Báo cáo khoa học: The hemoglobin genes of Drosophila ppt

The hemoglobin genes of Drosophila Thorsten Burmester 1 , Joachim Storf 1 , Anne Hasenja ¨ ger 2 , Sabine Klawitter 2 and Thomas Hankeln 2 1 Institute of Zoology, University of Mainz, Germany 2 Institute of Molecular Genetics, University of Mainz, Germany (Hemo-)globins (Hbs) are small globular proteins usu- ally consisting of a core of about 150 amino acids, which typically comprises eight a-helical segments (named A–H) and displays a characteristic 3-over-3 a-helical sandwich structure [1,2]. Hbs include an iron- containing heme (Fe 2+ -protoporphyrin IX), by which they reversibly bind gaseous ligands. Most Hbs are considered respiratory proteins because of their ability to transport or to store O 2 , thus enhancing the avail- ability of O 2 to the respiratory chain of the mitochon- dria [3]. Hbs are widespread among metazoan taxa [4–6] and it is not surprising that Hbs are among the best-investigated proteins in biological and biochemical sciences [1,2]. Recent studies have demonstrated that some Hbs may also detoxify NO (and possibly other noxious reactive nitrogen and oxygen species [RNS and ROS]) and or serve as O 2 sensing molecule [7–9]. For a long time Hbs were essentially unknown in most insects. Respiratory proteins had been regarded unnecessary in this taxon because of the well-devel- oped tracheal system, which enables an efficient diffu- sion of O 2 from the atmosphere to the inner organs [10]. Notable exceptions had been only some basal insects that possess hemocyanin as O 2 transport pro- tein in the hemolymph, which they inherited from the crustacean ancestor, but has been lost in the evolution Keywords Gene duplication; globin; insects; oxygen; phylogeny Correspondence T. Burmester, Institute of Zoology, University of Mainz, D-55099 Mainz, Germany Fax: +49 6131 39 24652 Tel: +49 6131 39 24477 E-mail: burmeste@uni-mainz.de Note The nucleotide sequences reported in this paper have been submitted to the GenBankTM ⁄ EMBL databases with the accession numbers AM086021 (D. virilis glob1 gene), AM086022 (D. virilis glob1 cDNA), AM086023 (D. pseudoobscura glob1 cDNA) and AM086024 (D. melanogaster glob2 cDNA). (Received 13 October 2005, revised 18 November 2005, accepted 23 November 2005) doi:10.1111/j.1742-4658.2005.05073.x We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yak- uba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the data- bases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds ⁄ dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system. Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae. Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved. In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0. Both genes are conserved in various drosophilid species, but only expres- sion of glob2 could be demonstrated by western blotting and RT-PCR. Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins. glob1 is closely related to the intracellular hemoglobin of the botfly Gastero- philus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade. Abbreviations AIC, Akaike information criterion; EPO, erythropoietin; Hb, hemoglobin; HRE, hypoxia responsive sequence elements; PIP, percent identity plot. 468 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS of higher neopteran insects [11]. Respiratory proteins belonging to the Hb-type were considered being con- fined to a few insect species that are adapted to hypoxic environments [6]. According to our present knowledge, the chironomid midges (Diptera:Nematocera) are the only insects that possess true extracellular Hbs in their circulatory system for O 2 transport [12,13]. Some aquatic Hemiptera and the larvae of the horse botfly Gasterophilus intestinalis harbour intracellular Hbs, which probably carry out myoglobin (Mb)-like O 2 storage-functions [6,14,15]. These cellular Hbs reach concentrations in the millimolar range and thus may easily be identified due to the red colour of the Hb- containing organs. Recently, however, we showed that a true Hb gene (glob1 or Hb1) is present and expressed in the fruitfly Drosophila melanogaster [16,17]. This finding was unprecedented because at the first glance D. melano- gaster is unlikely to face hypoxic conditions during its life cycle. The crystal structure of D. melanogaster glob1 identified a protein that displays a typical globin fold and shows conservation of the residues important for O 2 binding [18]. Although exhibiting a hexacoordi- nated binding scheme at the Fe 2+ ion in the deoxygen- ated state, the O 2 -binding kinetics of glob1 are similar to those of other insect intracellular Hbs (P 50 ¼ 0.14 Torr) [17]. In larval and adult Drosophila, glob1 is mainly expressed in tracheal cells and the fat body. These data indicate that Hbs may be in fact involved in O 2 metabolism and suggest that Hb genes may be much more widely distributed in insects than previ- ously thought. Here we investigate the occurrence and evolution of glob1 in other Drosophila species (Fig. 1). Moreover, we identified two additional putative globin genes of Drosophila, which we named glob2 and glob3. Results The Drosophila glob1 genes The D. melanogaster glob1 nucleotide and amino-acid sequences were used to search the databases of genom- ic DNA sequences available from various Drosophili- dae, as they are available at the EMBL ⁄ GenBank (http://www.ncbi.nlm.nih.gov) or at FlyBase (http:// flybase.net/), by the blast algorithm [19]. We identi- fied the full length glob1 coding sequences from the genomes of D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. virilis, D. mojavensis and D. grim- shawi, as well as a partial sequence from D. simulans that covers only the first coding exon (see Supplement- ary material). The D. virilis glob1 sequence was also independently obtained by screening a genomic DNA library with the D. melanogaster glob1 coding sequence as probe. The D. virilis glob1 sequence (Acc. No. AM086021), which covers the complete glob1 gene, comprises 6265 bp. It differs from the genomic sequence at FlyBase (contig 462) at 20 positions and displays four short indels (1–26 bp). In addition, the cDNA sequences, including their putative 5¢ ends, from D. virilis and D. pseudoobscura (Acc. Nos. AM086022 and AM086023) were obtained by RT-PCR and subsequent RACE experiments. We identified a single splice variant for each D. virilis and D. pseudoobscura glob1, which contains three coding exons and a single 5¢ noncoding exon (Fig. 2). The glob1 mRNAs of D. pseudoobscura and D. virilis cor- respond to transcript ‘A’ of the D. melanogaster glob1 gene, which consists of exons 1, 4, 5 and 6 [17]. In addition, the in silico analysis of the gene predicts the presence of a second splice variant in D. pseudoobscura, which corresponds to transcript ‘C’ of D. melanogaster glob1 (Exons 1, 3, 4, 5 and 6). However, the corres- ponding cDNA was not recovered by RT-PCR. The glob1 gene plus the upstream and downstream genomic regions were scanned for the presence of puta- tive hypoxia responsive sequence elements (HREs) (Fig. 2). HREs in hypoxia-regulated genes are defined by the binding motif of the hypoxia-inducible transcrip- tion factor HIF-1 (5¢-RCGTG-3¢) [20]. Typically, an HRE includes two HIF-1 motifs (in direct or inverted orientation) or one HIF-1 motif combined with an ery- thropoietin (EPO) box or a HIF ancillary sequence [21]. Six putative HREs are present in the D. melano- gaster and D. virilis glob1 genes, while the D . pseudo- obscura glob1 contains five such motifs. Interestingly, Fig. 1. Evolutionary history of Drosophilidae. The phylogenetic rela- tionships of drosophilid species were taken from Russo et al. [52], divergence times are indicated as calculated by Tamura et al. [24]. T. Burmester et al. Drosophila hemoglobins FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 469 the HREs are located in similar positions, although motif combinations are not always conserved. The degree of sequence conservation ± 4 kb upstream and downstream of the glob1 genes were evaluated with PipMaker, using the D. melanogaster glob1 gene region as reference (Fig. 3A). The putative glob1 coding exons (see below) are conserved and clearly discernable in all Drosophila species. Outside the melanogaster subgroup (D. simulans, D. yakuba, D. erecta), hardly any other region displays sequence similarities. The only exceptions are the noncoding exon 1, which is transcribed in D. melanogaster, D. pseudoobscura and D. virilis, and a region within the third intron, about 2 kb 5¢ of the translation start codon, which may harbour regulatory sequences. The D. melanogaster glob1 gene is located on the right arm of chromosome 3 at band 89A8 between the genes CG14877, which codes for a putative natriuretic pep- tide receptor, and CG31292, which has no putative ortholog outside the Drosophila species (see Supple- mentary material). In the other species of the melano- gaster subgroup (Fig. 1) for which the genome locations are available (D. simulans and D. yakuba), the glob1 gene is positioned on the same chromosome. The D. pseudoobscura glob1 gene is located on chromo- some 2, which is orthologous to chromosome 3R of the melanogaster subgroup [22,23]. Gene synteny ana- lyses of 10 neighbouring genes (five on each side) reveals the conservation of gene locations and orienta- tions in this region between D. melanogaster and D. pseudoobscura, which diverged about 55 million years ago (Fig. 1). Because of the absence of data from other genomes, gene synteny analyses was restricted to the two neighbouring gene in these species. This shows the presence of both CG14877 and CG31292 adjacent to the glob1 in all Drosophila species (see Supplement- ary material). Gene orientations were found to be con- served in all Drosophila genomes except D. ananassae, in which the putative CG31292 ortholog was found in reversed orientation. However, this may also reflect an assembly error. The Drosophila glob1 proteins and gene evolution Gene predictions and comparisons with the available cDNA sequences show that Drosophila glob1 genes consistently harbour two introns, which are located at positions D7.0 (i.e. between codons 6 and 7 of globin helix D) and G7.0 (Fig. 4). The introns are short, vary- ing from 64 to 73 bp for the first intron and between 59 and 198 bp for the second intron. Except of D. pseudoobscura glob1, the coding sequences of all Drosophila glob1 genes measures 462 bp (including the stop codon) and translate into polypeptides of 153 amino acids with estimated molecular masses of 17.1– 17.5 kDa. The D. pseudoobscura glob1 sequence has an insertion of three bp close to the 3¢ end, resulting in a protein of 154 amino acids. Otherwise no indel was observed in the coding region. The interspecies amino- acid differences sum up from two to 36 positions (75.2–98.7% identity and 91.5–100% similarity, consid- ering isofunctional replacements). The heme- and ligand-associated residues, i.e. the proximal and distal histidines in helix positions E7 and F8, LeuB10, PheCD1, ArgE10, and IleE11 [18], are invariant in the glob1 proteins of all Drosophila species (Fig. 4; Table 1). Only about a third of the amino-acid posi- tions in the glob1 proteins were found variable. Most of the observed variations are located in the loop regions, in particular in the CD, EF and FG corners, while the helices are well conserved. Rates of molecular evolution were estimated on the basis of a PAM substitution matrix assuming the Dro- sophila divergence times suggested by Tamura et al. [24]. We found that the glob1 proteins have evolved with an Fig. 2. Structure of Drosophila glob1 genes. The genomic sequences of glob1 genes of D. melanogaster, D. pseudoobscura and D. virilis are displayed. Exonic sequences are boxed and numbered 1–6 on top, the coding regions are hatched. Putative TATA boxes are indicated and the positions of potentially relevant hypoxia-responsive sequence elements in the gene region are shown as open squares. Drosophila hemoglobins T. Burmester et al. 470 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS average rate of 1.61 ± 0.56 · 10 )9 amino-acid replace- ments per site and per year (Table 2). The lowest rate was calculated from the D. melanogaster – D. yakuba comparison (0.50 · 10 )9 ), which is likely due to a mutational slow-down in the D. yakuba lineage, whereas on the other hand a faster average rate of evolution was observed in the Drosophila subgenus clade, partic- ularly in the Hawaiian species D. grimshawi. On the nucleotide level, 0.90 ± 0.31 · 10 )9 nonsynonymous (dn) and 10.97 ± 5.61 · 10 )9 synonymous (ds) replace- ments per site per year were inferred. Levels of selective constraints were measured by ds ⁄ dn ratios [25]. Strong selective pressure on a coding region favours synonymous substitutions (ds) over nonsynonymous substitutions that cause amino-acid replacements (dn). The high average ds ⁄ dn ratio of 14.7 indicates that significant purifying selection has been imposed on the glob1 gene. Identification of two novel globin genes of Drosophila By searching the D. melanogaster genome with the tblastn algorithm and by employing various inverteb- rate globin sequences as templates, we identified two novel putative globin genes, which we tentatively called glob2 and glob3 (Figs 5 and 6). The glob2 gene is anno- tated as orphan D. melanogaster gene CG15180, glob3 is known as CG14675. Reverse blastp searches were then performed with exclusion of the Drosophila sequences. Assuming the BLOSUM45 model we obtained the high- est sequence similarity of D. melanogaster glob2 with the Hb of the bivalve Barbatia virescens (Acc. No. BAA09587; e-value: 0.003), and of D. melanogaster glob3 with the globin D of the sea cucumber Caudina arenicola (Acc. No. AAB19247; e-value: 5 · 10 )5 ). The relatedness of glob2 and glob3 to other globins was cor- roborated by a random shuffling approach, as it is implemented in the prss program [26]. The analyses yield an e-value of 6.96 · 10 )10 for the D. melanogaster glob2–B. virescens Hb comparison and an e-value 1.02 · 10 )9 for D. melanogaster glob3 vs. globin D C. arenicola. These comparisons further confirm the identity of glob2 and glob3 as true globins. Analyses of Drosophila glob2 genes and proteins The predicted D. melanogaster glob2 coding sequence covers 669 bp (including stop codon), which was con- firmed by sequencing a cDNA fragment that had been obtained by RT-PCR experiments on adult D. melano- gaster total RNA (Acc. No. AM086024). In addition, six entries in the database of D. melanogaster expressed sequence tags (ESTs [27]); (BE976463, BE978352, BE978841, BE976596, CB305306, CO331008) demon- strate that glob2 is actually expressed. Moreover, an antiserum that had been raised against synthetic glob2 peptides detects a protein band of about 26 kDa in extracts from total flies (Fig. 7), which corresponds to the expected size of glob2 (see below). Comparison of cDNA and genomic sequences show that the glob2 gene contains two introns, which are A B C Fig. 3. Conservation of Drosophila globin gene regions. Percent identity plot (PIP) showing the comparisons of the Drosophila glo- bin 1 (Gb1; A), globin 2 (Gb2; B) and globin 3 (Gb3; C) genes and their ± 4 kb flanking regions. The D. melanogaster genes were used as reference sequences, in the upper row the exons are boxed, with the black boxes representing the coding sequences. Gene extensions and transcriptional orientations are indicated by arrows. Interspecies sequence identities are shown as horizontal bars on a 50–100% scale. GC-rich regions are indicted as shown on the right hand side. The boxes in dark grey indicate missing data. Species abbreviations are: Dsi, D. simulans; Dya, D. yakuba; Der, D. erecta; Dan, D. ananassae; Dps, D. pseudoobscura; Dvi, D. virilis; Dmo, D. mojavensis; Dgr, D. grimshawi. T. Burmester et al. Drosophila hemoglobins FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 471 positioned in B12.2 and G7.0 (Fig. 5). The introns are short, measuring 52 and 127 bp, respectively. The pre- dicted D. melanogaster glob2 protein covers 222 amino-acid positions (25.3 kDa), which is longer than glob1 and most other globins (typically  140–150 amino acids). This is mainly due to the 50 and 30 amino-acid extensions at the N- and C-termini, respectively. Nevertheless, except of an indel of six amino acids in the DF corner (positions D6 to E4), the globin core is well conserved. This also comprises the residues crucial for heme- and O 2 - binding, e.g. the PheCD1 and the proximal and distal histidines E7 and F8 (Table 1). The calculated pI of glob2 is 9.18, indi- cating a highly basic protein. This coincides with the analyses according to the Reinhardt’s method [28], which predicts glob2 to be nuclear protein (reliability 76.7%). Putative glob2 orthologs were identified from the genomic sequences of D. simulans, D. yakuba, D. erecta, D. ananassae and D. pseudoobscura (see Supplementary material; Fig. 5). They all display a similar length, varying from 218 to 223 amino acids, and all have short introns in positions B12.2 and G7.0. Except of an insertion of five amino acids in the CD loop in D. ananassae glob2 the different lengths of these proteins result from variations of the N- and C-terminal extensions. All glob2 proteins were predicted to reside in the nucleus. The rate cal- culations, as estimated as described above (Table 2), show that the Drosophila glob2 proteins evolve with 7.62 ± 3.89 · 10 )9 replacements per site and per year. This is about six times faster than in the glob1 proteins. The relaxed selective constraint is also indi- cated by a ds ⁄ dn ratio of 6.5. The PipMaker analy- sis showed discernable sequence similarities within Fig. 4. Comparison of Drosophila glob1 amino-acid sequences. The secondary structure elements of Drosophila glob1 (PDB entry 2BK9 [18]) is shown in the upper row. The alpha-helices are designated A through H and the globin consensus helix numbering is given in the lower row. Conserved amino acids are shaded in grey. The abbreviations are: DmeGb1, D. melanogaster globin 1; DyaGb1, D. yakuba globin 1; DerGb1, D. erecta globin 1; DanGb1, D. ananassae globin 1; DpsGb1, D. pseudoobscura globin 1; DviGb1, D. virilis globin 1; DmoGb1, D. mojavensis globin 1, and DgrGb1, D. grimshawi globin 1. Table 1. Functionally important residues in selected globins. Amino acids at key positions in human Hb, myoglobin, Chironomus Hb, G. intestinalis Hb, and Drosophila glob1–3 are given. Species A12 B10 CD1 E7 E10 E11 F8 Human Hba Trp Leu Phe His Lys Val His Human Hbb Trp Leu Phe His Lys Val His Human Mb Trp Leu Phe His Thr Val His Human Ngb Trp Phe Phe His Lys Val His Chironomus Hb Trp ⁄ Phe ⁄ Tyr Leu Phe His Arg Ile ⁄ Val His G. intestinalis Hb Trp Leu Phe His Arg Ile His Drosophila glob1 Trp Leu Phe His Arg Ile His Drosophila glob2 Trp Phe Phe His Ala Met His Drosophila glob3 Trp Phe ⁄ Tyr Phe His Arg ⁄ Ala ⁄ Thr Phe His Table 2. Substitution rates in Drosophila globins. Amino-acid evolu- tion was modeled according to the PAM matrix [49]. Corrected nonsynonymous (dn) and synonymous (ds) nucleotide substitutions per site were calculated by the method of Ota and Nei [51]. Evolu- tion rates are given as estimated replacements per site and per year. Substitution rates (x 10 )9 ) ds ⁄ dn Amino acids dn ds Globin 1 1.61 ± 0.56 0.90 ± 0.31 10.97 ± 5.61 14.7 Globin 2 7.62 ± 3.89 4.21 ± 2.02 14.59 ± 6.88 6.5 Globin 3 7.28 ± 1.53 3.86 ± 0.78 17.64 ± 7.27 4.6 Drosophila hemoglobins T. Burmester et al. 472 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS the melanogaster subgroup (Fig. 3B), while in D. ananassae and D. pseudoobscura only the coding exons are conserved. The gene of D. melanogaster glob2 was found localized on the right arm of chromosome 3 at band 83F4. The flanking genes are CG31482 on the proxi- mal side, which encodes for chain A of the Golgi- associated Pr-1 protein, and CG15184 on the distal side, for which no putative ortholog is available and which is poorly conserved even within the Drosophili- dae. We therefore used the next neighbouring gene CG15178 for synteny analyses (see below), which encodes a highly conserved EF hand calcium binding protein. In all species of the melanogaster subgroup, glob2 is located on chromosome 3R, and on the orthologous chromosome 2 of D. pseudoobscura. Gene synteny analyses of the neighbouring genes showed conservation of gene order within the melanogaster subgroup. However, there is no syntenic conservation of the glob2 regions between D. melano- gaster and D. pseudoobscura or D. ananassae. The D. melanogaster orthologs of the genes adjacent to D. pseudoobscura glob2 are located on chromosome 3R in regions 83C, which is the glob3 locus (see below), and 100D. Fig. 5. Comparison of Drosophila glob2 amino-acid sequences. The secondary structure of Drosophila glob1 is shown in the upper row, alpha-helices designations and shading was performed as described in Fig. 4. Abbreviations: DmeGb2, D. melanogaster globin 2; DsiGb2, D. simulans globin 2; DerGb2, D. erecta globin 2; DyaGb2, D. yakuba globin 2; DpsGb2, D. pseudoobscura globin 2; DmoGb2, D. mojavensis globin 2. Fig. 6. Comparison of Drosophila glob3 amino-acid sequences. The secondary structure in the upper row derives from Drosophila glob1, other decorations were performed as described in the legends to Figs 4 and 5. Abbreviations: DmeGb3, D. melanogaster globin 3; DsiGb3, D. simulans globin 3; DerGb3, D. erecta globin 3; DyaGb3, D. yakuba globin 3; DanGb3, D. ananassae globin 3; DpsGb3, D. pseudoobscura globin 3; DviGb3, D. virilis globin 3. Fig. 7. Western blot analyses. About 50 lg of total adult protein were applied per lane and the glob1 (lane 1) and glob2 (lane 2) pro- teins were detected using specific antibodies. T. Burmester et al. Drosophila hemoglobins FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 473 Analyses of Drosophila glob3 genes and proteins The putative coding sequence of the D. melanogaster glob3 gene measures 588 bp, which translates into a protein of 195 amino acids. However, we could not obtain any glob3 cDNA from larval or adult RNA by RT-PCR. Moreover, no entry in the Drosophila EST database was recovered that corresponds to glob3. Because at that stage we could not exclude that glob3 were a pseudogene, we searched the genomic sequences of the other Drosophila species. In fact, we obtained apparently conserved orthologs genes of glob3 from D. simulans, D. yakuba, D. erecta, D. pseudoobscura and D. virilis (see Supplementary material; Fig. 6). Gene prediction and comparison with glob2 suggest the presence of the two introns in B12.2 and G7.0 also Fig. 8. Phylogeny of Drosophila and other dipteran globins. An alignment of the Drosophila globins, G. intestinalis Hb and selected Hbs from chironomid midges was analysed by MrBayes 3.1, assuming a WAG model of evolution with gamma-distribution of rates. The Daphnia Hb domains represent the outgroup. Bayesian posterior probabilities are given at the nodes. The bar corresponds to 0.1 PAM distance. The abbreviations are: DmaHb1.1 and DmaHb1.2, first and second domain of the hemoglobin (U67067) of D. magna; DpuHb1.1 and DpuHb1.2, first and second domain of the hemoglobin (Q9U8H0) of D. pulex; Chironomus thummi thummi hemoglobins CttHbI (P02221), CttHbIIb (AF001292), CttHbIII (M17691), CttHbIV (P02230), CttHbVIIB6 (A30477), CttHbVIII (P02227), CttHbIX (P02223), CttHbX (P02228), CttHbE (P11582), CttHbZ (P29245); C. thummi piger hemoglobins: CtpHbV (X56271), CtpHbW (X56271), CtpHbY (X56271); GinHb, Gasterophilus intestinalis hemoglobin (AF063938); for abbreviations of the Drosophila globins refer to Figs 4, 5 and 6. Drosophila hemoglobins T. Burmester et al. 474 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS in the glob3 genes. As for glob2, the glob3 amino-acid sequences include extensions at the N- and C-terminal ends, and are longer than the typical globin proteins. Another similarity of glob2 and glob3 is the six-amino acids indel in the DF corner. The residues important for heme-contact and O 2 -binding globins are conserved (Fig. 6; Table 1). Reinhardt’s method [28] predicts the glob3 proteins to reside in the cytoplasm. The evolu- tion rate of the glob3 proteins was calculated to be 7.28 ± 1.53 · 10 )9 replacements per site and per year, which is in the same range as that of glob2 (Table 2). The glob3 genes also show relaxed selective constraints, as indicated by the ds ⁄ dn ratio of 4.6. pipmaker com- parisons clearly identify the glob3 coding exons (Fig. 3C), while otherwise there is little conservation except of closely related species. The 5¢ noncoding region was found GC-rich. The D. virilis sequence was too diverged to allow PipMaker analyses. Database searches show that the glob3 gene of D. melanogaster is located on chromosome 3R at band 83C4. On the proximal side of glob3, the gene CG10981 encodes a putative RING zinc finger protein, on the distal side resides the gene CG1208, which codes for a sugar-transporter (see Supplementary material). Gene orders and orientations in this region are identical in the species of the melanogaster group (D. melanogaster, D. simulans, D. yakuba, D. erecta, D. ananassae). Gene synteny is only partially conserved outside this taxon: in D. pseudoobscura, D. mojavensis and D. virilis, the genes located proximal to D. melano- gaster glob3 are not on the same contig as glob3. Gene synteny is only conserved on the distal side. While in D. melanogaster glob2 and glob3 are separated on chromosome 3R by 800 kb, they are neighbouring genes in D. pseudoobscura on chromosome 2 in a head to tail orientation separated by 388 bp. Drosophila hemoglobin phylogeny Because preliminary phylogenetic studies had demon- strated the monophyly of arthropod globins (data not shown), we constructed an alignment that covers a total of 33 insect globin amino-acid sequences and includes all Drosophila globins. The globin domains of the extracellular Hbs of the branchiopod crusta- ceans Daphnia magna and D. pulex were used as outgroup. As expected, each of the Drosophila glob1–3 clades is monophyletic (Fig. 8). The closest relative of Drosophila glob1 is the intracellular Hb of the horse botfly G. intestinalis [15]. This clade is associ- ated with the extracellular Hbs of the Chironomidae. Although such grouping is supported by only 0.84 Bayesian posterior probability, it was consistently recovered in all types of analyses. Glob2 and glob3 are highly supported sistergroups. Within the Dro- sophila clades, the globins generally follow the accep- ted phylogeny of the species, with the exception of D. pseudoobscura glob1, which was found to be asso- ciated with D. erecta glob1. However, the support value was low and additional Bayesian analyses with other evolution models or Neighbor joining trees did not resolve this clade (data not shown). Discussion Conservation of Drosophila glob1 and functional implications Until recently, it has been assumed that Drosophila has no Hb genes and even after the determination of the full D. melanogaster genome [29] their presence in this species was explicitly denied [30]. Nevertheless, we identified a true Hb gene in D. melanogaster [16], which is closely related to the intracellular Hb of the horse botfly G. intestinalis and to the extracellular Hbs of the Chironomidae (Fig. 8). While there is no doubt that the G. intestinalis and Chironomus Hbs carry out respiratory functions in transporting and storing O 2 [12,14], the actual physiological role of the Drosophila Hbs remains to be determined. Similarities in the O 2 affinities and tissue distributions of G. intestinalis Hb and D. melanogaster glob1 may be considered as sup- port for a respiratory function of glob1 in Drosophila, possibly facilitating the flow of O 2 from the trachea and tracheoles into the tissues [17]. This view may sup- ported by the high expression rate of glob1 mRNA, as reflected by  130 D. melanogaster ESTs (which corres- ponds to  0.03% of all D. melanogaster ESTs). More- over, a rough estimate of glob1 protein content by comparing the western blot signal intensities (Fig. 7) with known concentrations of recombinant glob1 sug- gests that glob1 corresponds to approximately 0.1% of total adult proteins. Taking into account that the expression of glob1 is largely restricted to the larval and adult tracheal system, as well as some regions of the fat body, this appears to be a significant amount, which is compatible with the idea that glob1 enhances local oxygen diffusion rates or acts as oxygen store. Alternatively, glob1 may function as a buffer system that protects the inner organs from an excess of O 2 [31,32]. Moreover, other, nonrespiratory functions of glob1, such as the detoxification of reactive oxygen species or NO, are also conceivable. An important role of glob1 in the flies’ metabolism is supported by the strong conservation of the gene and protein. Its evolution rate of 1.61 ± 0.56 · 10 )9 T. Burmester et al. Drosophila hemoglobins FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 475 amino-acid substitutions per site and per year is low compared to glob2 and glob3. The evaluation of D. melanogaster and D. pseudoobscura glob1 coding sequences shows a low number of nonsynonymous substitutions (ds) of 0.06, whereas the median of the predicted orthologs of these species is 0.14 [23]. This is also reflected by a higher than average amino-acid identity of 88.9%, whereas the mean identity of 10 987 orthologous protein pairs of D. melanogaster and D. pseudoobscura is 77.7%. Together with the high mean ds ⁄ dn ratio (14.7), these data indicate that glob1 is a slowly evolving gene that is under significant selective pressure. Recent studies employing the D. melanogaster SL2 cell line have demonstrated that glob1 expression levels decreased under low oxygen conditions [33]. As in other animals, hypoxia-response in Drosophila is medi- ated by binding of HIF-1, which consists of an a- (Similar in Drosophila) and a b-subunit (tango in Drosophila), to HREs [34]. Unexpectedly, HIF-1 was demonstrated to cause a hypoxia-dependent down- regulation of glob1 in the SL2 cells [33]. Here we have shown that the glob1 genes of D. melanogaster, D. viri- lis and D. pseudoobscura harbour several putative HREs. Although without experimental evidence we cannot conclude which HRE is functional, the posi- tional conservation of the HREs in distantly related Drosophila species suggest that these elements may actually mediate a hypoxia-response of glob1. Two novel globin genes in Drosophila with uncertain function While the function of glob1 may be related to oxygen supply, the physiological roles of glob2 and glob3 are presently unknown. Moreover, we failed to amplify any glob3 mRNA from D. melanogaster. Although we cannot exclude technical problems, we must consider the possibility that this gene is not expressed under normal physiological conditions. Latter assumption is also supported by the fact that no glob3 cDNA could be found in the > 300.000 EST sequences of D. mel- anogaster. Nevertheless, the conservation of glob3 in Drosophila evolution (Fig. 6) suggests that this gene is not a pseudogene, but is functional. It is conceivable that glob3 is only expressed under particular physio- logical conditions or only during a short period of development. glob2 is expressed in Drosophila, as confirmed by RT-PCR experiments, EST database entries and western blotting. The expression level is much lower than that of glob1, as reflected by only six EST sequences in the database and own estimations based on western blots (Fig. 7). A respiratory function of glob2 is therefore unlikely, and at present the phy- siological role of this protein must remain uncertain. There are, however, two interesting features provide some hints. First, glob2 was predicted to reside in the nucleus. A nuclear Hb has been observed, e.g. in alfalfa (Medicago sativa) and has been related to the NO metabolism [35]. Second, it is noteworthy that all ESTs derive from a testis specific D. melanogaster cDNA library. Three additional D. yakuba glob2 ESTs are also available (CV790354, CV790570, CV784980), which were obtained from testis cDNA, too. This might suggest a testis-specific function of glob2 as transcriptional regulator, which, however, requires further investigations. Insect globin phylogeny Globins are widespread in all kingdoms of life and display a complex evolutionary pattern [4,6,36]. The last common ancestor of all insects certainly har- boured a globin gene, although is must remain uncertain whether it had respiratory functions. Our analyses indicate that the insect H bs are monophyletic, and that a clade comprising of glob2 and glob3 is the earliest offshoot of the insect globins. Therefore, the separation of the glob2 ⁄ glob3 clade and other insect globins likely occurred early in the evolution of this taxon. On the other hand we cannot exclude the possibility that the high evolution rate of glob2 and glob3 has masked its actual relatedness and have led to a long-branch attraction effect. The rela- tionship of glob2 and glob3 is not only confirmed by the phylogenetic analysis, but also by the fact that they are neighbouring genes in D. pseudoobscura . The most parsimonious explanation is therefore that glob2 and glob3 arose by gene duplication in the Drosophila stemline. During the evolution of the melanogaster group the glob2 gene was moved to a different region of the 3R chromosome. This is com- patible with the notion that within the Drosophila genus there is a strong tendency for genes to remain on the same chromosome arm [22,23]. The Drosophila glob1 genes share a recent common ancestry with the G. intestinalis Hb, and it may be assumed that the genes are orthologs that diverged upon the separation of the species (about 80–100 mil- lion years ago [37,38]). As mentioned above, this gene orthology might be considered as support for a role in respiration also of Drosophila glob1. The respiratory function of the Chironomus Hbs has convincingly been proven (for a review, see [5]). The phylogenetic tree show that the Chironomus Hb genes most likely derive Drosophila hemoglobins T. Burmester et al. 476 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS from intracellular Hbs, as represented today by G. intestinalis Hb and Drosophila glob1. Therefore, with the evolution of the chironomid midges there was a shift in function from an intracellular O 2 -binding protein with possible O 2 supply function to an extra- cellular O 2 -transport protein. This event must have occurred after the separation of the Chironomidae (Nematocera) and the brachyceran flies. An ancient origin of the Chironomus Hbs, as originally proposed, e.g. by Goodman et al. [39,40], is not supported. Introns and evolution It is well established that intron positions may be phy- logenetically informative, particularly in case of globin genes. In fact, the relatedness and the basal position of glob2 and glob3 within the insect globins are supported by the presence of introns B12.2 and G7.0. In fact, these two intron positions are conserved in all verte- brate and most invertebrate globin genes, and almost certainly reflect the ancient gene structure of the eukaryote globins [4]. Therefore, the loss and gain of introns occurred in the clade of glob1 and chironomid globin genes only after they diverged from a common ancestor. The G7.0 intron has been retained in the glob1 and G. intestinalis Hb, while the B12.2 intron was lost in this clade. Instead, a novel intron had emerged in the common ancestor of glob1 and G. intestinalis Hb at position D7.0. None of the known chironomid globin genes have introns in the ‘classical’ positions B12.2 or G7.0. Taking into account the phylogenetic tree (Fig. 8), the most parsimonious explanation for this pattern is independent intron losses. While the B12.2 intron was lost in the clade leading to the last common ancestor of Drosophila glob1, G. intestinalis Hb and the chironomid Hbs, the G7.0 intron was deleted only before the radiation of the chironomid globins. While most present-day chironomid globin genes do not have any introns, some have acquired during evolution central introns at positions E9.1 and E15.0 [41]. The complex intron pattern in dipteran globin genes can only be inter- preted in terms of a dynamic evolution of gene struc- ture by intron loss and insertion [42,43]. Experimental procedures Animals D. melanogaster Oregon R, D. pseudoobscura and D. virilis were maintained at 18° or 25 °C on standard yeast-corn- meal-agar-sucrose medium sprinkled with active dry yeast. Propionic acid and 4-hydroxymethylbenzoate were added as mould inhibitors. D. virilis were a kind gift of C. Kra- emer (Institute of Molecular Genetics, Mainz). Database searches and sequence analyses The blast algorithm [19] was employed to search the databases of genomic DNA sequences available at GenBank (http://www.ncbi.nlm.nih.gov) and FlyBase (http://flybase. net). The significance of the observed amino-acid sequence similarities were evaluated using a Monte-Carlo shuffling approach applying the prss3 program (fasta package [26]);. Probability scores were estimated using the blosum50 matrix assuming a gap creation penalty of )12 and a gap length penalty of )2 with 1000 shuffles. The relevant nucleotide sequences of the genes were extracted from the databases and assembled by the aid of genedoc 2.6 [44]. DNA translation and analyses of primary structures were performed with the programs of the ExPASy Molecular Biology Server (http:// www.expasy.org). Interspecific comparison of gene sequences was performed with multipipmaker (http://bio.cse.psu.edu/ pipmaker/), which computes and visualizes local alignments in two or more sequences based on the blastz algorithm [45,46]. The D. melanogaster genes were used as templates. The multiple sequence alignments from pipmaker were visualized as ‘percent identity plots’ (PIP). Promoter predic- tions were carried out using the server at BDGP (http:// www.fruitfly.org/seq_tools/promoter.html) [47]. Molecular biology RNA from Drosophila species were extracted either with the GITC method [48] or by the ENZA total RNA extrac- tion kit (Peqlab). Various specific or degenerate oligonucleo- tide primers were used to amplify cDNA fragments by reverse transcription-PCR experiments, employing the Qiagen OneStep kit system or the Superscript II RNase H – (Invitrogen) according to the manufacturer’s instructions. The primer sequences are available from the authors upon request. The sequences of the PCR products were either obtained by direct sequencing or after cloning of the frag- ments into the pCR4-TOPO vector (Invitrogen). Sequences were obtained from both strands using a commercial sequencing service (GENterprise). Missing 5¢- and 3¢ ends of the cDNAs were obtained using the RACE system by Invitrogen. A kFIX II-library of D. virilis genomic DNA was kindly provided by H. Kress. Screening was performed with a D. melanogaster glob1 probe that had been labelled with 32 P-ATP employing the random labelling kit by Roche. Positive phage clones were grown on E. coli p2392 in standard l-medium until lysis [48]. Phage were then precipi- tated over night with 7% polyethylene glycol, 6% NaCl and suspended in 10 mm MgCl 2 . Contaminations by bacterial nucleic acids were removed by 30 min digestion with RNase A and DNase I. Phage DNA was obtained by T. Burmester et al. Drosophila hemoglobins FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 477 [...]... site, were obtained for each gene according to the method of Nei and Gojobori [25], as implemented in the snap program [51] To estimate the rates of evolution of globin genes and proteins, we followed the standard phylogeny of Drosophilidae as proposed by Russo et al [52], but assumed the divergence times of drosophilid taxa suggested by Tamura et al [24] These are on average about twice as old as those... and 139–153 (H2N-DEARKRAMSTALRTT-COOH) The peptides were coupled to KLH and used for immunization of rabbits Peptide syntheses and immunizations were carried out by EUROGENTEC The specific anti-glob2 IgG were purified from the serum employing the synthetic peptides coupled to SulfoLink columns (Pierce) according to the instructions of the manufacturer After elution, the antibodies were stored at 4 °C in... Sequence evolution and phylogenetic studies The globin DNA and amino-acid sequences were aligned by hand with GeneDoc [44] Pairwise protein distances according to the pam model [49] were calculated with the program protdist from the phylip 3.64 package [50] Corrected estimates of dn, the number of nonsynonymous nucleotide substitutions per site, and ds, the number of synonymous nucleotide substitutions... breathe discontinuously to avoid oxygen toxicity Nature 433, 516–519 Burmester T (2005) A welcome shortage of breath Nature 433, 471–472 Gorr TA, Tomita T, Wappner P & Bunn HF (2004) Regulation of Drosophila hypoxia-inducible factor (HIF) activity in SL2 cells: identification of a hypoxiainduced variant isoform of the HIFalpha homolog gene similar J Biol Chem 279, 36048–36058 Crews S (1998) Control of. .. element in two species of the obscura group of Drosophila Chromosoma 104, 129–136 Richards S, Liu Y, Bettencourt BR, Hradecky P, Letovsky S, Nielsen R, Thornton K, Hubisz MJ et al (2005) Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution Genome Res 15, 1–18 Tamura K, Subramanian S & Kumar S (2004) Temporal patterns of fruit fly (Drosophila) evolution... distribution in globin genes of Chironomus: evidence for recent intron gain Gene 205, 151–160 42 Logsdon JM, Stoltzfus A & Doolittle WF (1998) Molecular evolution: Recent cases of spliceosomal intron gain? Curr Biol 8, R560–R563 43 Roy SW (2003) Recent evidence for the exon theory of genes Genetica 118, 251–266 44 Nicholas KB & Nicholas HB Jr (1997) GeneDoc: analysis and visualization of genetic variation... phylogenies: an approach using the bootstrap Evolution 39, 783–791 56 Huelsenbeck JP & Ronquist F (2001) mrbayes: Bayesian inference of phylogenetic trees Bioinformatics 17, 754–755 Supplementary material The following supplementary material is available online: Table S1 Chromosomal localisation of Drosophila globin and flanking genes This material is available as part of the online article from http://www.blackwell-synergy.com... distinct globin families in animals Mol Biol Evol 22, 12–20 Burmester T, Massey HC Jr, Zakharkin SO & Benesˇ H (1998) The evolution of hexamerins and the phylogeny of insects J Mol Evol 47, 93–108 FEBS Journal 273 (2006) 468–480 ª 2006 The Authors Journal compilation ª 2006 FEBS 479 Drosophila hemoglobins T Burmester et al 38 Wiegmann BM, Yeates DK, Thorne JL & Kishino H (2003) Time flies, a new molecular... based on multiple protein alignments (data not shown) The appropriate model of amino-acid sequence evolution was selected by ProtTest [53] using the Akaike Information Criterion (AIC) Distance matrices were calculated with tree-puzzle 5.01 [54] Neighbor-joining trees were inferred 478 with the program neighbor from phylip [50] The reliability of the trees was tested by bootstrap analysis [55] with 100... in search of their role in the vertebrate globin family J Inorg Biochem 99, 110–119 10 Willmer P, Stone G & Johnston I (2000) Environmental Physiology of Animals Blackwell, Oxford, UK 11 Hagner-Holler S, Schoen A, Erker W, Marden JH, Rupprecht R, Decker H & Burmester T (2004) A respiratory hemocyanin from an insect Proc Natl Acad Sci USA 101, 871–874 12 Ewer RF (1942) On the function of hemoglobin . earliest offshoot of the insect globins. Therefore, the separation of the glob2 ⁄ glob3 clade and other insect globins likely occurred early in the evolution of. expected, each of the Drosophila glob1–3 clades is monophyletic (Fig. 8). The closest relative of Drosophila glob1 is the intracellular Hb of the horse botfly