The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila.
Citation: Haney RA, Feder ME (2009) Contrasting Patterns of Transposable Element Insertions in Drosophila Heat-Shock Promoters. PLoS ONE 4(12): e8486. https://doi.org/10.1371/journal.pone.0008486
Editor: Matthew W. Hahn, Indiana University, United States of America
Received: May 5, 2009; Accepted: November 22, 2009; Published: December 29, 2009
Copyright: © 2009 Haney, Feder. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by NSF IOB06-41278 (www.nsf.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The massive accumulation of comparative genomic data due to recent large-scale sequencing projects  can help test whether a putatively general pattern in a species is unique or is recurrent in evolution. Here we undertake such a test upon the remarkable abundance of a DNA transposon, the P element, in the proximal promoter regions of heat-shock genes in natural populations of the model organism Drosophila melanogaster . Previously we have ascribed this situation to the intersection of three processes. First, the promoters of heat-shock genes are specialized for rapid and massive transcription when induced by heat and other stresses. Specializations include a lack of nucleosomes and constitutively decondensed chromatin . Second, some mobile genetic elements, such as the D. melanogaster P transposable element (TE), have insertion site preferences related to the physical structure of candidate insertion sites rather than to a specific nucleotide sequence in the host DNA . Importantly, when the insertion site preference corresponds to a physical feature of a class of genes, as in this case, the entire class should be preferentially targeted by the transposable element. In heat-shock promoters, the constitutive decondensation of the chromatin and nucleosome-free regions correspond to the preference of P elements for such DNA . Finally, once inserted in the germ line, such TEs may persist due to genetic drift or positive selection – or decay and be lost due to unconstrained degeneration and/or negative selection.
In the specific case of P elements in heat-shock promoter regions, the ultimate products of these genes are proteins that are either beneficial or deleterious according to cellular context . Specifically, heat-shock proteins act as beneficial molecular chaperones in the stress response, but at high levels are deleterious in the absence of stress. Inasmuch as insertional mutagenesis of P elements typically reduces gene expression, both in general and for heat-shock promoter insertions, conditions favoring low levels of heat-shock proteins ought to select for the preservation of P element insertions in these genes. Indeed, the population frequencies of many of the P element bearing heat-shock alleles is consistent with positive selection , .
In principle, this intersection of processes ought to be general and demonstrable outside the case of D. melanogaster P elements whenever the underlying conditions obtain. Many aspects, including abundant and active transposable elements, “physical” insertion site preferences, and classes of genes with distinctive promoter architectures, are general. The insertional mutagenesis of the heat-shock genes in D. melanogaster, moreover, is a function of the physical disruption of the promoters by inserted DNA, as both non-P elements and random DNA of equivalent length have identical impacts on gene expression . Indeed, the only oddities in D. melanogaster are that the P element has invaded the genome only recently, and that this species can be distinctively prone to heat stress due to its life cycle . These oddities aside, an excess abundance of TEs in heat-shock promoters ought to be detectable across other species if the intersection of processes we hypothesize is general.
The recent sequencing of 11 additional genomes of Drosophila species provides a remarkable opportunity to test this expectation. In this study, we extend the work of  to enumerate TE insertions specifically into promoter regions across species of Drosophila by taking advantage of 12 species genome data together with experimental data on promoter location in D. melanogaster. First, we test whether the frequency of TE insertions varies between heat-shock gene promoters and promoters of genes that should have similar or divergent promoter architecture across Drosophila species. Second, we examine whether TE insertions in promoters are strongly biased towards P elements and occur only in P- bearing species, as in D. melanogaster . We also investigate characteristics of TE insertions that might be informative as to the forces involved in molding their distribution within promoter regions. We expect that differences in characteristics of TEs, such as length or distance from the transcription start site (TSS), or sequence conservation among shared elements, may be due to differences in the strength and/or direction of selection on TEs between genesets across species, and between heat-shock genes in D. melanogaster natural populations and those in the 12 species genomes.
Analysis and Distribution of TE Insertions
Hsp70 is uniformly a multi-copy gene in the genus Drosophila, with 50 proximal promoter regions (arbitrarily defined as 1000 bp immediately upstream of the TSS) in total computationally identified for the 12 species (Figure 1). In some cases these promoters overlap, thus reducing the total promoter sequence into which TEs could insert to the equivalent of 46 promoters. BLAST analysis of these regions identifies 6 insertions into a single copy within a species, 12 insertions common to >1 copy within a species, and 1 insertion common to 4 paralogs of two species (D. simulans and D. sechellia). In each common insertion, a given TE is shared and at approximately the same distance from the TSS. By parsimony, the common insertions represent insertion events that antedate gene duplication/conversion and species divergence, respectively.
Each numbered oval represents transposable element insertions. The position of the oval corresponds to the species, species pair, or species group in which the insertion was detected. Ovals at tips are insertions restricted to single species, while ovals at nodes are found in all species emanating from that node. The horizontal scale indicates the time at which the species are presumed to have diverged  but is otherwise not pertinent to the time of the TE insertions. Double slashes represent a compression of the time scale at the base of the tree. Numbers following species names in parentheses indicate the number of copies of Hsp70 per species.
Multi-copy genes such as Hsp70 present a distinctive target to transposable element insertion and therefore are not readily comparable to single-copy genes. As stated, single-copy genes whose promoters were to be compared were chosen a priori according to characteristics hypothesized to affect the frequency of TE insertions. Of the chosen genes, 36 conserved promoters were recognizable in all 12 species, 45 in only 8 species, and 36 in only 5 species, with equal numbers in each of the 3 sets of genes to be compared (Figure 2). This analysis also revealed that some genes thought to be single-copy have paralogs (based on coding sequence) in some species, of which 8 paralogs in 7 genes had a sufficiently conserved TSS to be included in the analysis (Table S1). BLAST analysis of the 980 individual promoter regions from the 117 distinct TSS of these genes recovered 71 TE insertions (Table S2). 33, 27, and 11 insertions were in the promoters conserved in the 12, 8, and 5 species, respectively. Of these TEs, 49 are present in only a single species (Figure 2). In every other case, the insertion site itself and a major portion of the inserted TE are similar in all species in which the TE appears (see Figure 3 for examples). Where the distance between the insertion site and the TSS is dissimilar in two species putatively sharing a TE, the dissimilarity often corresponds closely to the size of one or more indels. By parsimony, these TEs in >1 species thus represent insertion events that antedate species divergence rather than independent insertion events into multiple species. Only the most recently diverged species (D. melanogaster subgroup species/D. persimilis and D. pseudoobscura) shared insertions. Each of the more widely shared elements appears degenerate, varying extensively among species in both sequence and length (Table S2; Figure 3).
Species labels are omitted due to space considerations, but are as in Figure 1. Each numbered oval represents transposable element insertions. The position of the oval corresponds to the species, species pair, or species group in which the insertion was detected. Ovals at tips are insertions restricted to single species, while ovals at nodes are found in all species emanating from that node. Insertions are represented at each level of species sampling, and all genes screened at a given level were for the same number of species. The horizontal scale indicates the time at which the species are presumed to have diverged  but is otherwise not pertinent to the time of the TE insertions. Double slashes represent a compression of the time scale at the base of the tree.
Sequence conservation of TE and TSS flanking regions is indicated as % pairwise divergence based on Tamura-Nei distance calculated over a 100bp interval of CLUSTAL alignment. Pairwise sequence divergence of TE is based on three equal sized sections of CLUSTAL alignment for CG11143 and CG2127, where elements are of similar size. Solid lines indicate larger indels and dashed lines connect homologous sequence blocks. In CG2916 and CG2238, TE conservation is calculated over conserved blocks corresponding to the smallest element size amongst the three species. The synteny among elements and flanking sequence suggests that these elements are the product of singular insertion events prior to the divergence of the daughter species in which they are currently detectable.
Our goal is to compare numbers of TEs inserted in 3 sets of proximal promoter regions (hereafter “promoter sets”) presenting insertion targets of equal size but hypothesized to differ in susceptibility to gain and/or loss of TEs. Although the 3 promoter sets offer equal numbers of promoters and we have screened 1000 bp upstream of the TSSs in every case, their overall target sizes differ in several ways: (a) <1000 bp may separate TSSs, (b) proximal promoter sequences upstream of two TSSs may overlap, (c) inserted TEs occupy part of the 1000 bp screened, and (d) a 1000 bp window containing a TE is inherited. In each case, equating the target size to 1000 bp may overestimate it. We have corrected target size for (a) in every case. We compare the 3 promoter sets (and Hsp70) by estimating aggregate target size in several ways: conservatively [correcting for (b), (c), and (d)], semi-conservatively [correcting for (b) and (c) only], and liberally [correcting for (b) only].
The pattern for natural populations of D. melanogaster , i.e., vastly more promoters with TEs in that study's promoter sets I (n = 154) than in its promoter sets II (n = 7) and III (n = 2), was not evident in the similarly-defined but non-identical promoter sets of the present study regardless of how the aggregate target size is corrected. Promoter set I of the present study comprises 328–337 kB of promoter sequence depending on whether the correction is conservative, semi-conservative, or liberal (Table S3). This sequence harbors 28 insertions, or 83–85 per mB of target. Promoter set II comprises 324–340 kB of promoter sequence, and harbors 24 insertions or 70–74 per mB. Promoter set III comprises 330–343 kB of promoter sequence, and harbors 19 insertions or 55–57 per mB. These frequencies do not differ significantly (3-way G-test, p = 0.40, 0.37, and 0.39 depending on whether the target size correction is conservative, semi-conservative, or liberal, respectively.) By contrast, the promoters of Hsp70 comprise 40–48 kB of promoter sequence and harbor 19 insertions, or 395–476 per mB. As noted, not every promoter was recognizable in all 12 species. When the analysis is repeated for promoters recognizable in only 5 species (the D. melanogaster subgroup species) or only 8 species (all but D. willistoni, D. mojavensis, D. virilis and D. grimshawi), the result is essentially the same except that statistical significance is less (Table S3).
The pattern for natural populations of D. melanogaster  may differ from that in the present study for a second reason: the former study summed insertions into multiple alleles (minimally 1 per each of 48 populations), whereas the present study examines far fewer alleles (minimally 1 per each of 12 species). Accordingly, we have compared the distributions of TE insertions per allele for the 3 promoter sets of ; these differ (Kruskal-Wallis test, p<0.0001). Excluding D. melanogaster (because the sequenced strain was selected for the absence of one TE family) and comparing the 11 remaining sequenced species in the same way but for the promoter sets of the present study, we find that the distributions do not differ (p = 0.52).
Alternatively, the distributions of TE insertions in the genesets of the present study can be compared in phylogenetic context. The phylogenetic trees in Fig. 2 present 17 tips plus nodes (i.e., branchpoints of two clades) at the 12-species level, 13 at the 8-species level, and 8 at the 5-species level. Older nodes are excluded because no TE insertions are detectable at them, presumably because of degeneration over time. For all possible pairwise comparisons of the frequency distributions of TEs at tips plus nodes, the promoter sets do not differ (Kolmogorov-Smirnov two-sample test, 0.31>p>0.06), even without a Bonferroni correction.
Position of TE Insertions in Promoter Regions
The average distance between TE insertions and the TSS did not vary significantly among promoter sets (mean Promoter set I: 479 bp; Promoter set II: 515 bp; Promoter set III: 451 bp; Mann-Whitney U-test, I vs. II: p = 0.704, I vs. III: p = 0.624, II vs. III: p = 0.337). Insertions into promoters of Hsp70 are further from the TSS than are insertions into the three single-copy promoter sets (mean Hsp70: 658 bp), although no pairwise comparison is significant after Bonferroni correction (Mann-Whitney U-test, Hsp70 vs. I: p = 0.0016; Hsp70 vs. II: p = 0.016; Hsp70 vs. III: p = 0.0045). These patterns contrast to those for D. melanogaster natural populations . We calculated distances to the TSS for insertions listed in  and compared them to distances calculated in the present study. The distances differ most markedly for Hsp70 (Mann-Whitney U-test, p<0.0001), into which most TEs in D. melanogaster natural populations inserted within 400 bp of the TSS . No TEs in the 12 species' genomes inserted in this interval. For single copy heat-shock genes, the distances are more similar in the two studies (Figure 4), but in D. melanogaster natural populations are nearer the TSS ( p<0.0001).
Data for D. melanogaster natural populations was taken from . Due to shared insertions in all genesets, and gene duplication and the spatial arrangement of Hsp70 copies, some individual TE insertions occur in more than one promoter. Distance to TSS of shared insertions is shown for all promoters in which the insertion is found.
For the three single-copy promoter sets in the present study, TEs are inserted randomly across the entire region from the TSS to 1 kb upstream, whereas insertions into both D. melanogaster natural population heat-shock promoters and Hsp70 promoters of the 12 species are not (Figure 4). The latter sets deviate from a Poisson distribution (12 species hsp70: chi-square, p<0.0001, Hsp70 in D. melanogaster natural populations: p<0.0001; Set I promoters in D. melanogaster natural populations, p<0.0001), whereas those inserting into the genesets for the 12 species do not (Promoter set I: p = 0.743, Promoter set II: p = 0.279, Promoter set III: p = 0.272).
Extent of TE Sequence in Proximal Promoters with TE Insertions
The functional consequences of TE insertions may depend on their size. Lengths of TE sequence were similar in the three promoter sets (I: 158 bp mean, II: 175 bp, III: 97 bp). Length estimates intentionally excluded portions of TEs extending outside the 1000 bp window. Because promoter sets did not differ in TE insertion position, however, any bias due to this exclusion should apply equally to all promoter sets. Insertion sizes did not differ among promoter sets, even without Bonferroni correction (Mann-Whitney U-test, I vs. II: p = 0.142; I vs. III:p = 0.810; II vs. III: p = 0.066). The mean length of insertions in Hsp70 promoters (177 bp) only differed significantly from Promoter set III after Bonferroni correction (Mann-Whitney U-test, I v. Hsp70: p = 0.014, II v Hsp70: p = 0.271, III v. Hsp70: p = 0.0003).
Sequence conservation of TE insertions can elucidate the evolutionary forces acting on these elements. Few insertions recovered in this study are full-length, but instead are fragments. We compared these fragments with their best-hit consensus or species-specific centroid sequence . Although the average percent of consensus TE length varies among the three single copy promoter sets and Hsp70 promoters (average % of consensus: Promoter set I = 17.8%, II = 24.9%, III = 11.8%, Hsp70 = 19.5%), no pairwise comparisons among genesets on this metric are significant after Bonferroni correction (Mann-Whitney U-test, I vs. II: p = 0.089, I vs. III: p = 0.818, II vs. III: p = 0.013, I vs. Hsp70: p = 0.093, II vs. Hsp70: p = 0.465, III vs. Hsp70: p = 0.004).
The majority of TEs detected in this study are INE-1 type , which are small, non-autonomous miniature inverted-repeat elements (MITEs), totaling 26 of 71 (37%) insertions in the three single-copy promoter sets, and 11 of 19 insertions in the Hsp70 promoters (Table S2). Of the remainder, 25 are novel repetitive elements , 5 are LTR retrotransposons, 2 are Penelope retrotransposons, and 16 are DNA transposons.
First, as a caveat, we re-emphasize that the promoter sets chosen in the previous study of natural populations of D. melanogaster  and the present study of 12 sequenced genomes are similar but not identical. The dissimilarity arises because orthologues of the promoters examined in the prior work are not unambiguously recognizable in enough of the 12 species genomes for a powerful comparison of inserted TEs. The rationale for the selection of included promoters is in Table S1.
The whole-genome sequencing of Drosophila melanogaster  provided a first glimpse at the “transposome” of a complex eukaryote . First glimpses may not illuminate general patterns, however, because they reflect the distinctive features of species and strains chosen for sequencing. Thus, our initial finding of numerous TEs in proximal promoter regions of heat-shock genes of natural populations of D. melanogaster  was potentially idiosyncratic because it largely involved a TE (the P element) absent in both the strain chosen for sequencing and in some other Drosophila species , , . Is this finding unique to natural populations of D. melanogaster, or more general? Whole-genome sequences now available may answer this question.
The genome sequences for the 12 Drosophila species still yield the ascertainment bias inherent in the choice of strains for sequencing - a bias soon to diminish as resequencing projects progress . Nonetheless, they clearly indicate that the preponderance of P elements in proximal promoter regions of heat-shock genes is not universal in the genus Drosophila. First, in contrast to the natural populations of D. melanogaster screened by , the majority of the insertions recognized in the present study are MITEs. MITEs also abound in plants, in which they frequently insert in gene-rich regions and may figure prominently in the evolution of gene expression –. Indeed, these parallels between plants and Drosophila suggest that MITEs may play similar roles in diverse eukaryotes.
TE insertions into promoters in natural D. melanogaster populations differ from the aggregate pattern from the 12 single species genomes in a second key way. In the former, insertions are more numerous in promoters of single-copy heat-shock genes, where they cluster near the TSS, than in equally-sized control promoter sets. By contrast, for the 12 genomes as a group, insertions into promoters of single-copy heat-shock genes are not more numerous than into equally-sized control promoter sets, and are randomly distributed throughout the 1000 bp window screened.
The first of several explanations for the lack of concordance in results between D. melanogaster natural populations and the 12 species whose genomes were sequenced is that the Universal Fast Walking (UFW) technique of the former work failed to detect TEs that whole-genome sequencing revealed. Because UFW incorporates PCR, which can favor shorter amplicons, it is theoretically possible that UFW might be biased towards discovering only TEs close to the TSS. We disfavor this possibility for several reasons: (1) An actual bias has not been documented to date for UFW (K. Myrick and W. Gelbart, pers. com.), nor is one evident in other published uses of the technique. (2) Walser et al.  screened a full kB upstream of the TSS and reported more (i.e., not fewer) TEs than revealed through whole-genome sequencing.
A second explanation for the comparative rarity of TEs in Promoter set I of the present study is that extensive rescreening in the former (n = 48 populations) reveals rare TE insertion alleles not evident in the smaller sample from single genomic sequences. As the individual sequenced genomes sample only a limited proportion of the variation within-species, some mutations, especially low-frequency mutations such as some TE insertions, are unlikely to be discovered. We attempted to address this possibility in two ways. First, we reanalyzed the data of  by performing 20 random draws of 12 populations each. In every case, TE insertions into Promoter set I significantly outnumbered TE insertions into the other single-copy promoter sets. Presumably, if a similar pattern occurred in the sequenced species' genomes, a sample size of 12 would be sufficient to detect it. Second, we directly compared the distributions of TE insertions per allele for the 3 promoter sets of  and per genome for the 3 promoter sets of the 11 sequenced species. These comparisons recapitulated outcomes of comparisons in which TEs are simply aggregated for each Promoter set (see RESULTS).
A third explanation is that D. melanogaster is a conspicuous outlier in its number and dynamics of TEs in general. Interspecific comparisons of overall repetitive sequence content and TE composition ,  and of INE-1 element dynamics  in the 12 genomes do not support this explanation. The plurality of MITEs in the present study is consistent with their high copy number in all Drosophila genomes . MITEs are as numerous in the 3 promoter sets (25–26 MITEs per Mb using conservative and liberal target size corrections for the 3 promoter sets) as in average noncoding sequence of the 12 sequenced genomes (2.7–60.0 per Mb of noncoding sequence , ). Using as a basis the number of annotated repeat insertions in the 12 species genomes (Caspi, unpublished) obtained through a comparative alignment method , we estimated the total number of TE insertions per Mb in the sequenced genomes. These estimates indicate that TEs average 129–330 per Mb of average non-coding sequence vs. 69–72 for the 3 single-copy promoter sets (depending on whether the liberal or conservative correction for target size is applied) and 395–476 for the Hsp70 promoter set. Both comparisons are inexact, however, due to methodological differences and because only the present study differentiates between promoter vs. non-promoter noncoding sequence. Vertical acquisition of TE insertions (i.e., inheritance of a TE acquired prior to species divergence) in D. melanogaster, which the present study implicates, is both evident in other Drosophila species and is reported elsewhere . Finally, the promoter sets chosen for study may reflect some inadvertent bias unrelated to the main hypothesis, either in general or in D. melanogaster. For example, if heat-shock genes are more numerous due to gene duplication in D. melanogaster, the target for and/or tolerance of TE insertion would be larger in D. melanogaster populations than in other species. With respect to this example, the total number of orthologues for the D. melanogaster single-copy heat-shock promoters is no less in the other 11 species than in D. melanogaster (Table S4). An additional peculiarity is that the proportions of TEs other than MITES in the 3 genesets are not consistent with that found in the genomes as a whole , although sample sizes are small. LTR retrotransposons are underrepresented in the present study, and novel elements are overrepresented.
As the Introduction reviews in detail, the natural populations of D. melanogaster and some of the 12 species differ in ways relevant to the main hypothesis, including:
- activity of a TE (the P element) with a distinctive insertional mechanism and corresponding insertion site preference. This explanation receives some (but incomplete) support because P elements have not been detected in many of the 12 species , . P elements occur in D. willistoni, however, which appears similar to the other 11 species in overall TE distribution in this study. D. willistoni also has the second highest level of repeat coverage of the sequenced genomes . Further screening of single copy heat-shock genes in multiple D. willistoni populations has revealed no P element insertions (unpublished data). The D. willistoni situation shows that the presence of P elements, even within a genome where TEs appear to have been highly active, may not in itself be sufficient to yield the TE distribution characteristic of the D. melanogaster natural populations.
- recency of the horizontal transmission of P elements to D. melanogaster, which could contribute to the high number of insertions in D. melanogaster heat-shock promoters. Unless some mechanism (e.g., positive selection, duplication, gene conversion) preserves them, TEs may be subject to degeneration once inserted and immobilized, and may become unrecognizable.  reported substantial degeneration of P elements in the natural populations of D. melanogaster. In our sample, few insertions are complete TEs and most retain on average ∼20% of their canonical sequence. For TEs shared among species, times of lineage divergence place upper bounds on the insertion times: no insertion older than 12 MYA is recognizable. Recent invasions will be more recognizable, all else equal. The P element is believed to have invaded D. melanogaster within the last 100 years . The recency of P elements' invasion thus may make their presence conspicuous in D. melanogaster populations. However, recent activity alone cannot account for the restriction of the pattern to D. melanogaster. Other TEs, such as MITEs, have been recently active in many of the species , and are commonly found in promoter regions in this study. Yet, they are not in excess in single copy heat-shock promoters. In addition, recent activity of the P element has also been documented in D. willistoni , yet no P elements were found in any D. willistoni promoter region.
- habits and habitats that predispose D. melanogaster to heat shock in nature, accounting for distinctive patterns of heat-shock gene expression. D. melanogaster is cosmopolitan, encountering diverse macroclimates, and its non-adult stages occur in microhabitats (necrotic fruit) that are prone to thermal stress . These features may select for retention of TE insertions into heat-shock genes, depending on the TEs' impact on gene expression. A weakness of this candidate explanation, however, is that others of the 12 species (e.g., D. simulans, D. virilis) share many of these same attributes .
Thus, none of these possible distinctions in isolation provides a definitive explanation for the restriction of the excess of TE insertions in single-copy heat-shock promoter regions to natural populations of D. melanogaster. Possibly the particular combination of these characteristics produces the pattern. Nonetheless, a robust explanation is currently lacking.
The multi-copy promoter regions of Hsp70 are an exception to the findings for single copy heat-shock promoter regions. First, in both the natural populations of D. melanogaster and in the aggregate sequenced genomes of the 12 species, Hsp70 promoters harbor more TE insertions per promoter than any of the single-copy promoter sets. Second, the positions of natural and experimental TE insertions into Hsp70 promoters in the natural populations of D. melanogaster are nearly exclusive of the positions of insertions into Hsp70 in the sequenced genomes of the 12 species (Figure 4). Third, for the D. melanogaster natural populations, insertions into Hsp70 promoter regions include TEs other than P elements , .
In principle, Hsp70-specific hotspots could account for the numerous TE insertions into this gene's promoters. Indeed, the Hsp70 promoter presents a confluence of DNAse hypersensitivity, decondensed chromatin, nucleosome-free regions, and flanking GAGA elements [and, for P elements, preferred insertion sequence] , . Although other heat-shock promoters have not been investigated as thoroughly, they share these features , , which are thus not specific to Hsp70. Relative to single-copy heat-shock genes Hsp70 is more massively expressed, is perhaps more sensitive to induction, and obviously is multicopy. Insertion of TEs into promoters of single-copy heat-shock genes can severely reduce or eliminate expression of the affected gene, whereas insertion into a single Hsp70 promoter can leave many other copies unaffected, leading to reduced negative selection against insertions. Additionally, insertion of a TE into a single Hsp70 promoter creates allelic variation that is apparently responsive to positive selection , .
Why the positions of these insertions into Hsp70 promoters (Fig. 3) differ so dramatically between the D. melanogaster natural populations and the 12 species' sequenced genomes is inexplicable at present. As noted above, the prevalence of TEs near the TSS in D. melanogaster may reflect a bias in the UFW technique. As also noted above, such a bias is yet to be documented. Additionally, as the sequenced genomes are not prone to this bias yet contain no insertions in Hsp70 promoters within approximately 400 bp of the TSS, the insertion patterns still differ between Hsp70 promoters in the natural populations and in the sequenced genomes even if a bias exists.
The Hsp70 proximal promoter sequences, and presumably architectures, are grossly similar throughout the 12 species (unpublished data). At least in D. melanogaster, negative impact on Hsp70 expression declines with distance of the TE insertion from the TSS . By implication, therefore, large insertion-mediated reductions in a single Hsp70 copy are well-tolerated in natural populations of D. melanogaster, but not in strains/species whose genomes have been sequenced. No clearcut biological explanation for this implication presents itself.
In summary, the whole-genome sequencing of 12 species of Drosophila has elucidated both functional and evolutionary patterns of fundamental importance. In the present study, the genomic sequences clarify that both Drosophila melanogaster as a species and Hsp70 as a gene are distinctive in terms of their interaction with transposable elements. Nonetheless, the biological underpinnings of this distinctiveness remain elusive.
Materials and Methods
Promoter sets for analysis follow those of . Promoter set I includes heat-shock genes, in whose promoters P elements are abundant in D. melanogaster. Promoter set II excludes heat-shock genes but includes genes sharing one or more features with heat-shock genes, such as constitutively decondensed chromatin, lack of nucleosomes and presence of DNase hypersensitive sites in the proximal promoter region , regulation via polymerase II pausing, and high transcription in germline cells. Promoter sets I and II are expected to have similarly high exposure to transposable element insertion. Promoter set III members, by contrast, exhibit temporally limited transcription with low germline transcription, characteristics that should minimize transposon insertions. As will be described, the unambiguous identification of orthologs and/or their corresponding transcription start sites for all D. melanogaster promoters used in  was impossible. Table S1 indicates genes from  in which some or no orthologous transcription start sites could be identified. To maintain the power of the comparison, we added genes to each set until we achieved the following arbitrarily-chosen sample size: 12 in each set identifiable in all 12 species, 15 identifiable in only 8 species (D. melanogaster subgroup +D. ananassae, D. pseudoobscura and D. persimilis), and 12 identifiable in only the 5 D. melanogaster subgroup species. These promoters were included before scrutiny of their TEs.
We added to the original promoter sets through the incorporation of genes not included in , but with characteristics warranting inclusion –. Details of all characteristics used to classify genes are listed in Table S1, and column K shows all characteristics pertaining to a given gene.
Evaluation of Transcription Start Sites (TSS) in D. melanogaster
The search for TEs upstream of the TSS is contingent upon the unambiguous identification and localization of the TSS, which we describe here. All TSS from a total of 107 genes were evaluated first in D. melanogaster. The 107 genes (excluding Hsp70: see below) across the three genesets included 38 genes with multiple transcripts in D. melanogaster, for a total of 148 transcripts in the 107 genes. Twenty-seven of these multiple-transcript genes have transcripts that differ in putative transcription start sites (TSS) and thus presumably also differ in their associated promoter regions (Table S1). If a given gene contained more than one putative TSS and these start sites were separated by 500 bp or more (∼50% of data analyzed per gene per species), we retained both TSS and associated upstream promoter region for further analysis, for a total of 117 TSS/promoter regions considered (39 per geneset).
To determine the position of TSS for genes in D. melanogaster, we compared annotated transcripts and 5′ UTR to existing experimental data. First, 42 genes have an experimentally determined transcription start site (TSS) included in the Drosophila Core Promoter Database , or the Eukaryotic Promoter Database . An additional 60 TSS without DCPD or EPD entries had associated full-length or full-insert cDNA ,  catalogued in Flybase. Although no attempt was made to ensure that the cDNA represented the entire transcript, these cDNA provide some experimental support. The remaining 15 TSS were defined on the basis of an annotated UTR only.
From Flybase we extracted the coding region and sequence upstream from the start codon for each gene in D. melanogaster. Genomic sequence, experimentally determined promoter sequences with defined TSS, full-insert and full-length cDNA from Flybase, and annotated 5′ UTR or transcripts were aligned as available for each gene with CLUSTAL in Bioedit 184.108.40.206  and manually adjusted when necessary.
For the 42 experimentally determined TSS from the DCPD and EPD databases, only three agreed completely with the annotated 5′ UTR on TSS location. Only three full-insert or full-length cDNA catalogued in Flybase had the same TSS as the promoter database entry. However, in general the deviation from the experimentally confirmed promoter database TSS was small, averaging 20.91 bp (n = 37; SD = 32.6) for full-insert or full-length cDNA and 44.6 bp (n = 41; SD = 64.1) for annotated UTR. When the EPD/DCPD TSS was at odds with the Flybase annotated 5′ UTR entry or catalogued cDNA, we used the EPD/DCPD TSS.
Of the 60 additional TSS that were supported by a cDNA in Flybase, for 43 the 5′ end coincided with the annotated TSS position in Flybase. For those that did not coincide the average deviation was only 31.35 bp (N = 98; SD = 69.5). Whenever a full-length or full-insert cDNA was available, we based the TSS on cDNA. As will be seen, however, minor deviations in placement of the TSS would have little impact on the results.
Hsp70 TSS Determination
Coding and upstream sequence were obtained at Flybase for the six copies of Hsp70 in the D. melanogaster sequenced strain. A single EPD entry for Hsp70 was aligned to the sequences along with 5 full-insert or full-length cDNA. The five full-insert and full-length cDNA vary in putative TSS. First, of the two full-insert cDNA for Hsp70Bc, one (BT011541) is substantially longer than the other, and is highly divergent at the 5′ end, both from the other cDNA and from the genomic sequence. This sequence was excluded from the analysis. Of the remaining four cDNA, the TSS is identical in three, and 7 bp upstream in the fourth. Five of the six annotated UTRs or mRNAs indicate TSSs occurring within a narrow window of 16 bp. By contrast, the UTR annotated for Hsp70Aa is far longer, terminating 218 bp 5′ to the known TATA-box promoter , , and is hence assumed to be misannotated. All other cDNA and annotated UTR have TSS consistent with the position of the conserved TATA-box.
Orthology Determination and Sequence Extraction
To obtain orthologues of D. melanogaster genes from Drosophila species genomes, BLASTn and tBLASTn were performed at the Flybase website  with D. melanogaster coding sequences, gene region sequences, or protein sequences. BLAST was performed to D. melanogaster release 5.10, D. pseudoobscura release 2.3, D. virilis release 1.2, and release 1.3 for all other species. Of the 107 genes across three genesets examined in this study, excluding Hsp70 (which is known to be multicopy ), 70 have 1∶1 orthologues in all other sequenced genome species of Drosophila .
Of the remaining genes, we carefully screened using BLAST, Gbrowse orthology calls and reciprocal BLAST to the annotated D. melanogaster genome to determine the number of copies in each species. Only genes with predicted coding sequences in all species considered were included. Fourteen genes appeared to have more than one copy in at least one additional species at the phylogenetic depth at which they were analyzed (Table S1). We verified that duplicate copies had complete open reading frames by referring to predicted coding sequences in Flybase or Genscan . If a full-length or near full-length coding sequence was not present, the copy was discarded as a pseudogene; otherwise it was retained for further analysis. Twenty duplicates in the fourteen genes met this criterion and were retained for assessment of TSS conservation (see below).
In each species, we initially extracted 3000 bp upstream of each identified gene copy. Alignments of gene regions were performed with CLUSTAL in Bioedit  and more upstream sequence was acquired when necessary to account for the presence of length variation among species, particularly within 5′ UTR introns.
Hsp70 Data Collection and Alignment
We used BLASTn with the coding sequence of D. melanogaster Hsp70Aa as a query to each of the melanogaster subgroup genomes. All hits with an e-value of 0.0 were further examined to determine whether they constituted a putative copy of Hsp70, using a combination of Gbrowse orthology calls, synteny and relative position, and reciprocal BLAST to D. melanogaster. We included all verified copies found in each species in alignment. We identified four putative copies in the genomes of D. simulans, D sechellia and D. yakuba and six in D. erecta. While coding regions were complete for all D. erecta copies, gaps, often surrounded by variable length stretches of divergent sequence, interrupted the coding regions of 2 Hsp70 copies each from D. simulans and D. sechellia. All D. yakuba copies had gaps in the coding region, and two had divergent sequence surrounding the gap. Previous analysis had identified four copies in D. simulans, D. sechellia and D. yakuba, while copy number of Hsp70 in D. erecta was not assessed . We therefore assume that the coding region gaps in D. simulans, D sechellia and D. yakuba represent sequencing and assembly errors, and all are considered functional copies and included in further analysis, with one exception. The coding region gap in one of the copies of Hsp70 from D. simulans encompasses the 5′ end of the coding region and no alignment with the other Hsp70, including those from D. simulans, is found upstream of this gap. We are thus lacking the proximal promoter including the TATA box and putative TSS and cannot define the region to be analyzed for this copy, which is therefore excluded.
We followed the same procedure as outlined for the species of the melanogaster subgroup to collect putative orthologues of Hsp70 in the other seven Drosophila species for which genome data is available. In all species, multiple copies were recovered (D. ananassae: 5, D. pseudoobscura: 2, D. persimilis: 2, D. willistoni: 3, D. grimshawi: 3, D. virilis: 6, D. mojavensis: 6). In species where copy number of Hsp70 was previously examined ,  our findings were in agreement, with the exception of an additional copy found in D. willistoni.
Conservation of D. melanogaster TSS Across Species
For each distinct TSS in D. melanogaster, we examined conservation across species using the UCSC genome browser and threaded-blockset alignments produced by Multiz via the PromAn website . As in the UCSC genome alignments, this approach uses a BLASTz search to first identify homologous regions before aligning conserved blocks, and was shown to be more effective than other alignment algorithms at aligning highly diverged sequence, such as may be found in non-coding regions in between-species comparisons . We assume that conservation of sequence in the region encompassing the TSS implies functional conservation although this is not always necessarily the case .
Levels of putative TSS conservation varied among genes. Of the 117 TSS identified in D. melanogaster, 36 had a conserved block in all 12 species that included the D. melanogaster TSS. To keep species composition approximately constant across genesets, average out species-specific effects, discover general trends, and broaden sampling, we also analyzed 45 additional TSS that were conserved across only the eight species of the D. melanogaster and D. obscura groups, and 36 that were conserved only in the five species of the D. melanogaster subgroup. For Hsp70, all putative copies were conserved at the TSS and through the proximal promoter, and included a conserved TATA box motif . If data were missing for a species in the region encompassing the TSS, this was counted as lack of conservation. In some cases, only a single paralogue was alignable at the TSS, and this copy was retained for further analysis. Not including Hsp70, 8 paralogues in 6 genes were conserved at the TSS. These duplicate copies are not equally distributed among genesets (5 in geneset I, 3 in geneset III) but only account for a small additional amount of analyzed sequence (1-2% of total) per geneset and hence were included in analysis.
Alignments were trimmed to include the region 50 bp downstream from the TSS and 1000 bp upstream, a region that should encompass the majority of proximal promoter regulatory elements associated with a given gene . All alignments were converted to BLAST databases using the formatdb command in BLAST v. 2.2.16.
Discovery of Transposable Elements
Databases produced from alignments were submitted to BLASTn searches using (1) Repbase Drosophila transposable elements , (2) the curated D. melanogaster (version 9.4.1) set maintained at the Berkeley Drosophila Genome Project and (3) the non-redundant set of de novo PILER repetitive element predictions for the 12 Drosophila species genomes . Hits at 1e-05 were considered positive. In the case of multiple hits by related TE to the same location, only the highest scoring hit was retained. Results of searches with the three databases were manually compared and reconciled. Genome coordinates for TE sequence within all sampled promoters are included in Table S5.
To test for significance in pairwise and three-way comparisons in the observed number of distinct transposable element insertions in genesets versus an expected number based on a null hypothesis of equal probability of insertion into any individual promoter region included in the analysis, we used G-tests with the degrees of freedom scaled to the number of categories in a given comparison (Table S3). We used the Kruskal-Wallis test to compare the distribution of the number of TE insertions per line or species among genesets. A Kolmogorov-Smirnov two-sample test was used to determine if the phylogenetic distribution of insertions varied among genesets at each level of species sampling (Table S6).
We used non-parametric Mann-Whitney U-tests to determine if distance from the TSS, extent of TE sequence in promoter regions or percent element length relative to consensus or centroid length was different among genesets. As all sets of values compared had sufficient sample sizes, probability in the Mann-Whitney U-tests was assessed with the z statistic (Table S6).
We tested whether the distance of insertions from the TSS fit a Poisson distribution in the sampled interval by comparing observed and expected occurrences of downstream TE boundaries in 50 bp bins across the interval. Significance was assessed with a chi-square test with degrees of freedom dependent on the maximum number of TE insertions within an interval (Table S6).
The experiment-wide false positive rate was conservatively controlled using a standard Bonferroni correction for multiple tests.
Promoter regions and transcription start sites (TSS) sampled. The basis of TSS definition in D. melanogaster as defined in the text is listed in Column G. All characteristics pertaining to the inclusion of a promoter in a given promoter set are in Column K, and the key to these characteristics is below the table. The small number of analyzed duplicate promoters are identified in Column J.
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TE insertions in promoters of 12 species genomes. Each distinct insertion is listed with the promoter it was found, and all species in which it was identified. E-value refers to the best BLASTn hit. Element length indicates the amount of TE sequence in the defined promoter region. The identity of the TE is listed along with TE class (retrotranposon = I, DNA transposon = II).
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TE insertions per bp in each promoter set and 3-way G- test results at three levels of species sampling. Liberal, semi-conservative and conservative refer to method of estimating promoter set target size.
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Number of orthologues of D. melanogaster heat-shock genes in each Drosophila species
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Genomic location of TE insertion sequence in Drosophila promoters. For each insertion, in all promoters in which it is found, the genomic coordinates of both the promoter region and the TE sequence contained within it are tabled. Coordinates are from genome releases as detailed in the text. The first sheet contains insertions in promoters from all single copy genes, while insertions from the multicopy hsp70 genes are listed in the second sheet.
(0.06 MB XLS)
Conceived and designed the experiments: RAH MEF. Performed the experiments: RAH MEF. Analyzed the data: RAH MEF. Contributed reagents/materials/analysis tools: MEF. Wrote the paper: RAH MEF.
- 1. Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al. (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450: 203–218.
- 2. Walser JC, Chen B, Feder ME (2006) Heat-shock promoters: targets for evolution by P transposable elements in Drosophila. PLoS Genet 2: 1541–1555.
- 3. Lis J, Wu C (1993) Protein traffic on the heat-shock promoter: parking, stalling, and trucking along. Cell 74: 1–4.
- 4. Liao GC, Rehm EJ, Rubin GM (2000) Insertion site preferences of the P transposable element in Drosophila melanogaster. Proc Natl Acad Sci U S A 97: 3347–3351.
- 5. Shilova VY, Garbuz DG, Myasyankina EN, Chen B, Evgen'ev MB, et al. (2006) Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster. Genetics 173: 809–820.
- 6. Maside X, Bartolome C, Charlesworth B (2002) S-element insertions are associated with the evolution of the Hsp70 genes in Drosophila melanogaster. Curr Biol 12: 1686–1691.
- 7. McCollum AM, Ganko EW, Barrass PA, Rodriguez JM, McDonald JF (2002) Evidence for the adaptive significance of an LTR retrotransposon sequence in a Drosophila heterochromatic gene. BMC Evol Biol 2: 5.
- 8. Catania F, Kauer MO, Daborn PJ, Yen JL, Ffrench-Constant RH, et al. (2004) World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster. Mol Ecol 13: 2491–2504.
- 9. Schlenke TA, Begun DJ (2004) Strong selective sweep associated with a transposon insertion in Drosophila simulans. Proc Natl Acad Sci U S A 101: 1626–1631.
- 10. Aminetzach YT, Macpherson JM, Petrov DA (2005) Pesticide resistance via transposition-mediated adaptive gene truncation in Drosophila. Science 309: 764–767.
- 11. Ganko EW, Greene CS, Lewis JA, Bhattacharjee V, McDonald JF (2006) LTR retrotransposon-gene associations in Drosophila melanogaster. J Mol Evol 62: 111–120.
- 12. Rashkovetsky E, Iliadi K, Michalak P, Lupu A, Nevo E, et al. (2006) Adaptive differentiation of thermotolerance in Drosophila along a microclimatic gradient. Heredity 96: 353–359.
- 13. Gonzalez J, Lenkov K, Lipatov M, Macpherson JM, Petrov DA (2008) High rate of recent transposable element-induced adaptation in Drosophila melanogaster. PLoS Biol 6: e251.
- 14. Krebs RA, Feder ME (1997) Deleterious consequences of Hsp70 overexpression in Drosophila melanogaster larvae. Cell Stress Chaperones 2: 60–71.
- 15. Michalak P, Minkov I, Helin A, Lerman DN, Bettencourt BR, et al. (2001) Genetic evidence for adaptation-driven incipient speciation of Drosophila melanogaster along a microclimatic contrast in “Evolution Canyon,” Israel. Proc Natl Acad Sci U S A 98: 13195–13200.
- 16. Lerman DN, Feder ME (2005) Naturally occurring transposable elements disrupt hsp70 promoter function in Drosophila melanogaster. Mol Biol Evol 22: 776–783.
- 17. Feder ME (1997) Necrotic fruit: a novel model system for thermal biologists. J Therm Biol 22: 1–9.
- 18. Smith CD, Edgar RC, Yandell MD, Smith DR, Celniker SE, et al. (2006) Improved repeat identification and masking in Dipterans. Gene 389: 1–9.
- 19. Yang HP, Barbash DA (2008) Abundant and species-specific DINE-1 transposable elements in 12 Drosophila genomes. Genome Biol 9: R39.
- 20. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, et al. (2000) The genome sequence of Drosophila melanogaster. Science 287: 2185–2195.
- 21. Kaminker JS, Bergman CM, Kronmiller B, Carlson J, Svirskas R, et al. (2002) The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective. Genome Biol 3: RESEARCH0084.
- 22. Lansman RA, Stacey SN, Grigliatti TA, Brock HW (1985) Sequences homologous to the P mobile element of Drosophila melanogaster are widely distributed in the subgenus Sophophora. Nature 318: 561–563.
- 23. Stacey SN, Lansman RA, Brock HW, Grigliatti TA (1986) Distribution and conservation of mobile elements in the genus Drosophila. Mol Biol Evol 3: 522–534.
- 24. Begun DJ, Holloway AK, Stevens K, Hillier LW, Poh YP, et al. (2007) Population genomics: whole-genome analysis of polymorphism and divergence in Drosophila simulans. PLoS Biol 5: 2534–2559.
- 25. Bureau TE, Wessler SR (1994) Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6: 907–916.
- 26. El Amrani A, Marie L, Ainouche A, Nicolas J, Couee I (2002) Genome-wide distribution and potential regulatory functions of AtATE, a novel family of miniature inverted-repeat transposable elements in Arabidopsis thaliana. Mol Genet Genomics 267: 459–471.
- 27. Yang GJ, Lee YH, Jiang YM, Shi XY, Kertbundit S, et al. (2005) A two-edged role for the transposable element Kiddo in the rice ubiquitin2 promoter. Plant Cell 17: 1559–1568.
- 28. Xu L, Wang LJ, Liu T, Qlan WQ, Gao Y, et al. (2007) Triton, a novel family of miniature inverted-repeat transposable elements (MITEs) in Trichosanthes kirilowii Maximowicz and its effect on gene regulation. Biochem Biophys Res Commun 364: 668–674.
- 29. Kuang HH, Padmanabhan C, Li F, Kamei A, Bhaskar PB, et al. (2009) Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: New functional implications for MITEs. Genome Res 19: 42–56.
- 30. Caspi A, Pachter L (2006) Identification of transposable elements using multiple alignments of related genomes. Genome Res 16: 260–270.
- 31. Wang J, Keightley PD, Halligan DL (2007) Effect of divergence time and recombination rate on molecular evolution of Drosophila INE-1 transposable elements and other candidates for neutrally evolving sites. J Mol Evol 65: 627–639.
- 32. Kidwell MG (1983) Evolution of hybrid dysgenesis determinants in Drosophila melanogaster. Proc Natl Acad Sci U S A 80: 1655–1659.
- 33. Silva JC, Kidwell MG (2004) Evolution of P elements in natural populations of Drosophila willistoni and D. sturtevanti. Genetics 168: 1323–1335.
- 34. Markow TA, O' Grady PM (2007) Drosophila biology in the genomic age. Genetics 177: 1269–1276.
- 35. Zatsepina OG, Velikodvorskaia VV, Molodtsov VB, Garbuz D, Lerman DN, et al. (2001) A Drosophila melanogaster strain from sub-equatorial Africa has exceptional thermotolerance but decreased Hsp70 expression. J Exp Biol 204: 1869–1881.
- 36. Bellen HJ, Levis RW, Liao GC, He YC, Carlson JW, et al. (2004) The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167: 761–781.
- 37. Leibovitch BA, Lu Q, Benjamin LR, Liu YY, Gilmour DS, et al. (2002) GAGA factor and the TFIID complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter. Mol Cell Biol 22: 6148–6157.
- 38. Chen B, Shilova VY, Zatsepina OG, Evgen'ev MB, Feder ME (2008) Location of P element insertions in the proximal promoter region of Hsp70A is consequential for gene expression and correlated with fecundity in Drosophila melanogaster. Cell Stress Chaperones 13: 11–17.
- 39. Arbeitman MN, Furlong EE, Imam F, Johnson E, Null BH, et al. (2002) Gene expression during the life cycle of Drosophila melanogaster. Science 297: 2270–2275.
- 40. Boutanaev AM, Kalmykova AI, Shevelyov YY, Nurminsky DI (2002) Large clusters of co-expressed genes in the Drosophila genome. Nature 420: 666–669.
- 41. Muse GW, Gilchrist DA, Nechaev S, Shah R, Parker JS, et al. (2007) RNA polymerase is poised for activation across the genome. Nat Genet 39: 1507–1511.
- 42. Kutach AK, Kadonaga JT (2000) The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20: 4754–4764.
- 43. Schmid CD, Praz V, Delorenzi M, Perier R, Bucher P (2004) The Eukaryotic Promoter Database EPD: the impact of in silico primer extension. Nucleic Acids Res 32: D82–D85.
- 44. Rubin GM, Hong L, Brokstein P, Evans-Holm M, Frise E, et al. (2000) A Drosophila complementary DNA resource. Science 287: 2222–2224.
- 45. Stapleton M, Liao GC, Brokstein P, Hong L, Carninci P, et al. (2002) The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes. Genome Res 12: 1294–1300.
- 46. Hall T (1999) Bioedit sequence alignment editor. http://www.mbio.ncsu.edu/BioEdit/bioedit.html.
- 47. Corces V, Pellicer A (1984) Identification of sequences involved in the transcriptional control of a Drosophila heat-shock gene. J Biol Chem 259: 4812–4817.
- 48. Weber JA, Taxman DJ, Lu Q, Gilmour DS (1997) Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region. Mol Cell Biol 17: 3799–3808.
- 49. Crosby MA, Goodman JL, Strelets VB, Zhang PL (2007) FlyBase: genomes by the dozen. Nucleic Acids Res 35: D486–D491.
- 50. Bettencourt BR, Feder ME (2001) Hsp70 duplication in the Drosophila melanogaster species group: How and when did two become five? Mol Biol Evol 18: 1272–1282.
- 51. Burge C, Karlin S (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268: 78–94.
- 52. Bettencourt BR, Feder ME (2002) Rapid concerted evolution via gene conversion at the Drosophila hsp70 genes. J Mol Evol 54: 569–586.
- 53. Evgen'ev MB, Zatsepina OG, Garbuz D, Lerman DN, Velikodvorskaya V, et al. (2004) Evolution and arrangement of the hsp70 gene cluster in two closely related species of the virilis group of Drosophila. Chromosoma 113: 223–232.
- 54. Lardenois A, Chalmel F, Bianchetti L, Sahel JA, Leveillard T, et al. (2006) PromAn: an integrated knowledge-based web server dedicated to promoter analysis. Nucleic Acids Res 34: W578–583.
- 55. Blanchette M, Kent WJ, Riemer C, Elnitski L, Smit AF, et al. (2004) Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res 14: 708–715.
- 56. Wray GA, Hahn MW, Abouheif E, Balhoff JP, Pizer M, et al. (2003) The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol 20: 1377–1419.
- 57. Papatsenko DA, Makeev VJ, Lifanov AP, Regnier M, Nazina AG, et al. (2002) Extraction of functional binding sites from unique regulatory regions: the Drosophila early developmental enhancers. Genome Res 12: 470–481.
- 58. Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, et al. (2005) Repbase update, a database of eukaryotic repetitive elements. Cytogenet Genome Res 110: 462–467.
- 59. Russo CA, Takezaki N, Nei M (1995) Molecular phylogeny and divergence times of drosophilid species. Mol Biol Evol 12: 391–404.