Sophophoran subgenus. A more closely related outgroup, such as a representative of the subfamily Steganinae (e.g., Gitona bivisualiz, REMSEN and O'GRADY 2002), would provide greater resolution of the ancestral gene structure for Dbp80 for members of the genus Drosophila.
Expansion in the sizes of the introns in Dbp80 correlates with the surrounding content of repetitive sequences, yet RpL15 is impervious to this effect (see Figure 4 and Table 1). This difference is likely due to the contrasting biological functions that these genes encode and the fitness consequences that result from altering their structures. Dbp80 is a protein that belongs to a very large and diverse family of RNA helicases that exhibits great flexibility in gene structure (BOUDET et al. 2001). Additionally, many RNA helicases have overlapping, possibly redundant, functions in RNA metabolism (DE LA CRUZ et al. 1999), and this gene is not essential in D. melanogaster (GATFIELD et al. 2001; SCHULZE et al. 2005) or in any other organism in which it has been tested to date (KAMATH et al. 2003; ROLLENHAGEN et al. 2004). Therefore, purifying selection on Dbp80 apparently allows for variety in its gene structure. RpL15, on the other hand, encodes an essential housekeeping function, required at high levels of expression at all times. Such genes have been shown to experience a strong selection pressure to remain small and easily processed (CASTILLO-DAVIS et al. 2002), which is consistent with the highly conserved structure for this gene among the examined species regardless of the surrounding environment.
Model for interchromosomal relocation for Dbp80 and RpL15 in Drosophila:
Movement of genes between chromosomal elements of Drosophila is considered unusual, but has been reported for specific instances of genes belonging to multi-gene families (RANZ et al. 2003) and events of retrotransposition and pericentric inversion (LEMEUNIER and ASHBURNER 1976; BETRAN et al. 2002; RICHARDS et al. 2005). A special case of interelement gene movement is also exemplified by the transfer of genes from the ancestral Y chromosome into the autosomal genome of D. pseudoobscura (CARVALHO and CLARK 2005). Despite these examples, interelement movement is not considered the norm. The inference of repeated interelement movements involving Dbp80 and RpL15 is therefore quite striking. Both are intact, single-copy genes, and their movements appear to be confined to elements D and E, which compose the metacentric chromosome 3 of D. melanogaster.
At least two instances of interelement movement are required to account for the different positions of Dbp80 and RpL15 in the three focal species (D. virilis, D. pseudoobscura, and D. melanogaster), and the other species provide a context for inferring the pattern of movement (Figure 6). A conserved set of genes flanks RpL15 in species representing both subgenera, and Dbp80 is a component of this association in most members of the subgenus Sophophora (Figure 5). On the other hand, a unique set of genes is present flanking Dbp80 in the subgenus Drosophila (D. mojavensis and D. virilis), where Dbp80 is not adjacent to RpL15, thus implicating an initial interelement movement of Dbp80 during the divergence of the two subgenera. The genes reside on separate elements in An. gambiae, but a 1:1 relationship does not exist between the chromosomal arms of An. gambiae and D. melanogaster (ZDOBNOV et al. 2002). The direction of the interelement movement in the genus Drosophila therefore cannot be resolved without an appropriate (more closely related) outgroup to infer the ancestral arrangement of Dbp80 and RpL15.
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A second interelement movement is also necessary to account for the repositioning of the region, including both RpL15 and Dbp80 between element E (as in D. pseudoobscura) and element D (as in D. melanogaster) within the subgenus Sophophora. The position of RpL15 in the centromeric heterochromatin of D. melanogaster and D. virilis indicates that this is the ancestral location. A paracentric inversion is the simplest explanation for the relocation of both genes into the middle of element E in D. pseudoobscura, thus suggesting a possible route in the formation of intercalary heterochromatin. In D. melanogaster, a centromeric fusion between elements D and E, followed by a pericentric inversion, would cause the relocation of Dbp80 and RpL15 from element E to D in D. melanogaster. There is evidence for this course of events in D. melanogaster. Genes from elements D and E of D. melanogaster are present flanking RpL15 in the other species, which retrospectively reflects the redistribution of genes between these elements. This creates an impression of mosaicism in genomic regions associated with RpL15 across these lineages, but, in reality, the only true mosaic is D. melanogaster. In this species, genes formerly present on D and E have become redistributed as a result of a pericentric inversion having occurred after the fusion of these elements
The repetitive sequence context predominantly associated with both genes may also have facilitated the inferred movements: ectopic recombination between repetitive elements is not an unusual event and indeed has been exploited by geneticists for experimental purposes (GRAY et al. 1996). The Ulysses element that resides downstream of Dbp80 in D. virilis might have enabled just such a natural event involving a segment of euchromatin. The presence of this element is polymorphic (for instance, it is not present at this location in the strain sequenced by Agencourt) and may coincide in this position with an inversion breakpoint in the chromosome that can be identified in different strains of D. virilis (EVGEN'EV et al. 2000).
Chromosomal elements are dynamic genomic structures—caution advised for annotation:
While gene content of the euchromatic chromosomal arms for the most part appears to be conserved among Drosophila species, our analysis of the evolutionary history of Dbp80 and RpL15 demonstrates that a different picture may arise for genes contained within regions of centromeric heterochromatin. The static view of the chromosomal elements of Drosophila clearly must be applied cautiously with respect to genes residing in centromeric regions, especially in comparisons with the extensively annotated D. melanogaster genome, which has undergone two centric fusions involving four (of a total of six) chromosomal elements. This study implicates relocation of genes on chromosome 3 by a pericentric inversion, and the analysis of the genome sequence of D. pseudoobscura indicates the same has occurred for chromosome 2 (RICHARDS et al. 2005). Because of the dynamic repositioning of the two genes studied here, a wider examination of additional heterochromatic loci is needed to elucidate the constraints on these regions of the genome. This study also demonstrates that a full understanding of the genomic location of these genes requires experimental confirmation by in situ hybridization, as the standard assumptions (genes confined to Muller's elements) developed for conserved euchromatic genes do not apply to genes that have relocated into and out of heterochromatin.
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1 Present address: Department of Biology, Western Washington University, Bellingham, WA 98225-9160. 
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