* Committee on Evolutionary Biology and Department of Organismal Biology & Anatomy, The University of Chicago; Committees on Genetics and Molecular Medicine and Department of Organismal Biology & Anatomy, The University of Chicago
Correspondence: E-mail: m-feder{at}uchicago.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: transposable element hsp70 promoter transcriptional regulation gene expression
Although mobile genetic or transposable elements (TEs) have had a massive and ancient impact on genome organization and diversity (Kazazian 2004), the roles of TEs in ongoing adaptive evolution in eukaryotes are less clear, because transposition is typically both deleterious and effectively marginalized by evolutionary and genetic mechanisms (Franchini, Ganko, and McDonald 2004). Recent discoveries, however, distinctively correlate transposition with adaptive microevolution (Franchini, Ganko, and McDonald 2004). The components of this correlation are as follows: (1) transposition into regulatory regions of genes; (2) such genes segregating as alleles of TE-less genes in natural populations; (3) allelic variation in the regulation of gene expression; (4) allelic variation in gene expression affecting fitness, and thus exposing allelic frequency to natural selection; and (5) covariation of allelic frequency with natural variation in the agent of selection. Although each component has numerous examples, the experimental verification of all components in a single system has been elusive. For instance, the best recent examples (Kalendar et al. 2000; Daborn et al. 2002; Lerman et al. 2003; Schlenke and Begun 2004) are complete except for demonstrating that the TEs themselves (rather than sequence to which they are linked) encode the adaptive phenotype and how they do so. Here we supply this missing link by experimentally excising or replacing naturally occurring TEs in a common genetic background and showing that the expected changes in gene expression result.
The present study uses the Hsp70-encoding genes of Drosophila melanogaster as a model system. In this species, Hsp70 is a molecular chaperone that contributes significantly to inducible thermotolerance, which is key to reproductive success in the face of natural thermal stress (Feder et al. 1996; Feder, Blair, and Figueras 1997; Lerman et al. 2003). Hsp70, however, plays numerous additional roles in the cell, for which stringent regulation at low concentrations is essential (Lerman et al. 2003). Thus, repeatably and depending on circumstances, either high or low levels of Hsp70 expression evolve in both natural and experimental populations (Sorensen, Dahlgaard, and Loeschcke 2001; Feder et al. 2002). These levels are inversely correlated with the frequencies of hsp70 alleles in which distinct TEs have inserted in the proximal promoter, suggesting selection for or against TE-bearing alleles as a mechanism of action (Michalak et al. 2001; Zatsepina et al. 2001; Bettencourt et al. 2002; Lerman et al. 2003). Indeed, alleles with a Jockey insertion are associated with reduced hsp70 mRNA, and both Jockey and P-bearing alleles are associated with reduced Hsp70 protein (Lerman et al. 2003). The natural or evolutionary variation in Hsp70 level could be due to the transposable elements themselves, and/or to sequence variation that is linked to the TEs but external to them. If the former, the TEs could affect Hsp70 level via two non-exclusive mechanisms: First, diverse components of the transcriptional apparatus associate with the hsp70 promoter, and the proper organization and spacing of their binding sites are essential for full-strength transcription (O'Brien and Lis 1991; Lis and Wu 1993; Amin et al. 1994; Li et al. 1996; Mason and Lis 1997; Weber et al. 1997). Hence disruption of promoter architecture by TE insertion could reduce transcriptional activation (Xiao and Lis 1988; Lee et al. 1992; Amin et al. 1994; Shopland et al. 1995). Alternatively, variation in hsp70 transcription could stem from introduction of novel regulatory elements in the TEs themselves (Kazazian 2004). To characterize the effect of the TEs and elucidate the mechanism by which they affect Hsp70 levels, we placed hsp70 promoter alleles with or without TE insertions in luciferase expression constructs, replaced TE insertions in these constructs with intergenic DNA of equal length, and excised these sequences from constructs. Each TE reduced luciferase signal, indicating that the TEs can be sufficient to reduce gene transcription. Furthermore, each replacement insert similarly reduced luciferase signal in all four hsp70 constructs, suggesting that the corresponding TEs affect hsp70 promoter function primarily by altering promoter architecture.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Creation of Wild-type and TE-Bearing Reporter Constructs
The hsp70Ba promoter region from TE-bearing (TE+) and TE-lacking (TE) flies from the T, Arv/Zim, and 1997 Evolution Canyon ("Canyon 97") populations was amplified from single-fly DNA in a 100-µl reaction, as above (see table 1B in the Supplementary Material online). This amplification yields a 900 bp (for wild-type; 2,0992,354 bp for TE-disrupted promoters) product containing the promoter, 5' UTR, and first 190 bp of the hsp70Ba coding sequence.
To create luciferase reporter constructs, hsp70 promoters were re-amplified from the polymerase chain reaction (PCR) products described above (Canyon 2000 whole hsp70Bb gene and hsp70Ba promoter and partial coding sequence for the other three populations) with primers designed to introduce restriction sites to facilitate cloning (table 1C in the Supplementary Material online). Re-amplification of gene-specific PCR products ensured that the wild-type and TE-disrupted promoter constructs were derived from the same gene (rather than from different hsp70 gene copies). A 23 µg portion of these new PCR products was digested for 3 h at 37°C with 15 U each of Kpn1 and Bgl2 in a 40-µl reaction. Then, 4 µg of the pGL-3-Basic Vector (Promega; 4.8 kb), which contains the cDNA-encoding firefly luciferase (luc) but lacks eukaryotic promoter or enhancer sequences, was also digested with the same restriction enzymes. Digests were purified with the QIAquick Gel-Extraction kit (Qiagen), promoters were ligated into the pGL-3 vector, and maxi preps were purified with the Qiagen Plasmid Maxi Kit, yielding 300-µl purified plasmid at a concentration of 12 µg/µl. TE+ and TE constructs were confirmed by gel electrophoresis and sequencing. All constructs begin upstream of HSE4 and end 60 bp downstream of the transcription start site (fig. 1).
|
A 2-µg portion of each of the amplified promoter fragments was digested for 3 h at 37°C with Pac1, and the upstream and downstream promoter pieces (150 µg each) were ligated to each other at the Pac1 site with 3 U T4 Ligase. Successful upstream-downstream ligants were amplified with the aforementioned Kpn1- and Bgl2-containing primers and confirmed by gel electrophoresis. To create "Excision" reporter constructs, these ligants were digested with Kpn1 and Bgl2, ligated into pGL-3 (cut with Kpn1 and Bgl2), and purified as above. Preparations were confirmed by DNA sequencing. The excision constructs contained an 8-bp Pac1 site at the excision site, which was not present in wild-type promoters (the insertion of this site did not significantly reduce luciferase signal in the Canyon 2000, Arv/Zim, and T populations (see Results). The excision constructs differ from the TE-containing constructs in that the former contain an 8-bp Pac1 site where the TE was, and lack the 8 bp sequence duplicated at the TE insertion site.
Four random DNA sequences used to replace each of the naturally occurring TEs were obtained by amplification (table 1E in the Supplementary Material online) of intergenic sequences from D. melanogaster chromosome 3R (GenBank accession number AE003602). Designated by letter, these are: A (positions 6461866588 of the GenBank sequence), B (248199249655), C (253707255437), and D (262490263956). These positions are the limits of the respective intergenic sequences; each 3'-complimentary primer (table 1E in the Supplementary Material online) was designed so that the resulting amplicon would be the same length as the TE it was replacing.
Intergenic PCR products and the excision luciferase constructs described above were digested with Pac1 and cleaned with Qiagen spin columns. Three of the four random DNA sequences eventually were successfully ligated into the site from which each naturally occurring TE had previously been excised, and ligants (henceforth "Replacement" constructs) were purified and confirmed by gel electrophoresis. Because the Replacement constructs were the same length as the TEs they replaced, Replacement sequences from the same intergenic region were not identical (e.g., insert A for Canyon 97 was not identical to replacement sequence A for Arv/Zim, and so on).
Transient Transfection and Luciferase Assay
Drosophila Schneider S2 cells were cultured at 25°C in Sheilds and Sang M3 medium (Sigma) containing 10% heat-inactivated fetal bovine serum (Sigma); 3.5 ± 105 cells were seeded in 350 µl medium per well in 24-well plates the day before transfection. Cells were transiently transfected with Superfect Transfection Reagent (Qiagen). Then 1 µg plasmid DNA was co-transfected with 0.05 µg pRL-CMV (Promega) per well. The pRL-CMV vector contains the cDNA encoding Renilla luciferase (Rluc) and the CMV enhancer and early promoter.
After transfection, cells were incubated at 25°C for 6 h, transferred to 1.5-ml microcentrifuge tubes and placed in thermostatted waterbaths at either 36.5°C (heat shock) or 25°C (control) for 5 min, transferred to a 25°C waterbath for 1 h, and harvested by centrifugation. Eight samples per promoter (i.e., TE+, TE, Excision, Replacements) were subjected to heat shock, and four samples/promoter were subjected to control treatment for a total of 72 separate transfections for each of the populations (except in the T strains, for which only two replacement inserts were created). Cells were washed in phosphate-buffered saline (PBS), lysed in 150 µl Passive Lysis Buffer (Promega), and stored at 80°C overnight for analysis.
Luciferase signal was quantitated with the Dual-Luciferase Reporter Assay System (Promega), which allows sequential measurement of firefly and Renilla luciferase activity from the same sample of lysate. For each assay, 5 µl lysate (+ 5 µl PBS) was analyzed in a manual luminometer programmed to perform a 10-s measurement. Firefly luciferase background was determined by measuring luminescence activity of nontransfected control cells. Before quantitation of Renilla luminescent signal, firefly signal was quenched; nonetheless, residual luminescence from the firefly luciferase reaction can affect Renilla luminescence measurements. Accordingly, Renilla background luminescence was determined by preparing a lysate of heat-shocked cells transfected with the appropriate hsp70-luc reporter vector only (i.e., no pRL-CMV) and measuring firefly luciferase luminescence followed by apparent Renilla luminescence. Backgrounds were subtracted from raw firefly and Renilla luminescence measurements, respectively, and luciferase activity was expressed as the ratio of firefly/Renilla luminescence.
Several sets of S2 cells were used for transformations. These differed systematically in their normalized luminescence signal (data not shown). Accordingly, luminescence signals were compared only in transfected cells from the same source cell population, and all comparisons within a fly population (e.g., Arv/Zim etc.) were performed in a single experiment.
Statistics
Statistical analyses used StatView 4.5 (Abacus Concepts). Factorial analyses of variance (ANOVA) tested for group effect. Post hoc analyses tested for effect of promoter after heat-shock and control treatments, as well as for effect of heat shock in TE+, TE, Excision, and Replacement samples.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constructs, Controls, and Repeatability of Luciferase Assays
Renilla luciferase activity was positively correlated with the amount of transfected pRL-CMV. Luminescence in cells transfected with the pGL3-Basic vector only (i.e., with no promoter) was equal to firefly luciferase background measurements from nontransfected control cells or blank tubes after both heat-shock and control treatments (data not shown).
Control (no heat shock) Treatment
In the absence of heat-shock luciferase luminescence was low and unaffected by TE insertions in promoters extracted from each of the four populations (i.e., post-hoc comparisons are not significant [statistics not shown]; fig 2). Under these conditions, luciferase luminescence was also low and did not differ among constructs in which the TEs had been excised or replaced with the intergenic DNA amplicons A-D (fig. 2).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transposition events are frequent at both hsp70 gene clusters (Zatsepina et al. 2001; Bettencourt et al. 2002; Maside, Bartolome, and Charlesworth 2002), and, as the present study shows, TEs have interrupted the proximal promoters of two hsp70 genes. Lerman et al. (2003) hypothesized that the distinctive open chromatin conformation of hsp70 promoters facilitates their transpositional disruption.
While high levels of Hsp70 are important for tolerance of extreme temperatures (see Introduction), high levels of Hsp70 can be deleterious at moderately high but non-lethal temperatures (Krebs and Feder 1997a; 1997b; Bettencourt, Feder, and Cavicchi 1999; Zatsepina et al. 2001), presumably as a result of over-activation of its multiple functions in the cell (Lerman et al. 2003). Thus Lerman et al. (2003) hypothesized that selection maintains the TEs to reduce Hsp70 levels in Drosophila in non-stressful environments. The reduction in hsp70BaP allele frequency on the hotter and more variable south-facing slope (SFS; 1.2%) compared with the NFS (33.9%) in the 1997 Evolution Canyon populations is consistent with this hypothesis (Michalak et al. 2001), as is the lower hsp70BbP allele frequency for the SFS population (0%) than for the NFS (20%) population in the Evolution Canyon 2000 collection. An alternative hypothesis, that the absence of the hsp70BbP allele in the Canyon 2000 SFS population results from drift/founder effect, requires data on gene flow between these populations for testing.
Full-strength transcription of heat-shock genes requires the orderly interaction of the transcriptional holoenzyme/polymerase complex, the heat-shock transcription factor (HSF), and conserved elements in the heat-shock promoter. hsp70 genes normally have an open chromatin conformation (Costlow and Lis 1984), and TFIID, RNA polymerase II (Pol II), and the GAGA factor (GFA) all associate with the hsp70 promoter prior to heat shock (Lis and Wu 1993). While this constitutive binding and pre-assembly primes hsp70 genes for rapid response to heat shock, it is not sufficient for hsp70 induction because the Pol II complex pauses 2040 nucleotides downstream of the transcription start site (Weber et al. 1997). Resumption of elongation by Pol II, the rate-limiting step in hsp70 transcription (O'Brien and Lis 1991), requires cooperative binding of HSF trimers to HSEs (Amin et al. 1994). Heat-shock transcription factor and the TATA binding protein also interact cooperatively in vivo (Mason and Lis 1997), and GFA further stabilizes HSF-HSE engagement (Li et al. 1996; Mason and Lis 1997). Hence, disruption of promoter architecture, by TE insertion or any other means, might reduce transcriptional activation by altering cooperative protein-protein interactions at the hsp70 promoter. Prior work supports this hypothesis. Although HSEs 1 and 2 are sufficient for some hsp70 transcription, full-strength transcription requires all four (Xiao and Lis 1988; Lee et al. 1992; Shopland et al. 1995). The spacing between and stereoalignment of HSEs is also critical. Insertion of spacers of 15 and 1114 nucleotides between HSEs 1 and 2 reduces promoter activity by 90%, whereas spacers of 610 and 1618 nucleotides largely restore promoter activity (Amin et al. 1994).
As we show, all of the TEs affect hsp70 promoter function, despite differences in insertion site, length, and sequence. This effect was not a foregone conclusion, however, as the experimental manipulations of hsp70 promoter structure discussed above differ from the disruptions caused by the naturally occurring TEs in several important ways. For example, Amin et al. (1994) used constructs that contained only the minimal hsp70 promoteri.e., HSEs 1 and 2. Thus, changing the spacing between HSEs 2 and 3, where three of the TEs have inserted, was previously unexamined. In addition, while the maximum spacer length previously tested was 25 bp, the TEs described here are all much larger (>1 kb). Finally, the TEs are naturalnot anthropogenicmutagens.
In addition, that the TE insertions would decrease, rather than increase, expression of adjacent genes was unforeseen. Indeed, other TEs thought to play an adaptive role in regulatory evolution may increase gene expression. For example, Daborn et al. (2002) demonstrate that an Accord insertion in the 5' end of the D. melanogaster cytochrome P450 gene Cyp6g1 is associated with Cyp6g1 overtranscription, which confers insecticide resistance. Schlenke and Begun (2004) show that a 4,803-bp Doc insertion in the 5' flanking region of the D. simulans Cyp6g1 gene is also correlated with increased transcriptional abundance. Yet despite differences in insertion site, length, and sequence, all the TEs described here similarly reduced hsp70 promoter function. This reduction, and the absence of TE insertions that increase Hsp70 expression, may result from selection against high levels of Hsp70, which can be harmful in environments with infrequent extreme stress (Lerman et al. 2003).
Particular sequence elements within each of the TEs could in principle also be responsible for the impact of TEs on promoter activity. Some TEs carry regulatory motifs that alter expression of nearby genes. For example, insertion of the gypsy element, which contains an insulator that can disrupt regulatory interactions, is responsible for many spontaneous mutations in Drosophila (Modolell, Bender, and Meselson 1983; Geyer and Corces 1992; Hogga et al. 2001). Further, TE+ and TE promoters contain differences in addition to the TE that may contribute to the differences observed in promoter activity. For example, duplicated host DNA flanks each of the TE insertion sites. In the T population, this duplication results in an additional putative GAGA element, which may alter promoter activity, and the other insertion site duplications may also contain putative regulatory sequences. In addition, the Canyon 2000 and Arv/Zim populations contain single-nucleotide polymorphisms (SNPs; two and four SNPs, respectively) that are linked to the P element insertion. Although such noncoding regulatory variation is thought to underlie important phenotypic changes, distinguishing the impact of specific nucleotide variants from those of linked loci is often difficult (Rockman and Wray 2002). Indeed, differences in luciferase luminescence observed between TE+ and TE promoters might result from these SNPs, rather than from the TEs that they are linked to.
To distinguish between these alternative impacts of TEs, spatial disruption of the promoter versus addition of novel regulatory motifsand to remove the potentially confounding affect of the insertion site duplications and SNPswe created the Excision and Replacement luc constructs. All three intergenic replacement inserts in the Canyon 2000, Canyon 97, and Arv/Zim hsp70-luc promoters and one of the two inserts in the T promoter significantly reduced luciferase signal compared with both the wild-type (TE) and Excision constructs. These reductions were comparable to those observed in the TE+ constructs. The intergenic inserts also reduced heat inducibility of the hsp70-luc promoters, as the TEs themselves did. Taken together, 10 of 11 replacement inserts significantly reduced luciferase luminescence, as did all four TEs, indicating that the reductions in promoter activity caused by the TE insertions did not result primarily from TE-specific sequence, insertion site duplications, or from SNPs linked to the TEs. Rather, these results suggest that disruption of hsp promoter architecture is sufficient for reduced hsp transcription.
We have characterized naturally occurring TE insertions that disrupt the Drosophila hsp70 promoter and show that each of these insertions reduces hsp70 promoter activity. These TEs, moreover, reduce Hsp70 expression levels and affect organismal fitness measurements (Lerman et al. 2003). This study is the first direct demonstration that specific natural nucleotide polymorphisms underlie differences in Hsp70 expression. More broadly, these results suggest that TEs can underlie adaptive regulatory evolution in natural populations.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Claudia Schmidt-Dannert, Associate Editor
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amin, J., M. Fernandez, J. Ananthan, J. T. Lis, and R. Voellmy. 1994. Cooperative binding of heat shock transcription factor to the hsp70 promoter in vivo and in vitro. J. Biol. Chem. 269:48044811.
Bettencourt, B. R., M. E. Feder, and S. Cavicchi. 1999. Experimental evolution of Hsp70 expression and thermotolerance in Drosophila melanogaster. Evolution 53:484492.[ISI]
Bettencourt, B. R., I. Kim, A. A. Hoffmann, and M. E. Feder. 2002. Response to natural and laboratory selection at the Drosophila hsp70 genes. Evolution 56:17961801.[ISI][Medline]
Black, D. M., M. S. Jackson, M. G. Kidwell, and G. A. Dover. 1987. Kp Elements repress P-induced hybrid dysgenesis in Drosophila melanogaster. EMBO J. 6:41254135.[Abstract]
Costlow, N., and J. T. Lis. 1984. High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae. Mol. Cell Biol. 4:18531863.[ISI][Medline]
Daborn, P. J., J. L. Yen, M. R. Bogwitz, G. Le Goff, E. Feil, S. Jeffers, N. Tijet, T. Perry, D. Heckel, P. Batterham, R. et al. 2002. A single P450 allele associated with insecticide resistance in Drosophila. Science 297:22532256.
Feder, M. E., T. B. C. Bedford, D. R. Albright, and P. Michalak. 2002. Evolvability of Hsp70 expression under artificial selection for inducible thermotolerance in independent populations of Drosophila melanogaster. Physiol. Biochem. Zool. 75:325334.[CrossRef][ISI][Medline]
Feder, M. E., N. Blair, and H. Figueras. 1997. Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae. Funct. Ecol. 11:90100.[CrossRef][ISI]
Feder, M. E., N. V. Cartaño, L. Milos, R. A. Krebs, and S. L. Lindquist. 1996. Effect of engineering hsp70 copy number on Hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster. J. Exp. Biol. 199:18371844.
Franchini, L. F., E. W. Ganko, and J. F. McDonald. 2004. Retrotransposon-gene associations are widespread among D. melanogaster populations. Mol. Biol. Evol. 21:13231331.
Geyer, P. K., and V. G. Corces. 1992. DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6:18651873.[Abstract]
Hogga, I., J. Mihaly, S. Barges, and F. Karch. 2001. Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex. Mol. Cell 8:11451151.[ISI][Medline]
Kalendar, R., J. Tanskanen, S. Immonen, E. Nevo, and A. H. Schulman. 2000. Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proc. Natl. Acad. Sci. USA 97:66036607.
Kazazian, H. H. 2004. Mobile elements: drivers of genome evolution. Science 303:16261632.
Krebs, R. A., and M. E. Feder. 1997a. Natural variation in the expression of the heat-shock protein Hsp70 in a population of Drosophila melanogaster, and its correlation with tolerance of ecologically relevant thermal stress. Evolution 51:173179.[ISI]
Krebs, R. A., and M. E. Feder. 1997b. Deleterious consequences of Hsp70 overexpression in Drosophila melanogaster larvae. Cell Stress Chaperones 2:6071.[ISI][Medline]
Lee, H., K. W. Kraus, M. F. Wolfner, and J. T. Lis. 1992. DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev. 6:284295.[Abstract]
Lerman, D. N., P. Michalak, A. B. Helin, B. R. Bettencourt, and M. E. Feder. 2003. Modification of heat-shock gene expression in Drosophila melanogaster populations via transposable elements. Mol. Biol. Evol. 20:135144.
Li, B., J. A. Weber, Y. Chen, A. L. Greenleaf, and D. S. Gilmour. 1996. Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract. Mol. Cell. Biol. 6:54335443.
Lis, J., and C. Wu. 1993. Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell 74:14.[ISI][Medline]
Maside, X., C. Bartolome, and B. Charlesworth. 2002. S-element insertions are associated with the evolution of the hsp70 genes in Drosophila melanogaster. Curr. Biol. 12:16861691.[CrossRef][ISI][Medline]
Mason, P. B., and J. T. Lis. 1997. Cooperative and competitive protein interactions at the Hsp70 promoter. J. Biol. Chem. 272:3322733233.
Michalak, P., I. Minkov, A. Helin, D. N. Lerman, B. R. Bettencourt, M. E. Feder, A. B. Korol, and E. Nevo. 2001. Genetic evidence for adaptation-driven incipient speciation of Drosophila melanogaster along a microclimatic contrast in "Evolution Canyon," Israel. Proc. Natl. Acad. Sci. USA 98:1319513200.
Modolell, J., W. Bender, and M. Meselson. 1983. Drosophila melanogaster mutations suppressible by the suppressor of hairy-wing are insertions of a 7.3-kilobase mobile element. Proc. Natl. Acad. Sci. USABiol. Sci. 80:16781682.
O'Brien, T., and J. T. Lis. 1991. RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene. Mol. Cell. Biol. 11:52855290.[ISI][Medline]
Ohare, K., and G. M. Rubin. 1983. Structures of P-transposable elements and their sites of insertion and excision in the Drosophila melanogaster genome. Cell 34:2535.[ISI][Medline]
Omichinski, J. G., P. V. Pedone, G. Felsenfeld, A. M. Gronenborn, and G. M. Clore. 1997. The solution structure of a specific GAGA factor-DNA complex reveals a modular binding mode. Nat. Struct. Biol. 4:122132.[ISI][Medline]
Rasmusson, K. E., J. D. Raymond, and M. J. Simmons. 1993. Repression of hybrid dysgenesis in Drosophila melanogaster by individual naturally-occurring P-elements. Genetics 133:605622.
Rockman, M., and G. Wray. 2002. Abundant raw material for cis-regulatory evolution in humans. Mol. Biol. Evol. 19:19912004.
Schlenke, T. A., and D. J. Begun. 2004. Strong selective sweep associated with a transposon insertion in Drosophila simulans. Proc. Natl. Acad. Sci. USA 101:16261631.
Shopland, L. S., K. Hirayoshi, M. Fernandes, and J. T. Lis. 1995. HSF access to heat-shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID and RNA polymerase II binding sites. Genes Dev. 9:27562769.[Abstract]
Sorensen, J. G., J. Dahlgaard, and V. Loeschcke. 2001. Genetic variation in thermal tolerance among natural populations of Drosophila buzzatii: down regulation of Hsp70 expression and variation in heat stress resistance traits. Funct. Ecol. 15:289296.[CrossRef][ISI]
Weber, J. A., D. J. Taxman, Q. Lu, and D. S. Gilmour. 1997. Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region. Mol. Cell. Biol. 17:37993808.[Abstract]
Wilkins, R. C., and J. T. Lis. 1997. Dynamics of potentiation and activation: GAGA factor and its role in heat shock gene regulation. Nucleic Acids Res. 25:39633968.
Xiao, H., and J. T. Lis. 1988. Germline transformation used to define key features of heat-shock response elements. Science 239:11391142.[ISI][Medline]
Zatsepina, O. G., V. V. Velikodvorskaia, V. B. Molodtsov, D. Garbuz, D. N. Lerman, B. R. Bettencourt, M. E. Feder, and M. B. Evgenev. 2001. A Drosophila melanogaster strain from sub-equatorial Africa has exceptional thermotolerance but decreased Hsp70 expression. J. Exp. Biol. 204:18691881.
|